InteractiveFly: GeneBrief

Histone H2B: Biological Overview | References

BIOLOGICAL OVERVIEW

Histone post-translational modifications play an important role in regulating chromatin structure and gene expression in vivo. Extensive studies investigated the post-translational modifications of the core histones H3 and H4 or the linker histone H1. Much less is known on the regulation of H2A and H2B modifications. This study shows that a major modification of H2B in Drosophila is the methylation of the N-terminal proline, which increases during fly development. Experiments performed in cultured cells revealed higher levels of H2B methylation when cells are dense, regardless of their cell cycle distribution. dNTMT was identified as the enzyme responsible for H2B methylation. It was also found that the level of N-terminal methylation is regulated by dART8, an arginine methyltransferase that physically interacts with dNTMT and asymmetrically methylates Histone H3 arginine 2 (H3R2). These results demonstrate the existence of a complex containing two methyltransferases enzymes that negatively influence each other's activity (Villar-Garea, 2012).

In the eukaryotic nucleus DNA is packaged into chromatin by its association with the basic core histones. The folding of chromatin fibers has a major impact on many aspects of nuclear function and is regulated by the post-translational modification of the histone tail domains. In multicellular organisms malfunction of the enzymatic machinery that establishes these modifications leads to abnormalities such as failures in embryonic development, cancer and other diseases. Human H2B is phosphorylated at S14 by the caspase cleaved mammalian Mst-1 kinase (Cheung, 2003; Ajiro, 2010). H2A is phosphorylated at S1 (Bonenfant, 2007) where an ordered appearance and removal of distinct modifications is required for proper chromatin assembly. The modifications on the histone N-terminal tails are recognized by specialized proteins that selectively bind modified histones. The specific binding to particular modifications can then either lead to structural changes of chromatin or recruit enzymatic activities to specific loci, which in turn can either stimulate or inhibit a subsequent modification. Examples of this phenomenon are the stimulation of the acetylation of H3K14 by a phosphorylation of H3S10 and methylated at the N-terminal proline. Although the methylation of the terminal α-amino group has been found in H2B of a variety of organisms and a number of proteins other than histones. The two modifications that influence each other do not have to reside on the same molecule as it has been demonstrated that the ubiquitination of H2B by Rad6 facilitates the methylation of H3K4, suggesting a crosstalk of the two histone tails. Another example for such a crosstalk is the phosphorylation of H3S10 by the Pim1 kinase, which stimulates the acetylation (Villar-Garea, 2012).

Most of the global histone modification analyses done so far were performed on the two core histones H3 and H4 whereas the post-translational modifications of canonical H2A and H2B have been less well studied in metazoa. Only the ubiquitination of H2A and H2B has been suggested to have a specific function such as the silencing of genes (Wang, 2004) or the stimulation of H3K4 and H3K79 methylation, respectively (Lee, 2007; Briggs, 2002; Nakanishi, 2009). Human H2B is phosphorylated at S14 by the caspase cleaved mammalian Mst-1 kinase (Cheung, 2003). This phosphorylation has been proposed to mediate chromatin condensation during apoptosis, which is counteracted by the acetylation of the adjacent K15 (Ajiro, 2010). H2A is phosphorylated at S1 but so far this has not been shown to be regulated. The analysis of H2A and H2B methylation and acetylation in higher eukaryotes by mass spectrometry (MS) has been severely hampered by the multitude of different isoforms with similar molecular masses making it difficult to distinguish post-translational modifications from sequence variants. Drosophila melanogaster, in contrast to most higher organisms, has just a single H2B variant, which greatly facilitates the analysis of H2B PTMs (Villar-Garea, 2012).

Drosophila H2B is ubiquitinated at K120 (Weake, 2008; Bray, 2005), phosphorylated at S33 (Maile, 2004) and methylated at the N-terminal proline (Desrosiers, 1988). Although the methylation of the terminal α-amino group has been found in H2B of a variety of organisms and a number of proteins other than histones, its biological significance is largely unknown. Drosophila tissue culture cells show an increased proline methylation in response to heat shock and arsenite treatment. Also, the N-terminal methylation of mammalian Regulator of Chromosome Condensation 1 (RCC1) has been shown to be crucial for its binding to chromatin and for proper mitotic chromosomal segregation. A pair of ortholog enzymes that perform N-terminal methylation of proteins in humans and yeast, NRMT (METTL11A) and YBR261C/Tae1, respectively, has been also isolated (Webb, 2010; Tooley, 2010, Villar-Garea, 2012).

This study shows that N-terminal methylation of H2B in D. melanogaster is not only regulated by cellular stress such as heat shock but also changes during development. In tissue culture cells the proportion of methylated histone depends on cell density, but not on the cell cycle distribution. Knockdown experiments and in vitro assays demonstrate that Drosophila's ortholog of NRMT, dNTMT, mono- and di-methylates the N-terminus of H2B. Interestingly, dNTMT forms a complex with an arginine-specific H3 methyltransferase dART8, which targets H3R2. Although the interaction has no effects on the activity of the enzymes in vitro, modulation of dART8 levels in cells has a substantial effect on H2B N-terminal methylation in vivo. A knockdown of dART8 results in an increase of H2B methylation whereas over-expression leads to a reduction, suggesting a repressive effect of dART8 levels on the modification of the H2B N-terminus (Villar-Garea, 2012).

While studying the post-translational modifications of H2B in D. melanogaster this study found the methylation of its N-terminus as the major modification of this histone. In contrast to many other known N-terminal modifications, the methylation is highly regulated. The enzyme responsible for establishing this modification (dNTMT/CG1675) was identified and it was also confirmed that it physically interacts with another histone methyltransferase (dART8) specific for H3R2, thereby constituting a bifunctional methyltransferase complex. The two enzymes not only interact with each other physically but also have opposing functions as the reduction of dART8 protein levels results in an increase of N-terminal methylation and the overexpression in a strong decrease. This effect of the modulation of dART8 concentration on H2B methylation may be due to a negative crosstalk between the methylation of H3R2 and the N-terminal methylation of H2B or due to a competition of the two enzymes for the common cofactor SAM (Villar-Garea, 2012).

N-terminal methylation is a rare modification in eukaryotic proteins and has only recently been addressed functionally. The methylation of the N-terminus of human RCC1 has been shown to be important for its stable interaction with chromatin and its disturbance leads to mitotic defects in vivo. This regulation is in accordance with the structure of the nucleosome bound RCC1 molecule, where a N-terminal loop has been suggested to interact with the nucleosomal DNA. Interestingly, despite the strong conservation of RCC1 function in metazoans, the N-terminal methylation site of RCC1 is not conserved in the Drosophila ortholog of RCC1, suggesting that the stabilization of RCC1 on chromatin is either accomplished by a different mechanism or not required in Drosophila (Villar-Garea, 2012).

Eight proteins out of 36 that carry a recognition site for a presumptive N-terminal methyltransferases are conserved between humans and fruit flies. Those are three ribosomal subunits, two proteins associated with stress response and/or growth arrest, two with an unknown function and one member of the respiratory chain. Considering the conservation of the enzyme responsible for this modification, the low degree of overlap is very surprising. However, it is striking that frequently the human orthologous of proteins that carry a recognition site only in Drosophila can be found associated with putative targets in humans. So is for example RCC1 (methylated in humans but not in Drosophila) in a complex with H2B (methylated in Drosophila but not in humans) and many factors that are predicted to become N-terminally methylated are expressed in a testis specific manner in humans as well as in Drosophila. This suggests that the N-terminal methylation exerts its function on a protein complex as long as one subunit carries the modification (Villar-Garea, 2012).

What may be the function of H2B methylation in Drosophila? The observation that it increases during temperature stress as well as during differentiation points towards a role of H2B methylation in stabilizing chromatin. In both circumstances (heat shock and aging) the overall transcription is reduced and becomes restricted to a limited number of active genes. At the same time, only low levels of methylation are detected at early stages of embryonic development, where chromatin has been reported to be hyper-dynamic in other systems. In general, histones have been shown to have a higher turnover in dynamically transcribed regions compared to non-transcribed domains therefore lowering this turnover may have a repressive effect on general transcriptional activity. As N-terminal modifications have been shown to regulate protein turnover, H2B methylation might similarly stabilize the protein and contribute to a reduced overall transcription in differentiated or stressed cells. Alternatively, H2B methylation could also be a consequence of low transcriptional activity, which is the supported by the observation that it also increases when cells are treated with transcriptional inhibitors or with inhibitors of TopoII (Villar-Garea, 2012).

N-terminal modification of histones is not restricted to H2B in flies but has also been detected in H4, which is N-acetylated in virtually all eukaryotes, H2A, which is N-acetylated in human tissue culture cells and H2B from yeast, which is also acetylated at its N-terminal residue. None of the N-acetylations have been associated with a particular function and are thought to be constitutive modifications following histone synthesis. This description of a developmentally and stress-induced regulated N-terminal modification of H2B sheds new light on the potential function of this modification in chromatin metabolism (Villar-Garea, 2012).

The relative expression levels of dNTMT during different developmental stages correlate very well with the relative levels of H2B methylation. This suggests that the proportion of H2B methylation is regulated by the amount of enzyme present in the cell. However, in SL2 cells, it was also observed that the level of dART8 regulates H2B methylation, which suggests a second layer of control for H2B methylation. Although the two proteins interact physically, they do not methylate each other and the interaction does not lead to an alteration of dNTMTs activity. The observed interference of dART8 expression with H2B methylation can therefore not be explained by a direct effect mediated by the simple interaction of the two polypeptides. Expression studies show that the dNTMT expression is not downregulated by dART8 expression or upregulated in cells that lack dART8 (Villar-Garea, 2012).

Recently local SAM concentrations within the nucleus have been suggested to play an important role in regulating the activity of histone methyltransferases. The regulation of H2B methylation this study has observed in vivo by modulating dART8 levels may therefore be due to a competition of the two enzymes residing in the same complex for the limiting cofactor SAM. Several nuclear complexes have been shown to contain multiple methyltransferases activities that could potential be regulated by a similar mechanism. Alternatively, as dART8 methylates R2 at H3, a crosstalk between the two histone-tails where methylation of H3R2 inhibits H2B N-terminal methylation could also be an explanation for the reciprocal activities of the two enzymes. Based on these data, future studies are necessary that distinguish the possible mechanisms of how dART8 modulates dNTMTs activity within a single protein complex and analyze the role of the striking increase in H2B methylation during fly development (Villar-Garea, 2012).

The RING finger protein MSL2 in the MOF complex is an E3 ubiquitin ligase for H2B K34 and is involved in crosstalk with H3 K4 and K79 methylation

This study demonstrates that RING finger protein MSL2 in the MOF-MSL complex is a histone ubiquitin E3 ligase. MSL2, together with MSL1, has robust histone ubiquitylation activity that mainly targets nucleosomal H2B on lysine 34 (H2B K34ub), a site within a conserved basic patch on H2B tail. H2B K34ub by MSL1/2 directly regulates H3 K4 and K79 methylation through trans-tail crosstalk both in vitro and in cells. The significance of MSL1/2-mediated histone H2B ubiquitylation is underscored by the facts that MSL1/2 activity is important for transcription activation at HOXA9 and MEIS1 loci and that this activity is evolutionarily conserved in the Drosophila dosage compensation complex. Altogether, these results indicate that the MOF-MSL complex possesses two distinct chromatin-modifying activities (i.e., H4 K16 acetylation and H2B K34 ubiquitylation) through MOF and MSL2 subunits. They also shed light on how an intricate network of chromatin-modifying enzymes functions coordinately in gene activation (Wu, 2011).

This study describes a histone E3 ligase activity for the MOF-MSL complex. This activity mainly targets nucleosomal H2B K34, a site distinct from well-characterized H2B K120. Both MSL2 and MSL1, but not MOF and MSL3, are indispensable for the optimal activity. Importantly, H2B K34ub by MOF-MSL directly stimulates H3 K4 and K79 methylation through trans-tail regulation. It also affects H2B K120ub by regulating RNF20/40 chromatin association. The significance of MSL1/2-mediated H2B K34ub is underscored by data indicating that MSL1/2 activity is evolutionarily conserved in Drosophila DCC. Altogether, these results shed lights on the intricate network of chromatin-modifying enzymes that often function coordinately in gene activation (Wu, 2011).

Several early studies suggest that histone H2B can be ubiquitylated at sites other than K120 in vivo. For example, a proteomic study using a mouse brain sample identifies five ubiquitylation sites on histone H2B: K34, K46, K108, K116, and K120. Ubiquitylation of endogenous yeast H2B at multiple lysine residues in addition to K123 (K120 equivalent) has also been reported. Despite these observations, functions of these additional H2B ubiquitylation events as well as their respective Ub-conjugating enzymes and E3 ligases are largely unknown. Therefore, this study represents a step toward understanding the potential complexity of H2B ubiquitylation and their functions in transcription regulation beyond H2B K120ub. Comparing the activity of MSL1/2 to that of RNF20/40, several conclusions can be drawn: (1) MSL1/2 activity is lower than that of RNF20/40, contributing to low abundance of H2B K34ub in cells; (2) MSL1/2 has less strict substrate specificity, capable of weakly ubiquitylating H3 and H4 in vitro; and (3) MSL1/2 is mainly a monoubiquitin ligase, but it is able to add polyubiquitin chain to histones at lower efficiency. Given the distinct substrate specificity of MSL1/2 and RNF20/ 40, it is likely that similar to other histone modifications (e.g., methylation, acetylation), H2B ubiquitylation is catalyzed by a multitude of E3 ubiquitin ligases with distinct substrate preferences. Future studies on other RING finger-containing proteins will reveal yet-uncharacterized E3 enzymes and histone targets (Wu, 2011).

Like H2B K120ub, H2B K34ub plays important roles in regulating H3 K4/K79 methylation. It directly stimulates H3 K4 methylation by MLL and H3 K79 methylation by DOT1L in vitro. This result implies that trans-tail interactions between H2Bub and H3 methylation have certain plasticity in terms of H2B ubiquitylation site requirement. This is consistent with a previous report that disulfide mimics of ubH2B (uH2Bss) at K125 and K116 sites are able to stimulate DOT1L activity in vitro (Chatterjee, 2010). Given the demonstrated trans-tail regulation in this study, especially that H2B K34 resides between DNA gyres, it is important to further examine the basis for H2Bub-mediated stimulation of methyltransferase activity. It would be particularly interesting to test whether H2B K34ub stimulates H3 methylation by altering intrinsic nucleosome stability and/or nucleosome breathing mode (Böhm, 2011). In addition to a direct effect on H3 methylation, MSL1/2 also affects H3 methylation indirectly through regulating recruitment of RNF20/40 to chromatin and thus activity of RNF20/40. It was possible to differentiate these two effects by overexpression of RNF20/40 in MSL2 knockdown cells. Both immunoblot and ChIP assays support that MSL1/2 regulates H3 methylation through both direct and indirect mechanisms, further strengthening the case that MSL1/2 regulates an intricate network of histone modifications for transcription activation in cells (Wu, 2011).

Since it was not possible to detect any MOF-independent MSL1/2 heterodimer fraction on a size-exclusion column, it is likely that MSL1/2 always functions as an integral part of the MOF-MSL complex. Taking advantage of the fact that different histone modifying activities require different MOF-MSL components, it was possible to distinguish contributions of H2B K34ub and H4K16ac to transcription activation. MSL1 knockdown, which affects both H2B K34ub and H4 K16ac, has much more profound effects on HOX gene expression than MSL3 knockdown, which only affects H4 K16ac. It indicates functions of MOF-MSL in transcription regulation beyond HAT (Wu, 2011).

The finding that MOF-MSL regulates H2B K120ub and H3 K79me2, two important marks for transcription elongation, supports a role of MOF-MSL in later transcription events. Interestingly, the dMSL complex is implicated in the regulation of transcription elongation in Drosophila: (1) it is recruited to the bodies of X-linked genes, and (2) recruitment of the dMSL complex to ectopic loci with X chromosome enhancer sequences depends on active transcription, and (3) using global run-on sequencing (GRO-Seq), a recent study shows that the dMSL complex functions to facilitate progression of RNA Pol II across the bodies of active X-linked genes (Larschan, 2011). It would be interesting to test whether MOF-MSL in mammals regulates gene expression in the same manner and whether H2B K34ub (relative to H4 K16ac) contributes to the cascade of events associated with elongating Pol II. Future genome-wide analyses of H2B K34ub and MSL1/2 in mammalian cells will be very informative to further dissect the functions of different histone-modifying activities of MOF-MSL in transcription regulation (Wu, 2011).

Several remaining issues for H2B K34ub await future studies. First, how is H2B K34ub regulated in vivo? A series of elegant yeast genetic studies and in vitro biochemical studies show that H2B K120ub is extensively regulated by factors associated with Pol II transcription. They include the PAF complex, Pol II CTD, and Kin28 kinas. In addition, H2B K120ub is dynamically controlled by ubiquitin proteases such as Ubp8 and Ubp10. Given the robust activity of MSL1/2 in vitro and low H2B K34ub level in cells, it is likely that H2B K34ub is under extensive regulation. It is important to examine whether regulation of H2B K34ub and H2B K120ub is the same, or whether it involves different mechanisms despite their shared downstream effects on H3 methylation (Wu, 2011)

Second, what is the function of H2B K34ub in regulating chromatin structures? K34 resides in a highly conserved region in H2B from yeast to mammal. This region consists of a stretch of eight basic residues. Like the basic patch on the H4 N-terminal tail (14–20 aa), this H2B region is also proposed to play important roles in the formation of chromatin fibers by facilitating interactions with neighboring nucleosomes. This H2B basic patch overlaps with a conserved 'HBR' motif (30–37 aa) identified in S. cerevisiae, whose deletion leads to derepression of 8.6% of yeast genes. Given that both H2B K34 and H4 K16 are substrates of MOF-MSL and both reside within a basic patch, it is important to determine whether these two distinct histone modifications play any synergistic roles in regulating higher-order chromatin structures (Wu, 2011).

Finally, does dH2B K31ub play a role in Drosophila dosage compensation? Given the E3 ligase activity of the dMSL complex, it is important to test whether dH2B K31ub marks Drosophila male X chromosome and whether inactive dMSL2 leads to defects in expression of X-linked genes in male fly. It has been reported that dMSL1/2, but not other dMOF complex components, binds to large chromosome domains defined as high-affinity sites (HAS) or chromosome entry sites (CES) and initiates the spreading of H4 K16 acetylation mark along male X chromosome. It is important to examine whether the dMSL1/2 activity plays any role in setting up these HAS/CES sites in vivo. One common feature of HAS/CES is their relatively low nucleosome density compared to other chromatin regions. In light of the E3 activity of Drosophila dMSL1/2, it is important to test whether dMSL1/2 prefers to bind the nucleosome-free region or whether this is a result of nucleo- some destabilization caused by dH2B K31ub (Wu, 2011).

Mammalian SWI/SNF--a subunit BAF250/ARID1 is an E3 ubiquitin ligase that targets histone H2B

The mammalian SWI/SNF chromatin-remodeling complex facilitates DNA access by transcription factors and the transcription machinery. The characteristic member of human SWI/SNF-A is BAF250/ARID1, of which there are two isoforms, BAF250a/ARID1a and BAF250b/ARID1b. This study reports that BAF250b complexes purified from mammalian cells contain elongin C (Elo C), a BC box binding component of an E3 ubiquitin ligase. BAF250b was found to have a BC box motif, associate with Elo C in a BC box-dependent manner, and, together with cullin 2 and Roc1, assemble into an E3 ubiquitin ligase. The BAF250b BC box mutant protein was unstable in vivo and was autoubiquitinated in a manner similar to that for the VHL BC box mutants. The discovery that BAF250 is part of an E3 ubiquitin ligase adds an enzymatic function to the chromatin-remodeling complex SWI/SNF-A. The immunopurified BAF250b E3 ubiquitin ligase was found to target histone H2B at lysine 120 for monoubiquitination in vitro. To date, all H2B monoubiquitination was attributed to the human homolog of yeast Bre1 (RNF20/40). Mutation of Drosophila osa, the homolog of BAF250, or depletion of BAF250 by RNA interference (RNAi) in cultured human cells resulted in global decreases in monoubiquitinated H2B, implicating BAF250 in the cross talk of histone modifications (Li, 2010).

This study has shown that BAF250b interacts with Elo C and Cul2. The interaction with Elo C and Cul2 is dependent on a BC box in the CTD of BAF250b, whereas the Cul2 interaction requires both ARID and the BC box. In vivo, the single amino acid substitution in the BC box (BC*) resulted in the degradation of the BC box mutant BAF250b through autoubiquitination. The Cul2-dependent regulation of BC* supports the idea that BAF250b serves as an E3 ubiquitin ligase substrate recognition module in vivo. Histone H2B has been determined to be a substrate for the BAF250 complex in a nucleosomal context. It is proposed that this complex is assembled in a manner similar to that for the well-characterized VHL complex, which targets HIF1α. The ARID and the CTD of BAF250 have been shown to be important for transcriptional activation. BAF250CTD was also shown to interact with the glucocorticoid receptor to activate transcription. It is possible that the coactivator function of BAF250 is in part mediated through its association with Elo C and Cul2 (Li, 2010).

Until now, all ubiquitinated H2B (H2B-Ub) was thought to arise from the action of the heterodimeric RNF20/40 E3 ubiquitin ligase, although it has been noted that depletion of RNF20 by RNAi affected transcription of only a subset of genes. H2B-Ub has been shown to be required for transcriptional activation in vitro and associates with transcriptionally active genes in vivo. RNF20 is sufficient for ubiquitin ligase activity in vitro and shares approximately 30% homology with S. cerevisiae Bre1, which performs the analogous function in yeast. Interestingly, the yeast BAF250 ortholog Swi1 lacks an identifiable BC box necessary for interaction with Elo B/C. The Elo B/C interaction domain and the Swi1 ARID are also not highly conserved. Swi1 has not been identified in genetic screens for factors affecting H2B monoubiquitination, and multiple groups have reported on the elimination of H2B-Ub upon deletion of bre1 in yeast. Thus, the probability of Swi1 possessing E3 ubiquitin ligase activity seems low. Because yeast is a unicellular organism, the layers of epigenetic regulation necessary in mammals would not be required. The BAF250 E3 ubiquitin ligase activity therefore may reflect an evolutionary adaptation unique to the demands of developmental regulation in multicellular organisms (Li, 2010).

The BAF250-Elo B/C-Cul2-Roc1 complex would also be regulated by assembly and neddylation of the cullin subunit, whereas RNF20/RNF40 is presumably constitutively active. Dynamic regulation of the BAF250 E3 ubiquitin ligase assembly and activity by neddylation, the COP9 signalosome, and TIP120A/CAND1 complexes would allow the type of temporal regulation necessary in early development. Recent studies of developing cells have demonstrated that temporal regulation of different transcription factor complexes is important to cell fate decisions (Li, 2010).

Both BAF250a and BAF250b are expressed in mammalian embryonic stem cells. Chromatin immunoprecipitation studies suggest that Nanog, Oct4, and Sox2 occupy the BAF250b promoter, while E2F4 occupies the BAF250a promoter. The dependence of mammalian embryonic stem cells on BAF250a and -b argues that these are genuine trxG members. Although trxG and PcG proteins have antagonistic roles in controlling HOX gene expression, many of the trxG and PcG proteins studied have chromatin-remodeling and chromatin-modifying capabilities. Posttranslational modifications of histones, such as H3 lysine-4 trimethylation (H3K4me3) and H2B monoubiquitination (H2B-Ub), show a positive correlation with transcription activation. H2B-Ub is required for transcription from a chromatinized template in vitro and regulates H3K4me3 levels in vivo. In contrast, H2A monoubiquitination is considered a transcriptionally repressive mark set by Ring1a/b, part of Polycomb repressor complex 1. In accordance with Osa's antagonistic role in regard to Ring1a/b in HOX gene regulation, this study shows that BAF250a is a positive regulator of HoxA9 and that the BAF250b complex is specific for histone H2B in a nucleosomal context. Indeed, in vitro ubiquitination assays and in vivo knockdown experiments indicate that H2B is a target of the BAF250 E3 ubiquitin ligase. BAF250 is not essential to SWI/SNF targeting in vivo or to chromatin remodeling in vitro. Thus, it is not thought that BAF250 siRNA knockdown has disrupted SWI/SNF remodeling activity in general. Furthermore, Osa and Cul2 synergistically interact in genetic analysis carried out with the Drosophila wing discs, confirming the importance of association with Cul2 to the function of Osa in vivo. The discovery that BAF250 associates with Elo B/C, Cul2, and Roc1 to form an E3 ubiquitin ligase that specifically monoubiquitinates histone H2B in a nucleosomal context provides a mechanistic explanation for Osa's classification as a trxG member (Li, 2010).

The identification of a novel ubiquitination pathway mediated by the chromatin-remodeling complex SWI/SNF-A raises several important questions. Is the H2B-Ub mark set before or after remodeling? Does H2B-Ub affect chromatin remodeling? Is the H2B ubiquitination mediated by BAF250 gene specific? If so, what distinguishes such genes from those targeted by the E3 ligase RNF20/40? Genome-wide analysis of the occupancy of BAF250, RNF20/40, and H2B-Ub will help to identify genes targeted by the ubiquitin ligase activity of BAF250, which would facilitate future biochemical studies (Li, 2010).

dDsk2 regulates H2Bub1 and RNA polymerase II pausing at dHP1c complex target genes

dDsk2 (Ubqln2/Ubiquilin) is a conserved extraproteasomal ubiquitin receptor that targets ubiquitylated proteins for degradation. This study reports that dDsk2 plays a nonproteolytic function in transcription regulation. dDsk2 interacts with the dHP1c complex, localizes at promoters of developmental genes and is required for transcription. Through the ubiquitin-binding domain, dDsk2 interacts with H2Bub1 (monoubiquitylated H2B), a modification that occurs at dHP1c complex-binding sites. H2Bub1 is not required for binding of the complex; however, dDsk2 depletion strongly reduces H2Bub1. Co-depletion of the H2Bub1 deubiquitylase dUbp8/Nonstop suppresses this reduction and rescues expression of target genes. RNA polymerase II is strongly paused at promoters of dHP1c complex target genes and dDsk2 depletion disrupts pausing. Altogether, these results suggest that dDsk2 prevents dUbp8/Nonstop-dependent H2Bub1 deubiquitylation at promoters of dHP1c complex target genes and regulates RNA polymerase II pausing. These results expand the catalogue of nonproteolytic functions of ubiquitin receptors to the epigenetic regulation of chromatin modifications (Kessler, 2015).

This study reports that the extraproteasomal ubiquitin receptor dDsk2 interacts with the dHP1c complex, localizes at promoters and is required for transcription. Binding sites of the dHP1c complex are marked by H2Bub1 and the results suggest that, through the UBA domain, dDsk2 binds H2Bub1. However, reducing H2Bub1 levels affects binding of the complex only weakly, indicating that the interaction with H2Bub1 has only a minor contribution to recruitment. Actually, dDsk2 contains a single ubiquitin-binding site of low affinity (Kd~400 μM), which is in contrast to most ubiquitin receptors that contain several ubiquitin-binding sites that act synergistically to provide high-affinity binding. Similarly, the interaction of dDsk2 with H2Bub1 is of low specificity since selective recognition of ubiquitylated substrates is largely based on the recognition of the linkage type, length and anchoring site of a polyubiquitin chain, and, consequently, requires the presence of several ubiquitin-binding sites. In fact, the UBA domain of dDsk2 recognizes a monoubiquitylation in PTEN with a similar affinity as in H2B. On the other hand, binding of the dHP1c complex likely involves the recognition of specific DNA sequences since it depends on the zinc-finger proteins WOC and ROW. Noteworthy, dHP1c complex-binding sites are significantly enriched in a specific DNA sequence motif. However, although the interaction with H2Bub1 is weak, binding of the complex unexpectedly depends on dDsk2. Besides, dHP1c is dispensable for WOC and ROW binding, as well as for dDsk2 binding). These effects do not appear to be the consequence of changes in gene expression levels since dDsk2 mRNA levels do not significantly change on WOC, ROW or dHP1c depletion. Furthermore, dDsk2 depletion upregulates ROW and weakly downregulates dHP1c mRNA levels. Finally, it was reported that dHP1c interacts with RNA pol II, suggesting that binding of the dHP1c complex might depend on RNA pol II. However, arguing against this possibility, it was observed that binding of the complex at promoters is resistant to treatment with Actinomycin D. Altogether, these results suggest that WOC, ROW and dDsk2 constitute the actual binding module of the complex, being fully interdependent for binding to chromatin and required for binding of dHP1c. In this regard, the slight reduction of ROW and dHP1c protein levels observed on dDsk2 depletion is most likely due to their inability to bind chromatin, as described previously for dHP1c in ROW and WOC knockdowns, as well as for other chromatin-associated proteins when their binding to chromatin is impaired (Kessler, 2015).

These results suggest that the main function of dDsk2 in the dHP1c complex is to prevent H2Bub1 deubiquitylation by dUbp8/Nonstop, as H2Bub1 levels are strongly reduced on dDsk2 depletion and recovered after dUbp8/Nonstop co-depletion. Protection against deubiquitylation has also been reported for Rad23 and appears to be a common feature of many ubiquitin receptors. Simultaneous dDsk2 and dUbp8/Nonstop depletion also restores expression of target genes, whereas it has no effect on recruitment of the complex at promoters. Furthermore, overexpression of the ΔUBA–dDsk2 construct, which misses the UBA domain that mediates interaction with H2Bub1 in vitro, reduces H2Bub1 levels at promoters and downregulates expression of target genes. Altogether, these results strongly suggest that the contribution of dDsk2 to transcription regulation is mainly based on this protective function (Kessler, 2015).

dHP1c complex target genes show features associated with strong RNA pol II pausing. The results support a contribution of dDsk2 to pausing since its depletion reduces RNA pol II occupancy preferentially at TSS and strongly decreases NELF-E levels. dDsk2 depletion downregulates expression and, in good agreement, total RNA pol II occupancy across target genes is reduced. Interestingly, the majority of NELF target genes are also downregulated on NELF depletion in S2 cells51, and NELF potentiates gene expression in the Drosophila embryo. Actually, ~80% of dHP1c complex target genes that change expression in NELF knockdown conditions are downregulated. Furthermore, NELF-E depletion shows a similar reduction of total RNA pol II occupancy across target genes. Altogether, these observations suggest that disrupting RNA pol II pausing does not generally increase productive transcription, but results in reduced total RNA pol II occupancy and decreased expression. It is possible that premature pause release interferes with RNA pol II activation into elongation, resulting in abortive transcription that, in turn, could affect RNA pol II recruitment and/or re-initiation. Notice, however, that dDsk2 depletion does not affect the extent of histone acetylation detected at target promoters, suggesting that they retain the transcriptional active chromatin state (Kessler, 2015).

Notably, co-depletion of dUbp8/Nonstop, which rescues H2Bub1, also rescues the pausing defect caused by dDsk2 depletion and expression levels are restored, suggesting that dynamic regulation of H2Bub1 levels at promoters of dHP1c target genes plays a role in RNA pol II pausing. In this regard, work performed in yeast suggested that transcriptional activation involves sequential cycles of H2B ubiquitylation and deubiquitylation and that Ubp8 promotes Ctk1-dependent phosphorylation of Ser2 in the CTD33, a modification that is required for activation into the elongating Pol IIoser2 form. Nevertheless, H2Bub1 deubiquitylation at TSS does not appear to be sufficient by itself to induce pause release since dBre1 depletion, which also reduces H2Bub1 at TSS, has no significant effect in pausing. In this regard, it must be noted that dBre1 travels with the elongating RNA pol II along coding regions to induce H2Bub1, which stimulates Facilitates-Chromatin-Transcription (FACT) activity and, thus, facilitates elongation. On the other hand, dUbp8/Nonstop activity is mainly restricted to promoters since its depletion in dBre1-deficient cells has little effect in H2Bub1 levels at coding regions. On the contrary, dUbp8/Nonstop depletion in dDsk2-deficient cells strongly rescues H2Bub1 levels at coding regions. Interestingly, whereas dUbp8/Nonstop depletion restores expression of target genes in dDsk2-depleted cells, it has only a slight effect in dBre1-deficient cells. Altogether, these results suggest that dBre1 depletion impairs elongation and, thus, might prevent the release of paused RNA pol II by disturbing its actual engagement into elongation. Further work is required to better understand the mechanisms that regulate RNA pol II pausing, the actual contribution of dDsk2 and whether it involves H2Bub1 and/or additional factors also targeted by ubiquitylation (Kessler, 2015).

The dHP1c complex appears to have a particularly important contribution to nervous system development and function since target genes are enriched in related functions and knockdown conditions preferentially affect gene expression in the nervous system. Actually, WOC and ROW are highly expressed in the nervous system during embryo and larval development, and mutant larvae show brain defects. Furthermore, in humans, the WOC homologue DXS6673E/ZNF261 has been implicated in X-linked mental retardation. Interestingly, mutations in the human Dsk2 homologues (Ubqln-1/2) have been associated with Alzheimer's disease as well as other neurodegenerative diseases. Noteworthy, Ubqln-1/2 are detected in both the nucleus and the cytoplasm, and the development and progression of neurofibrillary tangles in Alzheimer's disease brains associate with an altered nuclear Ubqln-1 content. Whether the role of dDsk2 in transcription regulation is conserved in humans and contributes to disease remains to be determined (Kessler, 2015).

In summary, these results indicate that the ubiquitin receptor dDsk2 plays a nonproteolytic function in the regulation of H2Bub1 and RNA pol II pausing at promoters of dHP1c complex target genes. Ubiquitin receptors have been previously reported to play nonproteolytic functions in DNA repair and transcription elongation. Furthermore, in response to DNA damage, human Rad23B was found to interact with ubiquitylated p53, localize at chromatin and accumulate at the p21 promoter. In addition, in mouse embryonic stem cells, several components of the NER complex, including Rad23B, have been shown to act as an Oct4/Sox2 co-activator complex that associates with chromatin and is required for stem cell maintenance. Recruitment of NER factors to active promoters has also been reported in HeLa cells in the absence of DNA damage. However, in these cases, the precise function of the ubiquitin receptor has not been elucidated. In this regard, these results expand the catalogue of nonproteolytic functions of ubiquitin receptors to the epigenetic regulation of chromatin modifications and transcription initiation. It must also be noted that ubiquitylation participates in the regulation of multiple genomic functions and that the number of proteins containing ubiquitin-binding domains is large, ~100 in humans. Therefore, a role of ubiquitin-binding proteins as epigenetic regulators of chromatin emerges as a distinct possibility (Kessler, 2015).

Loss of Drosophila Ataxin-7, a SAGA subunit, reduces H2B ubiquitination and leads to neural and retinal degeneration

The Spt-Ada-Gcn5-acetyltransferase (SAGA) chromatin-modifying complex possesses acetyltransferase and deubiquitinase activities. Within this modular complex, Ataxin-7 anchors the deubiquitinase activity to the larger complex. This study identified and characterized Drosophila Ataxin-7 (CG9866) and found that reduction of Ataxin-7 protein results in loss of components from the SAGA complex. In contrast to yeast, where loss of Ataxin-7 inactivates the deubiquitinase and results in increased H2B ubiquitination, loss of Ataxin-7 results in decreased H2B ubiquitination and H3K9 acetylation without affecting other histone marks. Interestingly, the effect on ubiquitination was conserved in human cells, suggesting a novel mechanism regulating histone deubiquitination in higher organisms. Consistent with this mechanism in vivo, this study found that a recombinant deubiquitinase module is active in the absence of Ataxin-7 in vitro. When the consequences of reduced Ataxin-7 were examined in vivo, it was found that flies exhibited pronounced neural and retinal degeneration, impaired movement, and early lethality (Mohan, 2014).

The Spt-Ada-Gcn5-acetyltransferase (SAGA) chromatin-modifying complex is a highly conserved 2-MDa protein complex comprised of ∼20 subunits. The complex is arranged in a modular fashion and contains two enzymatic activities: an acetyltransferase activity associated with the GCN5/Pcaf subunit and a deubiquitinase activity associated with the yUbp8/dNon-stop/hUsp22 subunit. In yeast, the ataxin-7 homolog is Sgf73, and studies have shown that it anchors the deubiquitinase module (which includes Sgf73, Sgf11, Sus1, and Ubp8) to the SAGA complex. Crystal structures and biochemical analysis of the yeast deubiquitinase module have shown that the N terminus of Sgf73 extends deep into the deubiquitinase module, intertwining between the components of the module to ensure an active conformation for the deubiquitinase. Without Sgf73, the deubiquitinase is inactive. The carefully orchestrated addition and removal of ubiquitin on H2B are important regulators of transcription. H2B monoubiquitination is a prerequisite for di- and trimethylation of H3K4 and H3K79, modifications associated with transcriptionally active chromatin (Mohan, 2014).

This study identified the gene product of CG9866 as the Drosophila homolog of ataxin-7 (Ataxin-7). Biochemical analysis, including affinity purification, multidimensional protein identification technology (MudPIT) proteomic analysis, and gel filtration chromatography, establish that Ataxin-7 is a stable component of the SAGA chromatin remodeling complex. Analysis of SAGA from Ataxin-7-deficient flies revealed the loss of components from the SAGA complex, consistent with a role for Ataxin-7 in anchoring the deubiquitinase module to the complex. In contrast to the increased H2Bub observed upon loss of ataxin-7 in yeast, this study observed a decrease in H2B ubiquitination in Drosophila with no associated changes in histone methylation and a reduction in the levels of H3K9 acetylation but not K3K14 acetylation. This surprising change in H2B ubiquitination was confirmed in human cells in which knockdown of human Ataxin-7 also resulted in decreased H2B ubiquitination. It is hypothesized that this decrease reflects the release of an active deubiquitinase module from SAGA and, consequently, loss of SAGA-associated regulation of the deubiquitinase activity. Consistent with this model, it was found that the deubiquitinase is enzymatically active when the complex is reconstituted in vitro even in the absence of Ataxin-7. An examination of flies with reduced expression of Ataxin-7 showed that loss of Ataxin-7 results in neural and retinal degeneration, impaired movement, and decreased life span (Mohan, 2014).

This study presents the first study of Drosophila Ataxin-7. Ataxin-7 shares primary amino acid sequence conservation with human Ataxin-7 and, accordingly, is also a member of the SAGA chromatin-modifying complex. SAGA subunits are lost in the absence of Ataxin-7, resulting in a global decrease in the levels of H2B ubiquitination and H3K9 acetylation without affecting H3K14ac, H3K4me2/3, or H3K79me3. Because Ataxin-7 associates with the intact SAGA complex and loss of Ataxin-7 results in fragmentation of the complex, this study explored whether decreased levels of H2B ubiquitination were due to release of an active deubiquitinase module from SAGA. Indeed, the deubiquitinase module assembled in vitro from purified Non-stop, E(y)2, and Sgf11 is enzymatically active and is unaffected by the presence or absence of Ataxin-7. In vivo, disruption of Ataxin-7 expression leads to severe neural and retinal degeneration, limited life span, and defective locomotion. These defects are at least in part due to elevated deubiquitinase activity, since loss of one copy of the Non-stop deubiquitinase suppresses the lethality of Ataxin-7 mutants (Mohan, 2014).

These results suggest a more elaborate mode of regulation of histone ubiquitination in higher eukaryotes than that found in yeast. Studies in Saccharomyces cerevisiae showed that the deubiquitinase module comprised of Sgf73, Ubp8, Sgf11, and Sus1 is arranged so that each member of the module is in contact with the other three, and these contacts establish an enzymatically active module. Truncation of the Sgf73 N terminus led to an enzymatically inactive module. In contrast, in Drosophila and in human cells, the presence of Ataxin-7 is not necessary for deubiquitinase activity, and, instead, loss of Ataxin-7 results in increased deubiquitination and reduced levels of H2B ubiquitination (Mohan, 2014).

Interestingly, it wa found that this decrease in H2B ubiquitination does not coincide with a decrease in H3K4me2/3 or H3K79me3. Previously, it was shown that H2B ubiquitination was necessary for recruitment of methyltransferases to place these marks, and reduction of the Drosophila Bre1 E3 ubiquitin ligase results in loss of both H2B ubiquitination and H3 methylation. If indeed H2B ubiquitination is required for H3 methylation, then the mark would have been placed and then removed post-methylation by the mistargeted deubiquitinase module. This suggests that methylation may not be affected by the loss of Ataxin-7 because the deubiquitinase acts after ubiquitination-dependent methylation has occurred. It is also possible that a high level of H2B ubiquitination is not required to target methyltransferases. Recently, it was shown in yeast that loss of Chd1 results in reductions in H2B ubiquitination without a corresponding loss of H3K4 or H3K79 trimethylation, suggesting that very low levels of H2B ubiquitination are enough to target methyltransferases. Alternatively, it is possible that another mechanism exists for targeting methyltransferases when levels of ubiquitinated H2B are low or absent. During muscle differentiation, the RNF20 ubiquitin ligase is depleted in differentiated myotubes, resulting in the absence of H2B ubiquitination, yet trimethylation of H3K4 and H3K79 is detected on chromatin lacking H2B ubiquitination. Recently, it was shown that transcription factors such as p53 along with p300 can recruit the SET1 histone methyltransferase complex independent of H2B ubiquitination (Mohan, 2014).

Mutation of different SAGA subunits does not necessarily result in changes in expression of the same sets of genes. Disrupting expression of subunits in the acetyltransferase module versus the deubiquitinase module affects a curiously divergent set of genes, indicating that different genes have varying requirements for each catalytic activity of the complex. A dissociated active deubiquitinase module may play a role in this sophisticated regulation, since a free ubiquitin protease module could act on ubiquitinated chromatin independent of SAGA recruitment and regulation. In yeast lacking Sgf73, Sus1 is released from SAGA but is still recruited to genes, albeit at a reduced level. Moreover, early reports identified USP22 as an H2Aub deubiquitinase acting on polycomb-regulated genes. In principal, SAGA may release subcomplexes to participate in diverse functions. It was recently shown in S. cerevisiae that the proteasome is capable of pulling the enzymatically active DUB module, including Sgf73, Sgf11, and Sus1, from SAGA, indicating that separation of SAGA may normally occur. Further investigation into the modularity of SAGA and its implications for transcriptional regulation in vivo may aid in understanding how this complex might provide a sophisticated mechanism for chromatin modification and gene regulation. In addition to direct chromatin modification, Non-stop and its homologs have also been shown to act on nonhistone proteins, and it is possible that nonhistone targets play a significant role in SCA7 pathology (Mohan, 2014).

SCA7 and other polyglutamine expansion diseases have divergent pathologies. These differences suggest that the function of the expanded protein is critical to the etiology of each disease. Unfortunately, there is currently a limited understanding of the molecular functions of the wild-type proteins and what role loss or gain of function might play in the characteristic patterns of neural degeneration found upon polyglutamine expansion. In initial studies examining the wild-type function of other SCA proteins, loss of the wild-type protein did not entirely phenocopy the polyglutamine expansion disease. For example, Ataxin-1 knockout mice do not show cerebellar or brainstem degeneration but do exhibit cognitive defects and ataxia. The observations of this study suggest that loss of Ataxin-7 function may play a role in SCA7 disease progression. Polyglutamine-expanded Ataxin-7 is resistant to proteolysis, accumulating in cells. At the same time, the wild-type protein is only produced from one genomic copy, and the resulting protein is subjected to normal proteolysis. Furthermore, the Ataxin-7 N terminus extends into the deubiquitinase module, and this region is subject to polyglutamine tract expansion. Polyglutamine-expanded Ataxin-7 is present in the larger SAGA complex, but the deubiquitinase module was not specifically analyzed in this model. It will be interesting to examine whether the deubiquitinase module is also recruited to the complex under these conditions and, if so, whether it is enzymatically active. If the deubiquitinase module does not form around the expanded Ataxin-7, tracking the localization of this module may be insightful. It will also be interesting to examine the potential regulation of the deubiquitinase module independent of SAGA under wild-type conditions. Understanding what might trigger release of the module from the larger complex will be critical to understanding the role of SAGA in neural and retinal stability (Mohan, 2014).

dRYBP counteracts chromatin-dependent activation and repression of transcription

Chromatin dependent activation and repression of transcription is regulated by the histone modifying enzymatic activities of the trithorax (trxG) and Polycomb (PcG) proteins. To investigate the mechanisms underlying their mutual antagonistic activities this study analyzed the function of Drosophila Ring and YY1 Binding Protein (dRYBP), a conserved PcG- and trxG-associated protein. dRYBP is ubiquitylated and binds ubiquitylated proteins. Additionally dRYBP was shown to maintain H2A monoubiquitylation, H3K4 monomethylation and H3K36 dimethylation levels and does not affect H3K27 trimethylation levels. Further it was shown that dRYBP interacts with the repressive SCE (Ring) and dKDM2 (Lysine (K)-specific demethylase 2) proteins as well as the activating dBRE1 protein. Analysis of homeotic phenotypes and post-translationally modified histones levels show that dRYBP antagonizes dKDM2 and dBRE1 functions by respectively preventing H3K36me2 demethylation and H2B monoubiquitylation. Interestingly, the results show that inactivation of dBRE1 produces trithorax-like related homeotic transformations, suggesting that dBRE1 functions in the regulation of homeotic genes expression. These findings indicate that dRYBP regulates morphogenesis by counteracting transcriptional repression and activation. Thus, they suggest that dRYBP may participate in the epigenetic plasticity important during normal and pathological development (Fereres, 2014).

Linking H3K79 trimethylation to Wnt signaling through a novel Dot1-containing complex

Epigenetic modifications of chromatin play an important role in the regulation of gene expression. KMT4/Dot1 is a conserved histone methyltransferase capable of methylating chromatin on Lys79 of histone H3 (H3K79). This study reports the identification of a multisubunit Dot1 complex (DotCom), which includes several of the mixed lineage leukemia (MLL) partners in leukemia such as ENL, AF9/MLLT3, AF17/MLLT6, and AF10/MLLT10, as well as the known Wnt pathway modifiers TRRAP, Skp1, and β-catenin. The human DotCom is indeed capable of trimethylating H3K79 and, given the association of β-catenin, Skp1, and TRRAP, a role was sought for Dot1 in Wnt/Wingless signaling in an in vivo model system. Knockdown of Dot1 in Drosophila (Grappa) results in decreased expression of a subset of Wingless target genes. Furthermore, the loss of expression for the Drosophila homologs of the Dot1-associated proteins involved in the regulation of H3K79 shows a similar reduction in expression of these Wingless targets. From yeast to human, specific trimethylation of H3K79 by Dot1 requires the monoubiquitination of histone H2B by the Rad6/Bre1 complex. This study demonstrates that depletion of Bre1, the E3 ligase required for H2B monoubiquitination, leads specifically to reduced bulk H3K79 trimethylation levels and a reduction in expression of many Wingless targets. Overall, this study describes for the first time the components of DotCom and links the specific regulation of H3K79 trimethylation by Dot1 and its associated factors to the Wnt/Wingless signaling pathway (Mohan, 2010).

In eukaryotic organisms, gene expression patterns are spatiotemporally regulated in a manner that allows for specification of diverse cell types and their differentiation. This spatiotemporal expression is coordinated in part by transcription factors and chromatin modifiers, and by the activity of several signaling pathways, which contribute to gene expression by regulating the transcription factors. Understanding the relationship between chromatin events and signaling pathways is crucial to understanding gene regulation, development of the organism, and disease pathogenesis (Mohan, 2010).

The nucleosome, the basic unit of chromatin, consists of histones H2A, H2B, H3, and H4, and 146 base pairs (bp) of DNA. Crystal structure studies have demonstrated that the N-terminal tails of each histone protrude outward from the core of the nucleosome. These histone tails are subject to various post-translational modifications, including methylation, ubiquitination, ADP ribosylation, acetylation, phosphorylation, and sumoylation, and such modifications are involved in many biological processes involving chromatin such as transcription, genome stability, replication, and repair (Mohan, 2010).

Histones are methylated on either the lysine and/or arginine residues by different histone methyltransferases (HMTases). Histone lysine methylation can occur as mono-, di-, or trimethylated forms, and several lysine residues of histones have been shown to be multiply methylated. This includes methylation on Lys4, Lys9, Lys27, Lys36, and Lys79 of histone H3, and Lys20 of histone H4. Almost all of the lysine HMTases characterized to date contain a SET domain, named after Drosophila Su(var)3-9, Enhancer of zeste [E(z)], and trithorax (trx). SET domain-containing enzymes can catalyze the methylation of specific lysines on histones H3 and H4, and many SET domain-containing enzymes, such as Trithorax and Enhancer of zeste, are central players in epigenetic regulation and development (Mohan, 2010).

Histone H3 at Lys79 (H3K79) can be mono-, di-, and trimethylated by Dot1, which to date is the only characterized non-SET domain-containing lysine HMTase. Dot1 is conserved from yeast to humans. In yeast, telomeric silencing is lost when Dot1 is overexpressed or inactivated, as well as when H3K79 is mutated. Unlike other histone methylation patterns, the pattern of di- and trimethylation of H3K79 in yeast appears to be nonoverlapping. It was also first discovered in yeast that monoubiquitination of histone H2B on Lys123 (H2BK123) by the Rad6/Bre1 complex is required for proper H3K79 trimethylation by Dot1. In vivo analysis of the pattern of H2B monoubiquitination in yeast demonstrated that the H3K79 trimethylation pattern overlaps with that of H2B monoubiquitination, and that the H3K79 dimethylation pattern and H2B monoubiquitination appear to be nonoverlapping. This observation resulted in the proposal that the recruitment of the Rad6/Bre1 complex and the subsequent H2B monoubiquitination could dictate diversity between H3K79 di- and trimethylation on chromatin on certain loci within the genome. In addition to a role in the regulation of telomeric silencing in yeast, Dot1 has also been shown to be involved in meiotic checkpoint control and in double-strand break repair via sister chromatid recombination. A relationship has been found between cell cycle progression and H3K79 dimethylation, but not trimethylation, by Dot1. Consequently, to date, very little is known about a specific biological role of histone H3K79 trimethylation (Mohan, 2010).

In Drosophila, H3K79 methylation levels correlate with gene activity. Mutations in grappa, the Dot1 ortholog in Drosophila, show not only the loss of silencing, but also Polycomb and Trithorax-group phenotypes, indicating a key role for H3K79 methylation in the regulation of gene activity during development. Similarly, Dot1 in mammals has been implicated in the embryonic development of mice, including a role in the structural integrity of heterochromatin. Genome-wide profiling studies in various mammalian cell lines have suggested that Dot1 as well as H3K79me2 and H3K79me3 localize to the promoter-proximal regions of actively transcribed genes, and correlate well with high levels of gene transcription (Steger, 2008). It has also been proposed that Dot1 HMTase activity is required for leukemia pathogenesis (Mohan, 2010 and references therein).

The highly conserved Wnt/Wingless (Wnt/Wg) signaling pathway is essential for regulating developmental processes, including cell proliferation, organogenesis, and body axis formation. Deregulation or ectopic expression of members of the Wnt pathway has been associated with the development of various types of cancers, including acute myeloid and B-cell leukemias. In the canonical Wnt/Wg pathway, a cytoplasmic multiprotein scaffold consisting of Glycogen synthase kinase 3-β (GSK3-β), Adenomatous polyposis coli (APC), Casein kinase 1 (CK1), Protein phosphatase 2A, and Axin constitutively marks newly synthesized β-catenin/Armadillo for degradation by phosphorylation at the key N-terminal Ser and Thr residues. Binding of the Wnt ligands to the seven-transmembrane domain receptor Frizzled (Fz) leads to recruitment of an adaptor protein, Disheveled (Dvl), from the cytoplasm to the plasma membrane. Axin is then sequestered away from the multiprotein Axin complex, resulting in inhibition of GSK3-β and subsequent stabilization of hypophosphorylated β-catenin levels in the cytoplasm. Stabilized β-catenin translocates into the nucleus and binds to members of the DNA-binding T-cell factor/lymphoid enhancer factor (TCF/LEF) family, resulting in the recruitment of several chromatin-modifying complexes, including transformation/transcription domain-associated protein (TRRAP)/HIV Tat-interacting 60-kDa protein complex (TIP60) histone acetyltransferase (HAT), ISWI-containing complexes, and the SET1-type HMTase mixed lineage leukemia 1/2 (MLL1/MLL2) complexes, thereby activating the expression of Wnt/Wg target genes (Mohan, 2010 and references therein).

Although much is known about Dot1 as an H3K79 HMTase, biochemical studies isolating to homogeneity a Dot1-containing complex have not been successful during the past decade. This study reports the first biochemical isolation of a multisubunit complex associated with Dot1, which has been called DotCom. DotCom is comprised of Dot1, AF10, AF17, AF9, ENL, Skp1, TRRAP, and β-catenin. This complex is enzymatically active and can catalyze H3K79 dimethylation and trimethylation. Indeed, nucleosomes containing monoubiquitinated H2B are a better substrate for DotCom in the generation of trimethylated H3K79. Given the association of Skp1, TRRAP, and β-catenin with DotCom, and the fact that these factors have been linked to the Wnt signaling pathway in previous studies, this study investigated the role of the Drosophila homolog of Dot1, dDot1 (Grappa), for the regulation of Wg target genes. RNAi of dDot1 leads to a reduced expression of a subset of Wg target genes, including senseless, a high-threshold Wingless target gene. Furthermore, reduction by RNAi in the levels of the Drosophila homologs of other components of DotCom that regulate the pattern of H3K79 methylation in humans also showed a similar reduction in senseless expression and other Wg target genes. Importantly, DotCom requires monoubiquitination of H2B for H3K79 trimethylation, and, in Drosophila, the loss of Bre1, the E3 ubiquitin ligase, leads to reduction of H3K79 trimethylation and decreased expression of the senseless gene. Taken together, these data support a model in which monoubiquitinated H2B provides a regulatory platform for a novel Dot1 complex to mediate H3K79 trimethylation, which is required for the proper transcriptional control of Wnt/Wg target genes (Mohan, 2010).

Although H3K79 methylation is a ubiquitous mark associated with actively transcribed genes, and its presence is a clear indicator for the elongating form of RNA polymerase II, Dot1 itself has a very low abundance and is very hard to detect in cells. This indicates that Dot1 is an active enzyme with a very high specific activity toward its substrate, H3K79. Due to the low abundance of Dot1 in cells, its molecular isolation and biochemical purification have been hindered for the past decade. This study reports the biochemical isolation of a Dot1-containing complex (DotCom) and demonstrate a specific link between H3K79 trimethylation by DotCom and the Wnt signaling pathway. The study reports (1) the identification and biochemical isolation of a large macromolecular complex (~2 MDa) containing human Dot1, in association with human AF10, AF17, AF9, ENL, Skp1, TRRAP, and β-catenin; (2) the biochemical demonstration that the human DotCom is capable of trimethylating H3K79, and the analysis of the role of histone H2B monoubiquitination in the enhancement of this H3K79 trimethylase activity of the human DotCom; (3) identification of the role of the components of DotCom in the regulation of its H3K79 methylase activity; (4) demonstration of a role for the Drosophila homolog of Dot1 and its associated factors in the Wnt signaling pathway; and, finally, (5) the identification of a specific requirement of H3K79 trimethylation, but not mono- or dimethylation, in the regulation of Wnt target transcription, thereby linking H3K79 trimethylation to Wnt signaling (Mohan, 2010).

Dot1 was initially isolated from yeast, and these studies demonstrated that the enzyme is capable of mono-, di-, and trimethylating H3K79. Subsequent molecular and biochemical studies demonstrated that prior H2B monoubiquitination by the Rad6/Bre1 complex is required for proper H3K79 trimethylation by yeast Dot1. A recent analysis of the human homolog of Dot1 suggested that its HMTase domain is not capable of trimethylating H3K79, and that this enzyme can only dimethylate its substrate. It has also been demonstrated that reconstitution of monoubiquitinated H2B into chemically defined nucleosomes, followed by enzymatic treatment with Dot1, resulted only in dimethylation of H3K79. Since these observations are in contrast with the published studies in yeast, this study tested the enzymatic activity of purified human DotCom toward monoubiquitinated and nonmonoubiquitinated nucleosomes. The studies demonstrate that the human DotCom can indeed trimethylate H3K79, and that monoubiquitination of histone H2B enhances this enzymatic property of the human DotCom. Since the enzymatic studies employ antibodies generated toward mono-, di-, and trimethylated H3K79 to identify the products of the enzymatic reactions containing human Dot1, it was important to make certain that the observations are not the result of cross-reactivity between these antibodies. Therefore recombinant nucleosomes were generated and treated with human Dot1 in the presence and absence of SAM, and the products were analyzed by MS. The chemical analysis of the products from this enzymatic reaction confirmed that human Dot1 is capable of trimethylating H3K79. The hDot1-treated nucleosome samples were digested with Endoproteinase Arg-C because previous unpublished work on analyzing yeast histone modifications by MudPIT had shown that the trimethylated peptide containing H3K79 was not detected when digesting with trypsin. Notably, McGinty (2008) performed their digestions with trypsin, which might explain their failure to detect this modification by MS (Mohan, 2010).

These studies identified several factors -- including ENL, AF9, AF17, AF10, SKP1, TRA1/TRAPP, and β-catenin -- as components of the human DotCom. To test the role of these factors in regulating Dot1's catalytic activity, their levels were reduced via RNAi. These studies demonstrated that AF10 functions with Dot1 to regulate its catalytic properties in vivo. Significant differences in Dot1's H3K79 HMTase activity were not detected in vivo when reducing the levels of ENL, AF9, and AF17. Factors that significantly alter the H3K79 methylation pattern by Dot1 are also linked to its transcriptional regulatory functions at Wnt target genes (Mohan, 2010).

Since Dot1 also appears to interact with β-catenin, and given the known role for β-catenin, Skp1, and TRRAP in the Wnt signaling pathway, the role for Dot1 and the components of its complex were tested in Wnt signaling. Drosophila is an outstanding model system for the study of the Wnt signaling pathway. Given the power of genetics and biochemistry in Drosophila, the role of dDot1 and the members of its complex in wingless signaling were tested. From this study, it was learned that down-regulation of Drosophila Dot1 and Drosophila AF10 had the most significant effects in the regulation in the expression of the Wg target senseless. Given the fact that the molecular studies demonstrated that Dot1 and AF10 have the strongest effect in the regulation of H3K79 methylation in vivowe wanted to determine whether a specific form of H3K79 methylation is required for Wnt target gene expression was tested (Mohan, 2010).

Histone H2B monoubiquitination is required for proper H3K79 trimethylation. The E2/E3 complex Rad6/Bre1 is required for the proper implementation of H2B monoubiquitination on chromatin, and this complex is highly conserved from yeast to humans. Deletion of the Drosophila homolog of Bre1 results in the loss of H2B monoubiquitination and the specific loss of H3K79 trimethylation. Interestingly, reduction in the levels of H3K79 trimethylation results in a defect in expression of one of the Wnt target genes, senseless, although the H3K79 mono- and dimethylation in this mutant background appear to be normal. In addition to senseless, the role of H3K79 methylation at other Wnt targets was tested, and the same effect was observed for Notum and CG6234. Overall, these studies demonstrate a link between H3K79 trimethylation by the DotCom and the Wnt signaling pathway (Mohan, 2010).

Wnt/Wg signaling serves a critical role in tissue development, proliferation of progenitor cells, and many human cancers. The key player in the Wnt pathway is β-catenin, which is shuttled into the nucleus at the onset of activation of the pathway. Various proteins that interact with β-catenin in the nucleus -- such as CBP/p300, TRRAP, MLL1/MLL2, Brg1, telomerase, Hyrax, Pygopus, and CDK8 -- modulate the transcriptional output of Wg/Wnt target genes. These proteins probably provide the context specificity to Wnt response directing proliferation or differentiation effects of Wnt signaling. The finding that dDotCom is required for expression of a subset of Wg targets suggests that dDotCom might also facilitate Wg-regulated programs of transcriptional regulation in specific contexts. As most human cancers have elevated levels of Wnt signaling and require Wnt signaling for continued proliferation, DotCom might play a role in supporting the high rate of expression of Wnt target genes in such cancers (Mohan, 2010).

Several studies have found interactions between Dot1 and many translocation partners of MLL. While these associations suggest a link between Dot1 methylation and leukemogenesis, it was not clear how Dot1 methylation would participate in this process. Recently, GSK3, a regulator of β-catenin and Wnt signaling, was found to be essential for proliferation of MLL-transformed cells and for progression of a mouse model of MLL-based leukemia (Wang, 2008). These studies linking Dot1 H3K79me3 with Wnt signaling provide insight into the role of Wnt signaling and Dot1 methylation in MLL translocation-based leukemia (Mohan, 2010).

Drosophila stem cells share a common requirement for the histone H2B ubiquitin protease scrawny

Stem cells within diverse tissues share the need for a chromatin configuration that promotes self-renewal, yet few chromatin proteins are known to regulate multiple types of stem cells. A Drosophila gene, scrawny (scny), encoding a ubiquitin-specific protease, is required in germline, epithelial, and intestinal stem cells. Like its yeast relative UBP10, Scrawny deubiquitylates histone H2B and functions in gene silencing. Consistent with previous studies of this conserved pathway of chromatin regulation, scny mutant cells have elevated levels of ubiquitinylated H2B and trimethylated H3K4. These findings suggest that inhibiting H2B ubiquitylation through scny represents a common mechanism within stem cells that is used to repress the premature expression of key differentiation genes, including Notch target genes (Buszczak, 2009).

Stem cells are maintained in an undifferentiated state by signals they receive within the niche and are subsequently guided toward particular fates upon niche exit. Within ES cells and during differentiation, cell state changes are controlled at the level of chromatin by alterations involving higher order nucleosome packaging and histone tail modifications. Polycomb group (PcG) and Trithorax group (trxG) genes influence key histone methylation events at the promoters of target genes, including H3K27 and H3K4 modifications associated with gene repression and activation, respectively, but few other genes with a specific role in stem cells are known (Buszczak, 2009).

Histone H2A and H2B mono-ubiquitylation play fundamental roles in chromatin regulation, and H2A ubiquitylation has been linked to PcG-mediated gene repression and stem cell maintenance. The mammalian Polycomb repressive complex 1 (PRC1) component RING1B is a H2A ubiquitin ligase that is required to block the elongation of poised RNA polymerase II on bivalent genes in ES cells. Mutations in the PRC1 component, BMI-1, the ortholog of Psc in the mammalian PRC1, complexes with RING1B, and causes multiple types of adult stem cells to be prematurely lost. The role of H2B ubiquitylation in stem cells is unclear, however. In yeast, ubiquitylation of Histone H2B by the RAD6 and BRE1 ligases controls H3K4 methylation (H3K4me3), a process that requires the polymerase accessory factor PAF1. Conversely, H2B deubiquitylation by the ubiquitin-specific protease (USP) family member UBP10 is required for silencing telomeres, rDNA and other loci (Gardner, 2005). The Drosophila homolog of BRE1, dBRE1, also is needed for H3K4 methylation, suggesting that this pathway is conserved. Furthermore, the Drosophila ubiquitin-specific protease USP7 is part of a complex that selectively deubiquitylates H2B and genetically interacts with PcG mutations (van der Knaap, 2005). Mutations in another USP family member, Nonstop, increase H2B ubiquitylation and cause axon targeting defects in the eye (Buszczak, 2009).

In order to gain further insight into the role of H2B ubiquitylation in stem cells, a novel Drosophila gene, scrawny (scny) (CG5505), was identified, whose encoded USP family protein shares homology with human USP36 and among yeast USPs closely matches UBP10 within the core protease domain. Strains bearing scny insertions, except for a viable GFP protein trap (CA06690), were female sterile or lethal, and proved to be allelic. Transposon excision or expression of a scny-RB cDNA reverts the phenotype of tested alleles. An anti-SCNY antibody raised against a domain common to all SCNY isoforms recognizes wild type and SCNY-GFP on a Western blot. SCNY protein levels in homozygous third instar larvae are greatly reduced in lethal mutants, and SCNY expression is also lower in stem cell-enriched ovarian tissue from adults homozygous for the sterile d06513 allele. Consistent with a role in gene silencing, several scny mutations act as dominant suppressors of position effect variegation (Buszczak, 2009).

Further studies strongly suggested that SCNY functions in vivo as an H2B-ubiquitin protease. Recombinant full-length SCNY protein, but not a version bearing a point mutation in the protease domain, efficiently deubiquitylates histone H2B in vitro. scny^f01742 homozygous tissue contains levels of Ub-H2B that are elevated at least twofold compared to wild type. As expected if Ub-H2B is required for H3K4 methylation, clones of homozygous scny^e00340 mutant cells stain more strongly for H3K4me3 than heterozygous cells. Consistent with a direct rather than an indirect action on Ub-H2B levels, anti-SCNY antibodies co-immunoprecipitate H2B from Drosophila embryonic nuclear extracts. Moreover, epitope-tagged SCNY co-immunoprecipitates Drosophila PAF1, but not Cyclin T (or several other tested chromatin proteins) when co-expressed in S2 tissue culture cells. Together, these data support the view that SCNY participates in a conserved pathway of chromatin regulation linking H2B ubiquitylation with H3K4me3 methylation. Because the effects of scny mutation on Ub-H2B and H3K4me3 are opposite to those of dBre1 mutation, SCNY likely opposes dBRE1 action on H2B, just as UBP10 opposes BRE1 action on H2B in yeast (Buszczak, 2009).

Drosophila male and female gonads contain well characterized germline stem cells (GSCs) that allow the effects of genes on stem cell maintenance to be quantitatively analyzed. High levels of scny expression were observed in female and male GSCs using SCNY-GFP and identical staining was observed using anti-SCNY immunofluoresence. SCNY protein resides in cell nuclei and is enriched in nucleoli. In sterile or semi-fertile scny mutant adults, the numbers of germline stem cells surrounding the testis hub and within germaria were clearly reduced. The half-lives of female GSCs bearing clones of three different scny alleles were all sharply reduced. Later follicular development was also abnormal suggesting that scny continues to function after the stem cell stage. However, previous studies indicate that accelerated GSC loss is a specific phenotype, and hence that scny has a preferential requirement in GSCs (Buszczak, 2009).

A known mechanism of increased GSC loss is the premature activation of differentiation genes. Staining germaria with an antibody specific for multiple sites of histone H3 acetylation (H3-Ac) suggested that scny mutation affects the global chromatin organization of GSCs. Wild type GSCs contain lower levels of H3-Ac than slightly older germ cells within cysts. Presumptive GSCs located in the GSC niche in scny mutants frequently stained more strongly, suggesting that they have begun to upregulate general transcription. Some scny GSC-like cells also expressed bag-of-marbles (bam), a key cystoblast differentiation gene, and GSC-like cells in scny^d06513; bam^Δ86 mutant females persist in the germarium. However, it could not be completely ruled out that the observed increases in H3-Ac levels and bam expression were a result rather than a cause of the premature differentiation and loss of scny GSCs (Buszczak, 2009).

To determine if scny is also required in a very different type of stem cell, the epithelial follicle stem cell (FSC), the persistence of individual scny mutant FSCs was quantitatively. The half-life of FSCs mutant for scny^l(3)02331 was reduced more than 10-fold, while the scny^f01742 mutation also caused a sharp decline. However, mutant follicle cells continued to develop normally at later stages. Thus, scny is preferentially required to maintain FSCs as well as GSCs (Buszczak, 2009).

The largest population of Drosophila stem cells are the hundreds of multipotent intestinal stem cells (ISCs) that maintain the adult posterior midgut. ISCs signal to their daughters via Delta-Notch signaling to specify enterocyte vs. enteroendocrine cell fate, but the pathway must remain inactive in the ISCs themselves to avoid differentiation. Most ISCs (those about to produce enterocytes) express high levels of the Notch ligand Delta, allowing them to be specifically distinguished from other diploid gut cells. This study found that SCNY-GFP is expressed in ISCs suggesting that SCNY plays a role in these stem cells as well. While 7-day old normal adult midguts contain a high density of ISCs, as revealed by Delta staining, it was found that corresponding tissue from 7-day-old scny^f01742 or scny^f01742/scny^l(3)02331 escaper adults possess very few Delta-positive cells. ISCs are present in near normal numbers at eclosion, but are rapidly lost in the mutant adults, indicating that scny is required for ISC maintenance (Buszczak, 2009).

It is suspected that inappropriate Notch pathway activation was responsible for the premature ISC loss in scny mutants. dBre1 mutations strongly reduce Notch signaling, suggesting that Notch target genes are particularly dependent on H2B mono-ubiquitylation and H3K4 methylation. Consequently, scny mutations, which have the opposite effects on Ub-H2B and H3K4me3 levels, might upregulate Notch target genes, stimulating ISCs to differentiate prematurely. This idea was tested by supplementing the food of newly eclosed scny^f01742/scny^l(3)02331 adults with 8 mM DAPT, a gamma-secretase inhibitor that blocks Notch signaling and phenocopies Notch mutation when fed to wild type animals. scny^f01742/scny^l(3)02331 DAPT-treated adults remained healthy and the guts of 7-day old animals still contained many ISCs, although not as many as wild type. Tumors like those produced in wild type animals fed DAPT were not observed. Thus, in these animals endogenous stem cell loss can be slowed by drug treatment (Buszczak, 2009).

These experiments provide strong evidence that a pathway involving the ubiquitin protease Scrawny and the ubiquitin ligase dBRE1 controls the levels of Ub-H2B, and H3K4me3 at multiple target sites in the Drosophila genome. Although, other ubiquitin proteases also act on Ub-H2B in Drosophila, the direct interaction between SCNY and H2B, and the strong effects of scny mutations argue that it plays an essential, direct role in silencing genomic regions critical for cellular differentiation, including Notch target genes. SCNY interacts with the RNA polymerase accessory factor complex component, PAF1. Upregulation of H2B ubiquitinylation and H3 methylation in yeast is mediated by the PAF1 complex and is associated with elongating RNA Pol II. Drosophila PAF1 is required for normal levels of H3K4me3 at the hsp70 gene, and another PAF1 complex member, RTF1, is needed for H3K4 methylation and Notch target gene expression. Indeed, the pathway connecting Ub-H2B, H3K4me3 and gene silencing appears to be conserved in organisms as distant as Arabidopsis. A human protein closely related to SCNY, USP36, is overexpressed in ovarian cancer cells (Li, 2008), and the results suggest it may act as an oncogene by suppressing differentiation (Buszczak, 2009).

Above all, these experiments indicate that SCNY-mediated H2B deubiquitylation is required to maintain multiple Drosophila stem cells, including progenitors of germline, epithelial and endodermal lineages. In ES cells and presumably in adult stem cells, many differentiation genes contain promoter-bound, arrested RNA Pol II and are associated with Polycomb group proteins. It is envisioned that in the niche environment SCNY activity overrides that of dBRE1, keeping levels of Ub-H2B (and hence H3K4me3) low at key differentiation genes. Upon exit from the niche, the balance of signals shifts to favor H2B ubiquitylation, H3K4 trimethylation, and target gene activation. Thus, the control of H2B ubiquitylation, like H2A ubiquitylation, plays a fundamental interactive role in maintaining the chromatin environment of the stem cell state (Buszczak, 2009).

SAGA-mediated H2B deubiquitination controls the development of neuronal connectivity in the Drosophila visual system

Nonstop, which has previously been shown to have homology to ubiquitin proteases, is required for proper termination of axons R1-R6 in the optic lobe of the developing Drosophila eye. This study establish that Nonstop actually functions as an ubiquitin protease to control the levels of ubiquitinated histone H2B in flies. Nonstop is the functional homolog of yeast Ubp8, and can substitute for Ubp8 function in yeast cells. In yeast, Ubp8 activity requires Sgf11. In Drosophila, loss of Sgf11 function causes similar photoreceptor axon-targeting defects as loss of Nonstop. Ubp8 and Sgf11 are components of the yeast SAGA complex, suggesting that Nonstop function might be mediated through the Drosophila SAGA complex. Indeed, it was found that Nonstop does associate with SAGA components in flies, and mutants in other SAGA subunits display nonstop phenotypes, indicating that SAGA complex is required for accurate axon guidance in the optic lobe. Candidate genes regulated by SAGA that may be required for correct axon targeting were identified by microarray analysis of gene expression in SAGA mutants (Weake, 2008).

This study has identified a novel role for the coactivator complex dSAGA in Drosophila neural development. The Gcn5 (KAT2) HAT acts as the catalytic subunit of the yeast SAGA, SLIK, ADA and A2 multi-subunit protein complexes. Although many studies in multicellular organisms have focused on the HAT activity of the Gcn5 (KAT2) complexes, SAGA itself possesses a second catalytic activity. In addition to its HAT activity, yeast SAGA contains an H2B deubiquitinating enzyme, Ubp8. Ubp8 functions as part of a modular subunit domain within yeast SAGA that contains two additional proteins, Sgf11 and Sus1. Previous studies on histone deubiquitination have focused on its role in transcription in yeast. This study has characterized a role for histone deubiquitination in gene regulation in Drosophila (Weake, 2008).

Nonstop and Sgf11 constitute the H2B deubiquitination module within dSAGA. Moreover, both Nonstop and Sgf11 are required for correct axon targeting in the developing visual system. As in yeast, the two catalytic functions of SAGA are separable. However, mutations that differentially affect the two catalytic activities of dSAGA have overlapping but distinct effects on gene expression. Despite the differences in activity, both catalytic modules of dSAGA are required for correct axon targeting in the optic lobe. This implicates dSAGA in the regulation of pathways essential for neural development in higher eukaryotes (Weake, 2008).

This analysis of the axon-targeting defects in the nonstop, sgf11 and ada2b mutants indicates that the R-cell misprojection phenotype is associated in all three cases with a loss of glial cells from the lamina region of the optic lobe, and an increase in the number of glial cells at the dorsal and ventral margins of the lamina. Clonal analysis indicates that nonstop glial cells fail to migrate from these dorsal and ventral regions into the lamina plexus. This suggests that dSAGA may be required within glial cells to regulate pathways important for their migration. It is possible that the requirement of dSAGA may be due to its role in the ecdysone response as indicated by microarray analysis of genes downregulated in nonstop, sgf11 and ada2b mutant larvae. However, further studies on the role of dSAGA in these glial cells will be required to determine which pathway(s) are primarily responsible for the axon-targeting defect observed in these dSAGA mutants (Weake, 2008).

Mutations that affect the deubiquitination activity of dSAGA, such as nonstop and sgf11, appear to result in a more severe axon-targeting defect than those that affect the acetylation activity, such as ada2b. However, there is some variability in the degree of expressivity of the phenotype in all three mutant genotypes, and some ada2b mutants show phenotypes very similar to nonstop and sgf11. It is evident from studies on yeast SAGA that both enzymatic activities are required for optimal transcription upon gene induction. The variability in the ada2b mutant may be due to the large maternal contribution of Ada2b, and it is notable that the ada2b mutant larvae show considerably less developmental delay in comparison to nonstop, sgf11 and gcn5. Thus far, mutations in other components of dSAGA, such as wda and nipped-A, have not been examined for this phenotype because these do not reach the third instar larval stage of development (Weake, 2008).

Although in this study a specific effect was observed of the dSAGA deubiquitination module on histone H2B, it remains likely Nonstop and Sgf11 are also required for deubiquitination of other target proteins within the cell. Previous studies observed an increase in the level of three ubiquitinated proteins of 29, 55 and 200 kDa in extracts from nonstop third instar larval tissue relative to wild-type extracts. The smallest of these may correspond to ubiquitinated H2B, but the others may correspond to as yet unidentified potential targets of the deubiquitination module within dSAGA (Weake, 2008).

It is likely that the role of SAGA and H2B deubiquitination in neural development may be conserved in mammalian systems, as there is a striking degree of similarity between Sgf11 and ATXN7L3/ATXN7 in humans. ATXN7 is a subunit of the human STAGA and TFTC complexes. Polyglutamine expansions in the Spinocerebellar ataxia type 7 (sca7) gene, encoding ATXN7, result in a dominant neurodegenerative disorder that affects the retina. There is increased recruitment of STAGA/TFTC containing this polyQ-expanded ATXN7 at certain promoters in a mouse SCA7 model, resulting in hyperacetylation of H3. Interestingly, despite this increased promoter recruitment and hyperacetylation, these genes show decreased levels of transcription. However, in other studies incorporation of polyQ-expanded ATXN7 into STAGA reduces the acetyltransferase activity of the complex. The effect of the polyQ expansions in ATXN7 on the putative deubiquitination activity of mammalian STAGA/TFTC has not yet been examined (Weake, 2008).

Van der Knaap (2005) has demonstrated that another ubiquitin protease in flies, USP7, specifically deubiquitinates histone H2B in vitro. However, RNAi of USP7 had little effect on global levels of ubiquitinated H2B, and it was found that USP7 cannot functionally replace UBP8 in yeast. Yeast Ubp15, the putative homolog of USP7, also deubiquitinates H2B in vitro, but the biological role of this activity is presently unknown. In addition to Ubp8 in yeast, Ubp10 is also required for deubiquitination of H2B at the telomeres and at the rDNA locus. It is unknown whether the putative homolog of Ubp10 in flies, CG15817, is also required for H2B deubiquitination and gene silencing. It is intriguing that both Ubp10 and USP7 (via an interaction with the Polycomb complex) function in regulating heterochromatin structure, while Ubp8/Nonstop are required for active transcription. It remains to be determined whether alternative mechanisms for regulating histone ubiquitination at distinct chromatin environments exist within the genomes of higher eukaryotes (Weake, 2008).

Taken together, these results advance the understanding of the role of histone deubiquitination in transcription, while demonstrating a novel role for SAGA in regulating neural development in the visual system of Drosophila. These findings have implications for the use of Drosophila as a model system to understand the underlying mechanisms of neurodegenerative diseases (Weake, 2008).

Drosophila octamer elements and Pdm-1 dictate the coordinated transcription of core histone genes

This study reveals a set of divergent octamer elements in Drosophila core histone gene promoters. These elements recruit transcription factor POU-domain protein in Pdm-1, which along with co-activator dmOct-1 coactivator in S-phase (dmOCA-S), activates transcription from at least the Drosophila histone 2B (dmH2B) and 4 (dmH4) promoters in a fashion similar to the transcription of mammalian histone 2B (H2B) gene activated by octamer binding transcription factor 1 (Oct-1) and Oct-1 coactivator in S-phase (OCA-S). The expression of core histone genes in both kingdoms is coordinated; however, although the expression of mammalian histone genes involves subtype-specific transcription factors and/or co-activator(s), the expression of Drosophila core histone genes is regulated by a common module (Pdm-1/dmOCA-S) in a directly coordinated manner. Finally, dmOCA-S is recruited to the Drosophila histone locus bodies in the S-phase, marking S-phase-specific transcription activation of core histone genes (Lee, 2010).

Given that Oct-1 and Pdm-1 share similarities in their evolutionarily conserved POU-domains, as do the human OCA-S and putative dmOCA-S components, a similar trans-regulatory network is proposed in the Drosophila H2B gene activation pathway in which dmOCA-S abets the role of Pdm-1; thus, silencing individual dmOCA-S subunits may bring about phenotypes similar to those observed in Pdm-1-silenced cells. In particular, glyceraldehyde-3-phosphate dehydrogenase (p38/GAPDH) and lactate dehydrogenase (p36/LDH) were shown to be OCA-S components absolutely required for hH2B transcription (Zheng, 2003; Dai, 2008). DmGapdh1/2, dmLdh, and abnormal wing discs [Awd, the non-metastatic protein 23 in human (nm23) homolog] were subject to RNAi. The expression of these proteins was silenced to relative completion with 37 nm dsRNA doses, which was accompanied by coordinately repressed expression of dmH2B and dmH4 genes. The repressed dmH2B expression was attributed to reduced promoter activity of the dmH2B gene, for the activities of the ectopic dmH2B promoter-luciferase reporter were severely reduced upon silencing the protein expression of each of the tested dmOCA-S components (dmGapdh1/2, dmLdh, and Awd). Collectively, these results suggest an existence of a dmOCA-S co-activator in Drosophila that comprises of the above-tested proteins and functions in the dmH2B gene regulation pathway by abetting the role of Pdm-1 (Lee, 2010).

Although more than 800 million years apart in evolution, Drosophila and human share similarities in histone expression pathways, albeit with species-specific features. In Drosophila, core histone gene promoters contain multiple evolutionarily diversified Pdm-1 binding sites, contributing to the optimal histone expression​; on the other hand, a single prototype octamer ATTTGCAT mediates the hH2B transcription, and Oct-1 association with this element, which is conserved among vertebrates, is kept under strict sequence requirement. This strategy may be also employed by other Oct-1-dependent genes and their cognate coactivators. These species-specific features could imply significance for metazoan evolution (Lee, 2010).

Histone biosynthesis and DNA replication are coupled and essential for cell viability. Thus, impeding the function of Pdm-1/dmOCA-S would lead to S-phase defects and likely be detrimental to cell viability. In the case of dmGapdh and dmLdh, their roles in glycolysis and their moonlighting nuclear functions abetting the role of Pdm-1 also dictate negative consequences on the cellular ability to progress through the S-phase in a loss-of-function situation (Lee, 2010).

Efficient RNAi-mediated protein knockdown requires mRNA destruction and decay of the preexisting protein, which was realized at 72 h for Pdm-1 and 88A) with coordinately repressed expression of core histone genes; however, the cell cycle profiles were not affected or only marginally affected at this point, which was statistically insignificant. Thus, the histone expression defects at 72 h were primary defects. It was reasoned that a most likely scenario is that despite depriving cells of histone transcription at 72 h by a Pdm-1 deficiency, pre-existing histone protein levels are above a threshold that supports DNA replication. Indeed, H2B protein levels at 72 h were largely normal or slightly reduced. Beyond 72 h, however, Pdm-1-silenced cells also exhibited a prominent S-phase defect at 96 h, and cell viability defects manifested at 84 h with a more drastic viability decrease at 96 h (Lee, 2010).

The cell viability defects might be related to a mechanism coupling histone expression to S-phase progression. The more drastic histone expression defects at 84 h as compared with that at 72 h could be due to additive effects of a primary Pdm-1 loss-of-function and secondary cell viability and S-phase defects, which fed back. Histone transcription defects as a result of the Pdm-1/dmOCA-S deficiency would deprive cells of histone proteins in a long run, thus providing insufficient histone protein levels for chromatin assembly, which ultimately leads to DNA replication, S-phase, and cell viability defects. It is proposed that although histone transcription defects at 72 h were primary defects, the later-manifested cell viability and S-phase defects were secondary defects due to reduced histone protein levels as exemplified by that of H2B beyond 72 h (Lee, 2010).

Coordinated histone expression in diverse species is needed to maintain balanced expression of core histone genes, known to sustain genome integrity. In mammalian cells, eliminating OCA-S function by silencing p38/GAPDH expression led to H2B expression defect, and a lagged histone H4 expression defect manifested after a severe cell cycle arrest; eliminating H2B expression by gene deletion in yeast also led to cell cycle arrest and subsequent expression defects of other core histone genes. These observations led to a thought that coordinated histone expression was regulated through an S-phase feedback mechanism, which was later revised (Yu, 2009; Lee, 2010).

In Drosophila cells, the RNAi-mediated silencing of Pdm-1 or dmOCA-S components led to concerted and directly coordinated expression defects of all core histone genes and as primary transcription effects due to a missing Pdm-1/dmOCA-S function. In mammalian cells, the core histone genes employ distinct promoter elements and associated (co)factors, but their expression remains highly coordinated through an uncharacterized mechanism that is indirect but still does not involve S-phase feedback. Distinct histone expression coordination mechanisms in fly and mammalian cells might be of crucial relevance in metazoan evolution (Lee, 2010 and references therein).

The expression of metazoan-specific linker histone H1 gene is largely S-phase-specific, but the H1 expression output is not tightly coupled with that of core histone genes. The TATA-less Drosophila H1 gene utilizes a TRF-containing complex but not the prototype TBP-containing TFIID complex for its expression. This study found that Drosophila core histone genes, at least that of dmH2B and dmH4, contain TFIID in line with recruitment of TBP to dmH3 and dmH4 promoters and the idea that the basal transcription machineries of Drosophila core histone genes use the prototype TFIID. TRF-containing complexes have not been known to function in a cell cycle-dependent manner; it is of interest to investigate if the S-phase-specific Drosophila H1 expression is conferred upon by S-phase-specific factors that ought to be distinct from Pdm-1/dmOCA-S because the Drosophila H1 expression was not coordinately repressed with that of core histone genes in Pdm-1-deficient cells (Lee, 2010).

Mammalian histone genes are organized into Cajal bodies in a process facilitated by nuclear protein, ataxia-telangiectasia locus (NPAT), a cyclinE/cdk2 substrate that conveys the cyclinE/cdk2 signaling to histone transcription machineries. In contrast, Drosophila cells contain distinct nuclear domains dubbed HLB, which host all the histone genes and contain a cyclinE/cdk2-dependent phospho-epitope recognized by the MPM-2 monoclonal antibody in S-phase. Drosophila HLB are often in close proximity to, but never overlapped with Drosophila Cajal bodies (Lee, 2010 and references therein).

That cyclinE/cdk2 signaling is conserved from Drosophila to human, and that the HLB foci are associated with nascent histone transcripts prompted an investigation of S-phase-specific recruitment of dmOCA-S (represented by nuclear dmGapdh) to HLB. A higher percentage of HLB (~90%) foci as compared with dmOCA-S (~70%) foci in the early S-phase, i.e.,~80% of HLB foci are nuclear dmGapdh-positive, suggests that the dmOCA-S function (dmGapdh-HLB nuclear co-localization) is likely downstream of cyclinE/cdk2 signaling. The increase in the foci size and maximal degree of HLB-dmOCA-S co-localization coincided with the peak of dmH2B and dmH4 mRNA levels in the mid-S-phase, in line with the notion that the HLB foci size is proportional to histone expression levels (Lee, 2010 and references therein).

None of dmOCA-S components possesses a consensus sequence(s) for cdk; the fly cyclinE/cdk2 signaling might be conveyed to histone genes via an unidentified molecule(s) (dmNPAT), for which the phospho-epitope-containing protein recognized by MPM-2 monoclonal antibody is a potential candidate (Lee, 2010 and references therein).

Efforts to find dmNPAT have been fruitless; a dmNPAT gene might likely reside in so-far-unsequenced heterochromatic domains or possess sequences drastically divergent from vertebrate NPATs. Alternatively, an NPAT function may not have been acquired during insect evolution, necessitating histone genes to be organized into HLB and compelling cognate promoters to be regulated by diversified octamer elements and a Pdm-1/dmOCA-S module to ensure the directly coordinated expression of histone genes (Lee, 2010 and references therein).

The mechanistic aspects of co-activation by dmOCA-S seem to be similar to that of human OCA-S, in line with nuclear moonlighting transcription functions of metabolic enzyme conservation in metazoans. Conversely, some non-transcriptional functions of transcription factors or co-factors have been documented: e.g. in the cytoplasm, an isoform of co-activator OCA-B plays an essential non-transcriptional role for B-cell signaling (Lee, 2010).

Pdm-1 is a ubiquitous transcription factor for ubiquitous histone expression; the identified cell-specific roles of Pdm-1 in neuronal cell fate specification and Notch signaling might be due to cell-specific non-transcriptional functions of Pdm-1. Alternatively, cell-specific Pdm-1 phenotypes might be attributed to missing links between mutant pdm-1 alleles and alleles encoding cell-specific co-activators, especially in view of the fact that a given POU-domain is rich in separable surfaces that provide contacts for distinct ubiquitous or tissue-specific co-activators. Non-transcriptional development regulators may also interact with partners through different surfaces. Thus, it is not surprising that mutant alleles of even a ubiquitously expressed gene, be it specifying a non-transcription or transcription function, may lead to tissue-specific phenotypes (Lee, 2010).

The work toward dissecting cis- and trans-regulatory networks of the Drosophila histone transcription regulation pathway(s) will provide a new paradigm for studying transcriptional regulator changes implied in evolution and development as well as permit a broader range of questions to be asked about a cohort of genes involved in histone expression and related DNA replication-dependent transcription and its tight coupling with S-phase progression and about roles of the individual dmOCA-S components as novel S-phase-specific players in above processes in a genetically tractable organism (Lee, 2010).

Structure of the Drosophila nucleosome core particle highlights evolutionary constraints on the H2A-H2B histone dimer

The 2.45 A crystal structure of the nucleosome core particle was determined from Drosophila melanogaster and compared to that of Xenopus laevis bound to the identical 147 base-pair DNA fragment derived from human alpha-satellite DNA. Differences between the two structures primarily reflect 16 amino acid substitutions between species, 15 of which are in histones H2A and H2B. Four of these involve histone tail residues, resulting in subtly altered protein-DNA interactions that exemplify the structural plasticity of these tails. Of the 12 substitutions occurring within the histone core regions, five involve small, solvent-exposed residues not involved in intraparticle interactions. The remaining seven involve buried hydrophobic residues, and appear to have coevolved so as to preserve the volume of side chains within the H2A hydrophobic core and H2A-H2B dimer interface. Thus, apart from variations in the histone tails, amino acid substitutions that differentiate Drosophila from Xenopus histones occur in mutually compensatory combinations. This highlights the tight evolutionary constraints exerted on histones since the vertebrate and invertebrate lineages diverged (Clapier, 2008; full text of article).

Bre1 is required for Notch signaling and histone modification

Notch signaling controls numerous cell fate decisions during animal development. These typically involve a Notch-mediated switch in transcription of target genes, although the details of this molecular mechanism are poorly understood. dBre1 has been identified as a nuclear component required cell autonomously for the expression of Notch target genes in Drosophila development. dBre1 affects the levels of Su(H) in imaginal disc cells, and it stimulates the Su(H)-mediated transcription of a Notch-specific reporter in transfected Drosophila cells. Strikingly, dBre1 mutant clones show much reduced levels of methylated lysine 4 on histone 3 (H3K4m), a chromatin mark that has been implicated in transcriptional activation. Thus, dBre1 is the functional homolog of yeast Bre1p, an E3 ubiquitin ligase required for the monoubiquitination of histone H2B and, indirectly, for H3K4 methylation. These results indicate that histone modification is critical for the transcription of Notch target genes (Bray, 2005).

It has been suggested that the Paf1 complex (see Drosophila Paf1) functionally activates activation Rad6-Bre1 complex in ubiquitination of histone H2B at promoters (Wood, 2003). Monoubiquitination of histone H2B, catalyzed by Rad6-Bre1, is required for methylation of histone H3 on lysines 4 and 79, catalyzed by the Set1-containing complex COMPASS and Dot1p, respectively. The Paf1 protein complex, which associates with RNA polymerase II, is known to be required for these histone H3 methylation events. During the early elongation stage of transcription, the Paf1 complex is required for association of COMPASS with RNA polymerase II, but the role the Paf1 complex plays at the promoter has not been clear. Evidence that the Paf1 complex is required for monoubiquitination of histone H2B at promoters. Strains deleted for several components of the Paf1 complex are defective in monoubiquitination of histone H2B, which results in the loss of methylation of lysines 4 and 79 of histone H3. Paf1 complex is required for the interaction of Rad6 and COMPASS with RNA polymerase II. Finally, the Paf1 complex is shown to be required for Rad6-Bre1 catalytic activity but not for the recruitment of Rad6-Bre1 to promoters. Thus, in addition to its role during the elongation phase of transcription, the Paf1 complex appears to activate the function but not the placement of the Rad6-Bre1 ubiquitin-protein ligase at the promoters of active genes. A model is presented demonstrating the role of the Paf1 complex in the functional activation of the Rad6-Bre1 complex in ubiquitination of histone H2B at promoters (Wood, 2003; full text of article).

The hallmark of the Bre1 proteins is a C-terminal RING finger domain linked to an extensive N-terminal coiled-coil region. The 39 amino acid C3HC4 RING domain is flanked on both sides by 15 conserved amino acids, suggesting that the fly and mammalian proteins are true orthologs of yeast Bre1p (Hwang, 2003). RING domains are typically found in E3 ubiquitin ligases and frequently mediate the interaction with the E2 ubiquitin-activating enzyme while the other parts of the protein are involved in substrate recognition. The RING domains are therefore critical to catalyze the transfer of ubiquitin from the E2 to the substrate. To confirm the functional importance of the RING domain in dBre1, tests were performed to see whether an N-terminal fragment of dBre1 that lacks the RING domain (δ RING) could rescue dBre1 mutants. No rescue was observed with any of the 4 transgenic lines (from a total of 814 flies scored), confirming that the RING domain is essential for the function of dBre1 as it is for yeast Bre1p (Hwang, 2003; Wood, 2003; Bray, 2005).

To examine the subcellular location of full-length dBre1 and the derivative that lacks the RING domain, both forms of the protein were tagged with GFP at the N terminus. Both GFP-dBre1 and GFP-δRING are predominantly nuclear in embryonic and imaginal disc cells, although a low level of protein is also detectable in the cytoplasm. This nuclear-cytoplasmic distribution is similar to that of a δ RING derivative of human Bre1-B when it is overexpressed in mammalian cells. Thus dBre1 appears to be a nuclear protein, like its mammalian counterpart, and deletion of the RING domain does not alter its subcellular distribution even though it abolishes its ability to rescue the mutants (Bray, 2005).

The lethal allele E132 was fortuitously identified among a collection of mutants that modify the wing notching phenotype caused by Armadillo depletion. Genetic mapping of the lethality associated with E132 placed this at 64E8, and it was found to be allelic to an existing mutation, l(3)01640, caused by the P element insertion P1541. Using plasmid rescue of the P element, the site of insertion was localized to the first intron of the open reading frame CG10542, which encodes a predicted protein of 1044 amino acids. The insertion site is 48 nucleotides upstream of the translation initiation codon. Precise excision of P1541 restores viability, confirming that the P element insertion and, by inference, E132 are lethal alleles of CG10542. In support of this, ubiquitous overexpression of the full-length protein encoded by CG10542 rescues the lethality of E132 or P1541 mutant embryos and sustains development to give essentially normal adult flies (with a few minor defects including slightly reduced bristles). CG10542 encodes a conserved protein with close relatives in mammals, C. elegans, plants, and fungi. The Drosophila protein has been named dBre1, after its relative Bre1p in the yeast S. cerevisiae (Bray, 2005).

The hallmarks of the Bre1 proteins are a C-terminal RING finger domain linked to an extensive N-terminal coiled-coil region. The 39 amino acid C3HC4 RING domain is flanked on both sides by ~15 conserved amino acids, suggesting that the fly and mammalian proteins are true orthologs of yeast Bre1p. RING domains are typically found in E3 ubiquitin ligases and frequently mediate the interaction with the E2 ubiquitin-activating enzyme while the other parts of the protein are involved in substrate recognition. The RING domains are therefore critical to catalyze the transfer of ubiquitin from the E2 to the substrate. To confirm the functional importance of the RING domain in dBre1, tests were performed to see whether an N-terminal fragment of dBre1 that lacks the RING domain (ΔRING) could rescue dBre1 mutants. No rescue was observed with any of the 4 transgenic lines (from a total of 814 flies scored), confirming that the RING domain is essential for the function of dBre1 as it is for yeast Bre1p (Bray, 2005).

To examine the subcellular location of full-length dBre1 and the derivative that lacks the RING domain, both forms of the protein were tagged with GFP at the N terminus. Both GFP-dBre1 and GFP-ΔRING are predominantly nuclear in embryonic and imaginal disc cells, although a low level of protein is also detectable in the cytoplasm. This nuclear-cytoplasmic distribution is similar to that of a ΔRING derivative of human Bre1-B when it is overexpressed in mammalian cells. Thus dBre1 appears to be a nuclear protein, like its mammalian counterpart, and deletion of the RING domain does not alter its subcellular distribution even though it abolishes its ability to rescue the mutants (Bray, 2005).

To investigate the role of dBre1 in the fly, homozygous mutant clones were generated in the imaginal disc precursors of the adult structures. Surprisingly, it was found that the majority of defects were similar to those caused by defects in Notch signaling. Thus, adult flies bearing E132 or P1541 mutant clones show notches in the wing margin and aberrant spacing of wing margin bristles, wing blistering and vein defects, fusions of leg segments, and loss of notal bristles and rough eyes. Most of these phenotypes are characteristic of reduced Notch signaling and are distinct from those produced by loss-of-function of other signaling pathways, such as Wingless, Dpp, or Hedgehog signaling that also operate during imaginal disc development. The phenotypic data suggest therefore that dBre1 has a role in promoting Notch signaling (Bray, 2005).

To confirm this, the expression of Notch target genes was examined in dBre1 mutant clones. Since dBre1 mutant clones are considerably smaller than their matched wild-type twin clones, the Minute technique was used to compensate for the growth defect of the mutant clones. In wing imaginal discs, cut and Enhancer of split [E(spl)] are expressed along the prospective wing margin, and their expression depends directly on Notch signaling. Cut expression is absent in large E132 mutant clones, and is lost (3/11) or reduced (6/11) in most P1541 mutant clones. Likewise, E(spl) expression is lost cell autonomously from all E132 mutant clones in the wing. Conversely, expression of spalt, a target of Dpp signaling in the wing, is not reduced in P1541 and E132 mutant cells, indicating that the effects of dBre1 mutation are relatively specific. Similar results are obtained in the eye, where E(spl) expression is also disrupted in E132 clones. Expression in the neurogenic region at the furrow is lost, and elsewhere it is absent or severely reduced, except in the basal layer of undifferentiated cells where expression is independent of Notch. In addition, a derepression of the neuronal cell marker Elav was observed in eye disc clones. The latter indicates excessive neuronal recruitment due to diminished Notch-mediated lateral inhibition (note, however, that the phenotypes are not identical to those produced by complete absence of Notch, which in the eye results in loss of neuronal markers because Notch is needed to promote neural development by alleviating Su(H)-mediated repression. These results demonstrate that dBre1 functions in multiple developmental contexts and, specifically, that it is required for the subset of Notch functions that involve Su(H)-dependent activation of Notch target genes (Bray, 2005).

To further confirm the importance of dBre1 during Notch signaling, it was asked whether any genetic interactions could be detected between overexpressed dBre1 or ΔRING and mutations in Notch (N) or its ligand Delta (Dl). Indeed, overexpression of either protein in the wing disc results in adult phenotypes. In each of 5 ΔRING-expressing lines, mild if consistent mutant phenotypes were observed in both males and females, namely upward-curved wings (due to stronger expression in the dorsal wing compartment), tiny vein deltas, and a significant decrease in wing size. These defects are more severe after overexpression of ΔRING in dBre1 heterozygotes, indicating that ΔRING acts as a weak dominant-negative. Consistent with this, excess ΔRING significantly enhances the phenotypes of N/+ and Dl/+ heterozygotes, resulting in increased vein thickening and additional vein material and, in the case of N/+, also in more frequent wing notching. These genetic interactions support the link between dBre1 and Notch signaling (Bray, 2005).

Excess full-length dBre1 in wing discs causes vein defects whose strength, however, varies considerably between different dBre1-expressing lines, and between males and females (probably because the ms1096.GAL4 driver produces higher expression levels in males). In most lines (4/6), vein thickening and additional vein material were observe only in males, while female wings appear normal. These vein defects in male wings are suppressed to almost normal in dBre1 heterozygotes, suggesting that they are due to increased levels of functional dBre1 protein. The remaining 2 lines produce similar vein defects also in females. Unexpectedly, these defects are enhanced in N/+ and Dl/+ heterozygotes, suggesting that the overexpressed dBre1 interferes with Notch signaling, rather than enhancing it as might have been expected. This anomalous result could be explained if dBre1 is part of a multiprotein complex, in which case its overexpression might interfere with the function of this complex by titrating one of its components. Nevertheless, the genetic interactions between overexpressed dBre1 and Notch and Delta further underscore the link between dBre1 and Notch signaling (Bray, 2005).

To test whether dBre1 directly influences Notch-dependent transcription, Drosophila S2 cells were transfected with Flag-tagged or untagged dBre1, and the activity of a Notch-specific reporter containing 4 Su(H) binding sites [NRE, a luciferase derivative of Gbe+Su(H)_m8] was measured in the presence or absence of low levels of N^ICD. As a control, a reporter was used with mutant Su(H) binding sites [NME, or Gbe+Su(H)_mut]. These experiments reveal a significant stimulation of the NRE reporter by dBre1, especially in the presence of N^ICD. The degree of stimulation is similar to that observed when the ubiquitin ligase Hdm2 is added to transcription assays of Tat activity. dBre1 also elicits a slight stimulation of NME. The fact that overexpressed dBre1 has stimulatory effects on Notch in the transfection assays but not in imaginal discs presumably reflects differences either in the levels of dBre1 or in the amounts of other limiting factors in the two cell contexts. Nevertheless, the transfection assays reveal an intrinsic potential of dBre1 in stimulating the transcription mediated by Su(H) and its coactivator N^ICD (Bray, 2005).

All these results point to a role of dBre1 in promoting Notch signaling. Since other ubiquitin ligases have been shown to influence the levels of specific protein components of the Notch pathway, whether there were any alterations to Notch, Delta, or Su(H) levels in dBre1 mutant clones was investigated. While there are no detectable changes in Notch or Delta staining in dBre1 mutant cells, the levels of Su(H) staining are enhanced slightly but consistently, and cell autonomously, in mutant clones of both dBre1 alleles, regardless of the location of these clones within the disc. This is also obvious in clones induced early in larval development in a non-Minute background in which the mutant dBre1 clones remain small. As an aside, these clones reveal that individual dBre1 mutant cells are enlarged, reminiscent of the yeast bre1p mutant which also shows a 'large cell'phenotype. This phenotype has not been observed in cells lacking Notch signaling, so this aspect of dBre1 function appears distinct from its role in the Notch pathway, and suggests that there are additional molecular targets. Nevertheless, the elevated levels of Su(H) in the dBre1 mutant clones identify Su(H) as one molecular target of dBre1 and suggest that, in the wild-type, dBre1 may expose Su(H) to ubiquitin-mediated degradation. The effects on Su(H) are consistent with the cell-autonomous action of dBre1 on Notch target gene expression, but the fact that removal of dBre1 has a stabilizing effect on Su(H) appears to contradict its stimulating effect on Notch-dependent transcription. Since Su(H) functions as both a repressor and an activator, this may be explained if loss of dBre1 specifically stabilizes the repressor complex. Alternatively, the effect of dBre1 mutations on Su(H) may reflect an indirect bystander activity of dBre1 (Bray, 2005).

Finally, it was asked whether dBre1 has a similar molecular function as its relative yeast Bre1p. The latter is required for the monoubiquitination of histone H2B, which is a prerequisite for the subsequent methylation of histone H3 at K4 by SET1-containing complexes. H3K4 methylation appears to be a chromatin mark for transcriptionally active genes, and yeast bre1p mutants show defects in the transcription of inducible genes that have been ascribed to the lack of H2B ubiquitination and H3K4 methylation at the promoters of these genes. Since there are no in vitro assays for H2B ubiquitination and no antibodies that detect this modified form of H2B, effects of dBre1 mutations on the linked H3K4 methylation were investigated. Wing discs bearing dBre1 mutant clones were stained with an antibody specific for trimethylated H3K4 (H3K4m). This revealed a significant reduction of H3K4m in P1541 mutant clones. More strikingly, in clones of the stronger E132 allele, H3K4m is barely detectable. In contrast, staining of these clones with an antibody against H3K9m does not show any changes in the mutant territory, indicating that the effect in dBre1 mutant clones on the methylation of H3K4 is relatively specific. It is noted that, in wild-type wing discs, there is slight modulation of trimethylated H3K4, with higher levels at the dorsoventral boundary where Notch is activated. However, Notch mutant cells retain robust H3K4m staining, although occasionally show slightly lowered levels compared to adjacent wild-type cells. Thus, the reduced H3K4m staining in dBre1 mutant cells is primarily due to an activity loss of dBre1 rather than due to loss of Notch signaling. Based on its effects on tri-methylated H3K4, it is concluded that dBre1 is indeed the functional homolog of yeast Bre1p. Furthermore, it appears that the activity of dBre1 is essential for the bulk of trimethylated H3K4 in imaginal disc cells (Bray, 2005).

In yeast, H2B ubiquitination and H3K4 methylation are associated with sites of active transcription, but the only identified natural target gene is GAL1. In Drosophila, the target genes of dBre1 evidently include genes regulated by Notch, given the requirement of dBre1 for their transcription. It is therefore conceivable that Su(H) may have a role in targeting dBre1 to their promoters (although it was not possible to detect direct binding or coimmunoprecipitation between dBre1 and Su(H). It is puzzling that dBre1 has a slight destabilizing effect on Su(H), despite being an activating component of Notch signaling. It is believed that this could be a bystander effect of dBre1: evidence suggests that the Bre1p-mediated monoubiquitination of H2B leads to a transient recruitment of proteasome subunits to chromatin, and that the subsequent methylation of H3K4 depends on the activity of these proteasome subunits. Their transient presence at specific target genes may have a destabilizing effect on nearby DNA binding proteins, and the mildly increased levels of Su(H) in dBre1 mutant cells could therefore reflect a failure of proteasome recruitment due to loss of H2B monoubiquitination (Bray, 2005).

Perhaps the most interesting implication of the results is that the dBre1-mediated monoubiquitination of H2B and methylation of H3K4 may be critical steps in the transcription of Notch target genes. Indeed, it appears that the Notch target genes belong to a group of genes whose transcription is particularly susceptible to the much reduced levels of H3K4m in dBre1 mutant cells. Based on the dBre1 mutant phenotypes, there are likely to be other genes in this group, including for example genes controlling cell survival and cell size. Nevertheless, it would appear that the transcription of Notch target genes is particularly reliant on the activity of dBre1. Other examples are emerging where the transcriptional activity of a subset of signal responsive genes is particularly sensitive to the function of a particular chromatin modifying and/or remodelling factor. This sensitivity presumably reflects the molecular mechanisms used by signaling pathways to activate transcription at their responsive enhancers. Understanding why Notch-induced transcription is particularly susceptible to loss of dBre1 function will require knowledge of these underlying molecular mechanisms (Bray, 2005).

Drosophila stem cells share a common requirement for the histone H2B ubiquitin protease scrawny: Scrawny interacts with PAF1 and likely opposes BRE1 action on H2B thus participating in a conserved pathway of chromatin regulation linking H2B ubiquitylation with H3K4me3 methylation

Stem cells within diverse tissues share the need for a chromatin configuration that promotes self-renewal, yet few chromatin proteins are known to regulate multiple types of stem cells. A Drosophila gene, scrawny (scny), encoding a ubiquitin-specific protease, is required in germline, epithelial, and intestinal stem cells. Like its yeast relative UBP10, Scrawny deubiquitylates histone H2B and functions in gene silencing. Consistent with previous studies of this conserved pathway of chromatin regulation, scny mutant cells have elevated levels of ubiquitinylated H2B and trimethylated H3K4. These findings suggest that inhibiting H2B ubiquitylation through scny represents a common mechanism within stem cells that is used to repress the premature expression of key differentiation genes, including Notch target genes (Buszczak, 2009).

Stem cells are maintained in an undifferentiated state by signals they receive within the niche and are subsequently guided toward particular fates upon niche exit. Within ES cells and during differentiation, cell state changes are controlled at the level of chromatin by alterations involving higher order nucleosome packaging and histone tail modifications. Polycomb group (PcG) and Trithorax group (trxG) genes influence key histone methylation events at the promoters of target genes, including H3K27 and H3K4 modifications associated with gene repression and activation, respectively, but few other genes with a specific role in stem cells are known (Buszczak, 2009).

Histone H2A and H2B mono-ubiquitylation play fundamental roles in chromatin regulation, and H2A ubiquitylation has been linked to PcG-mediated gene repression and stem cell maintenance. The mammalian Polycomb repressive complex 1 (PRC1) component RING1B is a H2A ubiquitin ligase that is required to block the elongation of poised RNA polymerase II on bivalent genes in ES cells. Mutations in the PRC1 component, BMI-1, the ortholog of Psc in the mammalian PRC1, complexes with RING1B, and causes multiple types of adult stem cells to be prematurely lost. The role of H2B ubiquitylation in stem cells is unclear, however. In yeast, ubiquitylation of Histone H2B by the RAD6 and BRE1 ligases controls H3K4 methylation (H3K4me3), a process that requires the polymerase accessory factor PAF1. Conversely, H2B deubiquitylation by the ubiquitin-specific protease (USP) family member UBP10 is required for silencing telomeres, rDNA and other loci. The Drosophila homolog of BRE1, dBRE1, also is needed for H3K4 methylation, suggesting that this pathway is conserved. Furthermore, the Drosophila ubiquitin-specific protease USP7 is part of a complex that selectively deubiquitylates H2B and genetically interacts with PcG mutations. Mutations in another USP family member, Nonstop, increase H2B ubiquitylation and cause axon targeting defects in the eye (Buszczak, 2009).

In order to gain further insight into the role of H2B ubiquitylation in stem cells, a novel Drosophila gene, scrawny (scny) (CG5505), was identified, whose encoded USP family protein shares homology with human USP36 and among yeast USPs closely matches UBP10 within the core protease domain. Strains bearing scny insertions, except for a viable GFP protein trap (CA06690), were female sterile or lethal, and proved to be allelic. Transposon excision or expression of a scny-RB cDNA reverts the phenotype of tested alleles. An anti-SCNY antibody raised against a domain common to all SCNY isoforms recognizes wild type and SCNY-GFP on a Western blot. SCNY protein levels in homozygous third instar larvae are greatly reduced in lethal mutants, and SCNY expression is also lower in stem cell-enriched ovarian tissue from adults homozygous for the sterile d06513 allele. Consistent with a role in gene silencing, several scny mutations act as dominant suppressors of position effect variegation (Buszczak, 2009).

Further studies strongly suggested that SCNY functions in vivo as an H2B-ubiquitin protease. Recombinant full-length SCNY protein, but not a version bearing a point mutation in the protease domain, efficiently deubiquitylates histone H2B in vitro. scny^f01742 homozygous tissue contains levels of Ub-H2B that are elevated at least twofold compared to wild type. As expected if Ub-H2B is required for H3K4 methylation, clones of homozygous scny^e00340 mutant cells stain more strongly for H3K4me3 than heterozygous cells. Consistent with a direct rather than an indirect action on Ub-H2B levels, anti-SCNY antibodies co-immunoprecipitate H2B from Drosophila embryonic nuclear extracts. Moreover, epitope-tagged SCNY co-immunoprecipitates Drosophila PAF1, but not Cyclin T (or several other tested chromatin proteins) when co-expressed in S2 tissue culture cells. Together, these data support the view that SCNY participates in a conserved pathway of chromatin regulation linking H2B ubiquitylation with H3K4me3 methylation. Because the effects of scny mutation on Ub-H2B and H3K4me3 are opposite to those of dBre1 mutation, SCNY likely opposes dBRE1 action on H2B, just as UBP10 opposes BRE1 action on H2B in yeast (Buszczak, 2009).

Drosophila male and female gonads contain well characterized germline stem cells (GSCs) that allow the effects of genes on stem cell maintenance to be quantitatively analyzed. High levels of scny expression were observed in female and male GSCs using SCNY-GFP and identical staining was observed using anti-SCNY immunofluoresence. SCNY protein resides in cell nuclei and is enriched in nucleoli. In sterile or semi-fertile scny mutant adults, the numbers of germline stem cells surrounding the testis hub and within germaria were clearly reduced. The half-lives of female GSCs bearing clones of three different scny alleles were all sharply reduced. Later follicular development was also abnormal suggesting that scny continues to function after the stem cell stage. However, previous studies indicate that accelerated GSC loss is a specific phenotype, and hence that scny has a preferential requirement in GSCs (Buszczak, 2009).

A known mechanism of increased GSC loss is the premature activation of differentiation genes. Staining germaria with an antibody specific for multiple sites of histone H3 acetylation (H3-Ac) suggested that scny mutation affects the global chromatin organization of GSCs. Wild type GSCs contain lower levels of H3-Ac than slightly older germ cells within cysts. Presumptive GSCs located in the GSC niche in scny mutants frequently stained more strongly, suggesting that they have begun to upregulate general transcription. Some scny GSC-like cells also expressed bag-of-marbles (bam), a key cystoblast differentiation gene, and GSC-like cells in scny^d06513; bam^Δ86 mutant females persist in the germarium . However, it could not be completely ruled out that the observed increases in H3-Ac levels and bam expression were a result rather than a cause of the premature differentiation and loss of scny GSCs (Buszczak, 2009).

To determine if scny is also required in a very different type of stem cell, the epithelial follicle stem cell (FSC), the persistence of individual scny mutant FSCs was quantitatively. The half-life of FSCs mutant for scny^l(3)02331 was reduced more than 10-fold, while the scny^f01742 mutation also caused a sharp decline. However, mutant follicle cells continued to develop normally at later stages. Thus, scny is preferentially required to maintain FSCs as well as GSCs (Buszczak, 2009).

The largest population of Drosophila stem cells are the hundreds of multipotent intestinal stem cells (ISCs) that maintain the adult posterior midgut. ISCs signal to their daughters via Delta-Notch signaling to specify enterocyte vs. enteroendocrine cell fate, but the pathway must remain inactive in the ISCs themselves to avoid differentiation. Most ISCs (those about to produce enterocytes) express high levels of the Notch ligand Delta, allowing them to be specifically distinguished from other diploid gut cells. This study found that SCNY-GFP is expressed in ISCs suggesting that SCNY plays a role in these stem cells as well. While 7-day old normal adult midguts contain a high density of ISCs, as revealed by Delta staining, it was found that corresponding tissue from 7-day-old scny^f01742 or scny^f01742/scny^l(3)02331 escaper adults possess very few Delta-positive cells. ISCs are present in near normal numbers at eclosion, but are rapidly lost in the mutant adults, indicating that scny is required for ISC maintenance (Buszczak, 2009).

It is suspected that inappropriate Notch pathway activation was responsible for the premature ISC loss in scny mutants. dBre1 mutations strongly reduce Notch signaling, suggesting that Notch target genes are particularly dependent on H2B mono-ubiquitylation and H3K4 methylation. Consequently, scny mutations, which have the opposite effects on Ub-H2B and H3K4me3 levels, might upregulate Notch target genes, stimulating ISCs to differentiate prematurely. This idea was tested by supplementing the food of newly eclosed scny^f01742/scny^l(3)02331 adults with 8 mM DAPT, a gamma-secretase inhibitor that blocks Notch signaling and phenocopies Notch mutation when fed to wild type animals. scny^f01742/scny^l(3)02331 DAPT-treated adults remained healthy and the guts of 7-day old animals still contained many ISCs, although not as many as wild type. Tumors like those produced in wild type animals fed DAPT were not observed. Thus, in these animals endogenous stem cell loss can be slowed by drug treatment (Buszczak, 2009).

These experiments provide strong evidence that a pathway involving the ubiquitin protease Scrawny and the ubiquitin ligase dBRE1 controls the levels of Ub-H2B, and H3K4me3 at multiple target sites in the Drosophila genome. Although, other ubiquitin proteases also act on Ub-H2B in Drosophila, the direct interaction between SCNY and H2B, and the strong effects of scny mutations argue that it plays an essential, direct role in silencing genomic regions critical for cellular differentiation, including Notch target genes. SCNY interacts with the RNA polymerase accessory factor complex component, PAF1. Upregulation of H2B ubiquitinylation and H3 methylation in yeast is mediated by the PAF1 complex and is associated with elongating RNA Pol II. Drosophila PAF1 is required for normal levels of H3K4me3 at the hsp70 gene, and another PAF1 complex member, RTF1, is needed for H3K4 methylation and Notch target gene expression. Indeed, the pathway connecting Ub-H2B, H3K4me3 and gene silencing appears to be conserved in organisms as distant as Arabidopsis. A human protein closely related to SCNY, USP36, is overexpressed in ovarian cancer cells, and the results suggest it may act as an oncogene by suppressing differentiation (Buszczak, 2009).

Above all, these experiments indicate that SCNY-mediated H2B deubiquitylation is required to maintain multiple Drosophila stem cells, including progenitors of germline, epithelial and endodermal lineages. In ES cells and presumably in adult stem cells, many differentiation genes contain promoter-bound, arrested RNA Pol II and are associated with Polycomb group proteins. It is envisioned that in the niche environment SCNY activity overrides that of dBRE1, keeping levels of Ub-H2B (and hence H3K4me3) low at key differentiation genes. Upon exit from the niche, the balance of signals shifts to favor H2B ubiquitylation, H3K4 trimethylation, and target gene activation. Thus, the control of H2B ubiquitylation, like H2A ubiquitylation, plays a fundamental interactive role in maintaining the chromatin environment of the stem cell state (Buszczak, 2009).

Catalysis-dependent stabilization of Bre1 fine-tunes histone H2B ubiquitylation to regulate gene transcription.

Monoubiquitylation of histone H2B on Lys123 (H2BK123ub1) plays a multifaceted role in diverse DNA-templated processes, yet the mechanistic details by which this modification is regulated are not fully elucidated. This study shows, in yeast, that H2BK123ub1 is regulated in part through the protein stability of the E3 ubiquitin ligase Bre1. Bre1 stability is controlled by the Rtf1 subunit of the polymerase-associated factor (PAF) complex and through the ability of Bre1 to catalyze H2BK123ub1. Using a domain in Rtf1 that stabilizes Bre1, it was shown that inappropriate Bre1 levels lead to defects in gene regulation. Collectively, these data uncover a novel quality control mechanism used by the cell to maintain proper Bre1 and H2BK123ub1 levels, thereby ensuring proper control of gene expression (Wozniak, 2014).

Structural analysis of nucleosomal barrier to transcription

Thousands of human and Drosophila genes are regulated at the level of transcript elongation and nucleosomes are likely targets for this regulation. However, the molecular mechanisms of formation of the nucleosomal barrier to transcribing RNA polymerase II (Pol II) and nucleosome survival during/after transcription remain unknown. This study shows that both DNA-histone interactions and Pol II backtracking contribute to formation of the barrier and that nucleosome survival during transcription likely occurs through allosterically stabilized histone-histone interactions. Structural analysis indicates that after Pol II encounters the barrier, the enzyme backtracks and nucleosomal DNA recoils on the octamer, locking Pol II in the arrested state. DNA is displaced from one of the H2A/H2B dimers that remains associated with the octamer. The data reveal the importance of intranucleosomal DNA-protein and protein-protein interactions during conformational changes in the nucleosome structure on transcription. Mechanisms of nucleosomal barrier formation and nucleosome survival during transcription are proposed (Gaykalova, 2015).

The data identify the transition from the +48 to +49 position as the key step during transcription through a nucleosome where Pol II makes a choice between arrest and further transcription. In the minimal system containing only Pol II and a nucleosome, this choice is dictated primarily by the sequence of nucleosomal DNA that determines the affinity of DNA-histone interactions and the probability of Pol II backtracking. In particular, R3 DNA sequence at the +(99-102) region is critical for escape from the +48 position (Gaykolova, 2015).

The arrest at position +48 becomes nearly irreversible on the 601 nucleosome due to Pol II backtracking along DNA and partial recoiling of nucleosomal DNA on the octamer after backtracking. The ability of Pol II to backtrack most likely depends on the DNA sequence immediately upstream of the active center of the enzyme and is typically reversible on DNA. However, recoiling of nucleosomal DNA on the octamer 'locks' Pol II and makes backtracking irreversible; a similar mechanism has been proposed previously (Gaykolova, 2015).

The choice between the arrest and further transcription through the nucleosome can be affected by transcription factors and histone chaperones. Thus, elongation factor TFIIS that facilitates transcription through chromatin in vitro can facilitate escape from the arrested state. Histone chaperone FACT facilitates transcription through chromatin via transient interaction with the open surface of histone octamer. Thus, depending on the sequence of nucleosomal DNA and presence of dedicated proteins, various steps during transcription through a nucleosome can be rate limiting and possibly regulated in vivo (Gaykolova, 2015).

After overcoming the nucleosomal barrier, further Pol II transcription is typically accompanied by nucleosome survival mediated by formation of a small intranucleosomal DNA loop on the surface of the histone octamer (Ø loop). The high efficiency of nucleosome survival has been puzzling because transcription by various RNA polymerases is accompanied by uncoiling of an extended DNA region (up to 80 bp) from the octamer, which is expected to induce immediate loss of an H2A/H2B dimer. The observed stability of the +42 complex containing a H2A/H2B dimer that is exposed into the solution suggests that binding of the H2A/H2B dimer to the H3/H4 tetramer could be allosterically stabilized and provides an explanation for the remarkable nucleosome stability of nucleosomal structure during various processes including transcription, replication, and ATP-dependent chromatin remodeling. Although one dimer is constitutively displaced from nucleosomes during transcription in vitro, this displacement likely occurs after transcription through the position +49 because the EC +49 contains both H2A/H2B dimers (Gaykolova, 2015).

The +42/48 pausing described in this study is related to nucleosomal pausing observed in Drosophila and yeast. In yeast, the primary nucleosomal barrier during transcript elongation by Pol II is encountered when the active center of the enzyme is +(40-55) bp in the nucleosome. A similar barrier is encountered by Pol II in Drosophila during transcription through nucleosomes localized more than ~400 bp from the transcription start site. However, there are additional nucleosomal barriers at the positions -7 and +20 bp in Drosophila, characteristic for nucleosomes localized immediately downstream of the transcription start site (Gaykolova, 2015).

Recent studies suggest that the density of Pol II complexes along the transcribed genes likely dictates the critical outcomes of transcription through chromatin-the height of nucleosomal barrier and the extents of histone displacement and exchange. At the same time, multiple factors including histone modifications, histone variants, histone chaperones, and chromatin remodelers further modify the histone dynamics on transcribed genes. Some of these factors interact with tumor suppressors and present important targets for the development of anticancer drugs (Gaykolova, 2015).

Search PubMed for articles about Drosophila

Ajiro, K,, et al. (2010) Reciprocal epigenetic modification of histone H2B occurs in chromatin during apoptosis in vitro and in vivo. Cell Death Differ. 17: 984-993. PubMed ID: 20057502 P>Böhm, V., Hieb, A.R., Andrews, A.J., Gansen, A., Rocker, A., To ́ th, K., Luger, K. and Langowski, J. (2011). Nucleosome accessibility governed by the dimer/tetramer interface. Nucleic Acids Res. 39: 3093–3102. PubMed ID: 21177647

Bonenfant, D., et al. (2007). Analysis of dynamic changes in post-translational modifications of human histones during cell cycle by mass spectrometry. Mol. Cell Proteomics 6: 1917-1932. PubMed ID: 17644761

Bray, S., Musisi, H. and Bienz, M. (2005). Bre1 is required for Notch signaling and histone modification. Dev. Cell 8(2): 279-86. PubMed ID: 15691768

Briggs, S. D., et al. (2002). Gene silencing: trans-histone regulatory pathway in chromatin. Nature 418: 498. PubMed ID: 12152067

Buszczak, M., Paterno, S. and Spradling, A. C. (2009). Drosophila stem cells share a common requirement for the histone H2B ubiquitin protease scrawny. Science 323: 248-251. PubMed ID: 19039105

Chatterjee, C., McGinty, R.K., Fierz, B. and Muir, T.W. (2010). Disulfide- directed histone ubiquitylation reveals plasticity in hDot1L activation. Nat. Chem. Biol. 6: 267-269. PubMed ID: 20208522

Cheung, W. L., et al (2003). Apoptotic phosphorylation of histone H2B is mediated by mammalian sterile twenty kinase. Cell 113: 507-517. PubMed ID: 12757711

Clapier, C. R., et al. (2008). Structure of the Drosophila nucleosome core particle highlights evolutionary constraints on the H2A-H2B histone dimer. Proteins 71(1): 1-7. PubMed ID: 17957772

Dai R. P., Yu F. X., Goh S. R., Chng H. W., Tan Y. L., Fu J. L., Zheng L. and Luo Y. (2008). Histone 2B (H2B) expression is confined to a proper NAD+/NADH redox status. J. Biol. Chem. 283: 26894-26901. PubMed ID: 18682386

Desrosiers, R. and Tanguay, R. M. et al. (1988), Methylation of Drosophila histones at proline, lysine, and arginine residues during heat shock. J. Biol. Chem. 263: 4686-4692. PubMed ID: 3127388

Fereres, S., Simon, R., Mohd-Sarip, A., Verrijzer, C. P. and Busturia, A. (2014). dRYBP counteracts chromatin-dependent activation and repression of transcription. PLoS One 9: e113255. PubMed ID: 25415640

Gardner, R. G., Nelson, Z. W. and Gottschling, D. E. (2005). Ubp10/Dot4p regulates the persistence of ubiquitinated histone H2B: distinct roles in telomeric silencing and general chromatin. Mol. Cell Biol. 25(14): 6123-39. PubMed ID: 15988024

Gaykalova, D. A., Kulaeva, O. I., Volokh, O., Shaytan, A. K., Hsieh, F. K., Kirpichnikov, M. P., Sokolova, O. S. and Studitsky, V. M. (2015). Structural analysis of nucleosomal barrier to transcription. Proc Natl Acad Sci U S A 112: E5787-5795. PubMed ID: 26460019

Hwang, W. W., et al. (2003). A conserved RING finger protein required for histone H2B monoubiquitination and cell size control. Mol. Cell 11: 261-266. PubMed ID: 12535538

Kessler, R., Tisserand, J., Font-Burgada, J., Reina, O., Coch, L., Attolini, C. S., Garcia-Bassets, I. and Azorin, F. (2015). dDsk2 regulates H2Bub1 and RNA polymerase II pausing at dHP1c complex target genes. Nat Commun 6: 7049. PubMed ID: 25916810

Larschan, E., Bishop, E.P., Kharchenko, P.V., Core, L.J., Lis, J.T., Park, P.J. and Kuroda, M.I. (2011). X chromosome dosage compensation via enhanced transcriptional elongation in Drosophila. Nature 471: 115-118. PubMed ID: 21368835

Lee, J. S., et al. (2007). Histone crosstalk between H2B monoubiquitination and H3 methylation mediated by COMPASS. Cell 131: 1084-1096. PubMed ID: 18083099

Lee, M. C., Toh, L. L., Yaw, L. P. and Luo, Y. (2010). Drosophila octamer elements and Pdm-1 dictate the coordinated transcription of core histone genes. J. Biol. Chem. 285(12): 9041-53. PubMed ID: 20097756

Li, J., et al. (2008). Differential display identifies overexpression of the USP36 gene, encoding a deubiquitinating enzyme, in ovarian cancer. Int. J. Med. Sci. 5(3): 133-42. PubMed ID: 18566677

Li, X. S., Trojer, P., Matsumura, T., Treisman, J. E. and Tanese, N. (2010). Mammalian SWI/SNF--a subunit BAF250/ARID1 is an E3 ubiquitin ligase that targets histone H2B. Mol. Cell Biol. 30(7): 1673-88. PubMed ID: 20086098

Maile T, et al. (2004). TAF1 activates transcription by phosphorylation of serine 33 in histone H2B. Science 304: 1010-1014. PubMed ID: 15143281

Mohan, M., et al. (2010). Linking H3K79 trimethylation to Wnt signaling through a novel Dot1-containing complex (DotCom). Genes Dev. 24(6): 574-89. PubMed ID: 20203130

Mohan, R. D., Dialynas, G., Weake, V. M., Liu, J., Martin-Brown, S., Florens, L., Washburn, M. P., Workman, J. L. and Abmayr, S. M. (2014). Loss of Drosophila Ataxin-7, a SAGA subunit, reduces H2B ubiquitination and leads to neural and retinal degeneration. Genes Dev 28: 259-272. PubMed ID: 24493646

Nakanishi, S., et al. (2009), Histone H2BK123 monoubiquitination is the critical determinant for H3K4 and H3K79 trimethylation by COMPASS and Dot1. J. Cell Biol. 186: 371-377. PubMed ID: 19667127

Steger, D. J., et al. DOT1L/KMT4 recruitment and H3K79 methylation are ubiquitously coupled with gene transcription in mammalian cells. Mol Cell Biol. 2008;28:2825–2839.

Tooley, C. E., et al. (2010). NRMT is an alpha-N-methyltransferase that methylates RCC1 and retinoblastoma protein. Nature 466: 1125-1128. PubMed ID: 20668449

van der Knaap, J. A., et al. (2005). GMP synthetase stimulates histone H2B deubiquitylation by the epigenetic silencer USP7. Mol. Cell 17(5): 695-707. PubMed ID: 15749019

Villar-Garea, A., et al. (2012). Developmental regulation of N-terminal H2B methylation in Drosophila melanogaster. Nucleic Acids Res. 40(4): 1536-49. PubMed ID: 22053083

Wang, H., et al. (2004). Role of histone H2A ubiquitination in Polycomb silencing. Nature 431: 873-878. PubMed ID:

Webb, K. J., et al. (2010). Identification of protein N-terminal methyltransferases in yeast and humans. Biochemistry 49: 5225-5235. PubMed ID: 20481588

Weake, V. M., et al. (2008). SAGA-mediated H2B deubiquitination controls the development of neuronal connectivity in the Drosophila visual system. EMBO J. 27(2): 394-405. PubMed ID: 18188155

Wood, A., et al. (2003). Bre1, an E3 ubiquitin ligase required for recruitment and substrate selection of Rad6 at a promoter. Mol. Cell 11: 267-274. PubMed ID: 12535539

Wozniak, G. G. and Strahl, B. D. (2014). Catalysis-dependent stabilization of Bre1 fine-tunes histone H2B ubiquitylation to regulate gene transcription. Genes Dev 28: 1647-1652. PubMed ID: 25085417

Wu, L., et al. (2011). The RING finger protein MSL2 in the MOF complex is an E3 ubiquitin ligase for H2B K34 and is involved in crosstalk with H3 K4 and K79 methylation. Mol. Cell 43(1): 132-44. PubMed ID: 21726816

Yu F. X., Dai R. P., Goh S. R., Zheng L. and Luo Y. (2009). Logic of a mammalian metabolic cycle: an oscillated NAD+/NADH redox signaling regulates coordinated histone expression and S-phase progression. Cell Cycle 8: 773-779. PubMed ID: 19221488

Zheng L., Roeder R. G., Luo Y. (2003). S phase activation of the histone H2B promoter by OCA-S, a coactivator complex that contains GAPDH as a key component. Cell 114: 255–266. PubMed ID: 12887926

date revised: 11 June 2022

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