InteractiveFly: GeneBrief

Heterochromatin Protein 1c: Biological Overview | References

BIOLOGICAL OVERVIEW

Heterochromatin protein 1 (HP1) proteins are conserved in eukaryotes, with most species containing several isoforms. Based on the properties of Drosophila HP1a, it has proposed that HP1s bind H3K9me2,3 (Histone 3 methylate on lysine 9) and recruit factors involved in heterochromatin assembly and silencing. Yet, it is unclear whether this general picture applies to all HP1 isoforms and functional contexts. Evidence reported here suggests that Drosophila HP1c regulates gene expression as follows: (1) it localizes to active chromatin domains, where it extensively colocalizes with the poised form of RNApolymerase II (RNApol II), Pol IIo(ser5), and H3K4me3, suggesting a contribution to transcriptional regulation; (2) its targeting to a reporter gene does not induce silencing but, on the contrary, increases its expression, and (3) it interacts with the zinc-finger transcription factors WOC (Without children) and Relative-of-WOC (ROW). Although HP1c efficiently binds H3K9me2,3 in vitro, its binding to chromatin strictly depends on both WOC and ROW. Moreover, expression profiling indicates that HP1c, WOC, and ROW regulate a common gene expression program that, in part, is executed in the context of the nervous system. From this study, which unveils the essential contribution of DNA-binding proteins to HP1c functionality and recruitment, HP1 proteins emerge as an increasingly diverse family of chromatin regulators (Font-Burgada, 2008).

The contribution of chromatin to the regulation of genomic functions is well established. Most frequently, regulation by chromatin involves the establishment of specific patterns of post-translational histone modifications, which result in recruitment of regulatory nonhistone proteins. Heterochromatin protein 1 (HP1) constitutes one of the best-studied examples, where a regulatory nonhistone protein is recruited to chromatin through the recognition of a specific histone modification, di- or trimethylation of Lys 9 on the histone H3 tail (H3K9me2,3). This interaction, which involves the N-terminal chromodomain of HP1, is known to play a fundamental role in the formation and maintenance of heterochromatic domains (Font-Burgada, 2008).

Except in budding yeast, HP1 is widely conserved in eukaryotes, with most species containing several isoforms. HP1 proteins are characterized by a common structural organization consisting of two conserved domains, the N-terminal chromodomain and the C-terminal chromo-shadow domain, which are spaced by a variable nonconserved hinge domain. The existence of multiple isoforms suggests functional specialization, with different isoforms playing different functions. For instance, in Drosophila, three of the five HP1 isoforms (HP1a, HP1b, and HP1c) are ubiquitously expressed, while the other two (HP1d/Rhino and HP1e) are predominantly expressed in the germline (Vermaak, 2005). Moreover, ubiquitously expressed HP1 isoforms show differential chromosomal distributions, as HP1a is mainly associated to heterochromatin, while HP1c is excluded from centromeric heterochromatin and HP1b is found both at euchromatic and heterochromatic domains (Smothers, 2001). A similar situation is observed in mammals, where the patterns of localization of the three HP1 isoforms (HP1α, HP1β, and HP1γ) overlap only partially and show differential dynamics during differentiation and cell cycle progression (Minc, 1999; Hayakawa, 2003; Dialynas, 2007). In contrast, in the nematode C. elegans, two HP1 isoforms exist (HPL1 and HPL2), showing preferential euchromatic association, partially nonoverlapping expression and localization patterns, and distinct mutant phenotypes (Coustham, 2006; Schott, 2006). Interestingly, the only HP1 protein (TLF2/LHP1) of Arabidopsis thaliana appears to localize exclusively to euchromatin, where it colocalizes with H3K27me3 (Font-Burgada, 2008).

The molecular mechanisms that determine the differential distribution of the various HP1 isoforms, and their differential functional properties, remain largely unknown. Most knowledge about the mechanisms of action of HP1 proteins derives from studies addressing the functional properties of Drosophila HP1a or mammalian HP1α. From these studies, a general picture emerges by which, through the chromodomain, HP1 proteins bind chromatin regions enriched in H3K9me2,3 while, through the chromo-shadow domain, recruiting different factors resulting in various functional outcomes; namely, heterochromatin assembly and gene silencing. It is uncertain whether this general picture applies to all HP1 proteins and possible scenarios. Actually, Drosophila HP1a is known to play a more complex role(s) in the regulation of gene expression, as it is required for the expression of several heterochromatic genes, and certain euchromatic genes. Moreover, Drosophila HP1a is recruited to developmentally regulated genes and heat-shock-induced puffs in an RNA-dependent manner, and in an erythroid cell line, murine HP1α was found associated to actively transcribed genes (Font-Burgada, 2008).

This study reports on the functional characterization of HP1c, a Drosophila HP1 protein of largely unknown properties. The results show that HP1c extensively colocalizes with poised RNA polymerase II (RNApol II) and H3K4me3, a modification that correlates with active chromatin domains. Moreover, targeting HP1c to a reporter construct does not induce silencing but, on the contrary, results in increased expression of the reporter gene. This study also reports on the interaction of HP1c with the zinc-finger proteins WOC (without children) (Wismar, 2000; Warren, 2001), and Relative-of-WOC (ROW), which are putative transcription factors. HP1c efficiently binds H3K9me2,3 in vitro, but its binding to chromatin strictly depends on both WOC and ROW. Moreover, expression profiling indicates that HP1c, WOC, and ROW extensively cooperate to regulate gene expression, especially in the context of the nervous system. These results unveil the essential contribution of sequence-specific DNA-binding proteins to functionality of HP1c and its recruitment to chromatin (Font-Burgada, 2008).

Altogether, these results indicate that HP1c, WOC, and ROW are components of a distinct multiprotein complex. HP1c-WOC interaction is likely to be direct, since WOC contains a canonical PxVxL motif (¹⁵³⁶PHVLL¹⁵⁴⁰), which is known to mediate binding to the chromo-shadow domain of HP1 proteins (Lechner, 2000; Smothers, 2000). This motif is located within the highly conserved C-terminal HC domain of WOC, being also present in the three human homologs. ROW also contains several variant PxVxL motifs, suggesting that it might also bind directly to HP1c. In agreement with these observations, euchromatic localization of HP1c depends on the C-terminal chromo-shadow domain (Smothers, 2001), strongly suggesting that it mediates interaction with WOC and ROW (Font-Burgada, 2008).

This study also shows that binding of HP1c to chromatin depends on WOC and ROW that, in contrast, are reciprocally required for binding to chromatin. Domain structure and organization indicate that WOC and ROW are sequence-specific DNA-binding proteins. These results indicate that chromosomal association of HP1c is largely determined by the recognition of specific DNA sequences, which is in contrast to the situation observed in the case of Drosophila HP1a, or mammalian HP1α, where chromosomal association was found to depend on the recognition of a specific pattern of histone modifications; namely, H3K9 methylation. Several HP1 proteins, including HP1a and HP1α, were reported to interact with a number of transcription factors, replication proteins, and chromatin assembly complexes. Yet, it is unclear whether these interactions mediate recruitment of HP1 to specific sites, and/or in response to particular processes, or they actually take place after recruitment to regulate their functions. What is striking in the case of Drosophila HP1c is that its binding to chromatin is strictly dependent on DNA-binding proteins. The results, however, also show that, through the chromodomain, HP1c efficiently binds H3K9me2,3 in vitro. Actually, overexpression of HP1c-lacI leads to its mislocalization to heterochromatin, likely reflecting binding to H3K9me2,3. In this context, it must be noticed that HP1c shows a partial colocalization with H3K9me3. However, binding of HP1c at these sites is in general weak, being also obliterated in the absence of WOC and ROW. HP1 proteins have been reported to interact with different histone methyltransferases (HMTs), being involved in their recruitment to specific sites. Therefore, it is possible that H3K9 methylation at these sites is actually the consequence of HP1c binding. Altogether, these observations indicate that recognition of H3K9me2,3 is not a major determinant of the association of HP1c with chromatin in vivo. Consistent with this interpretation, replacing the chromodomain of HP1c by that of HP1a does not alter its chromosomal distribution. The interaction with WOC and ROW does not appear to hinder the chromodomain from binding H3K9me2,3 since overexpression of HP1c-lacI brings both HP1c and WOC to heterochromatin. Several other possibilities can account for the inability of HP1c to bind H3K9me2,3 in vivo. The interaction of HP1c with WOC and ROW might be of higher affinity than the interaction with H3K9me2,3. In addition, post-translational modifications could regulate these interactions. It is also possible that HP1c is actively excluded from heterochromatin. Whether binding to H3K9me2,3 plays a role at any stage during development or cell cycle progression remains, however, to be determined (Font-Burgada, 2008).

HP1a and HP1b also localize to euchromatin, yet they show strong binding to heterochromatin. Therefore, WOC and ROW could also play a role in binding of HP1a and HP1b to euchromatin. In fact, euchromatic localization of HP1b is decreased in both woc^RNAi and row^RNAi mutants. This effect, however, is much weaker than that observed in the case of HP1c. In contrast, binding of HP1a to euchromatin is not grossly altered in woc^RNAi and row^RNAi mutants, though its association to some specific loci, such as at the 31C region, appears to be affected. In contrast, binding of HP1a and HP1b to heterochromatin, which depends on H3K9me2,3, is not significantly affected in woc^RNAi and row^RNAi mutants. Similarly, mislocalization of overexpressed HP1c-lacI to heterochromatin, which likely reflects binding to H3K9me2,3, is not affected either in woc^RNAi and row^RNAi mutants (Font-Burgada, 2008).

Altogether, these observations suggest that, in Drosophila, HP1 proteins could be recruited to chromatin by at least two independent mechanisms: (1) recognition of H3K9me2,3, which is instrumental in heterochromatin binding, and (2) interaction with sequence-specific DNA-binding proteins, which mediate euchromatic localization of HP1c and, perhaps, of HP1b and HP1a to some specific loci. Actually, the interaction of HP1 proteins with DNA-binding proteins might be more frequent than anticipated. In fact, in C. elegans, HPL-2 was found to interact with LIN-13 (Coustham, 2006), a sequence-specific DNA-binding protein containing multiple zinc-finger domains (Font-Burgada, 2008).

HP1c localizes at multiple active chromatin domains and cooperates with WOC and ROW, which show features characteristic of transcription factors, to regulate gene expression. Moreover, targeting HP1c to a reporter construct promotes expression of the reporter gene. This effect is specific of HP1c, since targeting both HP1a and HP1b induce silencing. These results indicate that, rather than as a silencing factor, HP1c acts as a transcriptional regulator that is recruited to chromatin by sequence-specific DNA-binding proteins (Font-Burgada, 2008).

Other HP1 proteins have also been shown to contribute to the regulation of gene expression, yet their presence is generally associated to silencing. These include Drosophila HP1a, which is required for proper expression of most heterochromatic genes as well as a few euchromatic genes. Mammalian HP1γ has also been shown to localize at active genes in a murine erythroid cell line (Vakoc, 2005). In these cases, presence of HP1 appears to be implicated in stabilizing RNA transcripts, or in another RNA-processing event occurring during elongation. In the case of HP1c, however, colocalization with the poised RNApol II form, Pol IIo^ser5, is much stronger than with the elongating form, Pol IIo^ser2, suggesting that HP1c acts at the promoter level rather than during elongation. Consistent with this hypothesis, HP1c shows a much stronger colocalization with H3K4me3, a modification that occurs at promoters, than with H3K36me3, which occurs all through transcribed regions and incorporates during elongation. In full agreement with these results, WOC also shows extensive colocalization with Pol IIo^ser5, which is stronger than with Pol IIo^ser2 (Raffa, 2005). These results favor a contribution to the regulation of genes containing poised RNApol II. Actually, recent studies show that the presence of poised RNApol II at promoters is more frequent than anticipated, particularly on developmental control genes. Interestingly, a high proportion of genes coregulated by HP1c, WOC, and ROW act during development and morphogenesis (Font-Burgada, 2008).

The precise molecular mechanism(s) underlying the contribution of HP1c to transcription regulation remains to be determined. However, a contribution to RNApol II recruitment appears unlikely since, in the absence of WOC, RNApol II recruitment is not affected (Raffa, 2005). A contribution to the regulation of poised RNApol II is also uncertain, since no gross changes in the levels of Pol IIo^ser5 and Pol IIo^ser2 are observed in woc-null mutants by either immunostaining (Raffa, 2005) or Western analysis. It is possible, however, that HP1c/WOC/ROW act only on a reduced subset of genes containing poised RNApol II. It must also be noticed that HP1c likely participates both in promoting and inhibiting transcription. In fact, among the 158 genes that are differentially expressed in the same direction in hp1c^RNAi, woc^RNAi, and row^RNAi mutants, the number of up-regulated and down-regulated genes is similar, 80 in front of 78. Moreover, consistent with a contribution to repression, out of the 35 genes that are coregulated by HP1c, WOC, and ROW in the context of the nervous system, a significantly higher number of genes are found up-regulated than down-regulated in the mutants, 22 versus 13. In contrast, targeting HP1c to a reporter, though modestly, increases its expression. Altogether, these observations suggest that, depending on the actual functional/promoter context, HP1c can be engaged in either promoting or inhibiting transcription (Font-Burgada, 2008).

The patterns of differentially expressed genes observed in woc^RNAi and row^RNAi mutants show a very strong correlation, indicating that WOC and ROW share a common gene expression program. In addition, these results show that genes regulated by HP1c are also regulated by both WOC and ROW. In contrast, a high proportion (78.3%) of differentially expressed genes change expression both in woc^RNAi and row^RNAi but not in hp1c^RNAi, suggesting that WOC and ROW could also regulate gene expression independently of HP1c. However, the extensive colocalization observed between WOC, ROW, and HP1c argues against this possibility. In contrast, the results show that HP1c protein levels are significantly decreased in woc^RNAi and row^RNAi mutants. In this scenario, stronger synergistic effects would be expected in woc^RNAi and row^RNAi mutants than in hp1c^RNAi, which could result in more genes being differentially expressed. Consistent with this hypothesis, a major proportion of genes that are differentially expressed in all three mutants shows stronger changes in woc^RNAi and row^RNAi than in hp1c^RNAi. Actually, out of the 158 genes that are differentially expressed in the same direction in all three mutants, 77 had smaller changes in hp1c^RNAi than in woc^RNAi or row^RNAi, a statistically significant higher number than the 52.6 genes expected under the assumption that the magnitude of change is the same in all three mutants (Font-Burgada, 2008).

Clustering analysis suggests that the gene expression program coregulated by HP1c, WOC, and ROW is executed, at least in part, in the context of the nervous system. In agreement with this hypothesis, expression of woc and row is high in the nervous system during embryogenesis and larval development, and mutant larvae have reduced brains (Wismar, 2000). Furthermore, one of the human homologs of WOC, DXS6673E/ZNF261, is implicated in a form of X-linked mental retardation (Font-Burgada, 2008).

Others have reported that WOC regulates telomere function, since it is required to prevent chromosomal end-to-end fusions (Raffa, 2005). The physical and functional interactions between WOC, HP1c, and ROW suggest that HP1c and ROW might also regulate telomere function. Actually, at telomeres, colocalization of WOC with HP1c and ROW is also most extensive, with all detectable αWOC bands overlapping with αHP1c and αROW bands and vice versa. However, the incidence of telomere fusions in hp1c^RNAi and row^RNAi mutants is low, being similar to that observed in control flies, carrying an UAS-hairpin construct against an unrelated gene, GFP. This is likely the consequence of both the hypomorph character of the mutations and hyperactivation of the RNAi pathway, which is known to regulate telomere function. Consistent with this hypothesis, the frequency of telomere fusions is also low in woc^RNAi. The contribution of ROW to telomere function was also analyzed in row^l(2)SH2172, which corresponds to a very strong mutation caused by a P-element insertion at the ATG-start codon. The incidence of telomere fusions is significantly higher in homozygous row^l(2)SH2172 flies than in control wild-type flies, confirming its contribution to telomere function. The use of currently unavailable hp1c-null mutations is also likely to confirm the contribution of HP1c to telomere function (Font-Burgada, 2008).

HP1a is also known to regulate telomere function. Several observations, however, indicate that the contribution of WOC/ROW/HP1c is not related to that of HP1a. On one hand, su(var)2-5 mutants show much stronger effects than either woc or row mutants. Furthermore, both telomere length and expression of the telomeric retrotransposons Het-A and TART are increased in su(var)2-5 mutants, but they are not affected in woc-null mutants. In addition, expression profiling data show that, in hp1c^RNAi, woc^RNAi, and row^RNAi mutants, expression of Het-A and TART is not significantly affected. Similarly, other genes known to contribute to telomere function do not change expression in hp1c^RNAi, woc^RNAi, and row^RNAi mutants (data not shown). Altogether, these observations suggest that the contribution of WOC/ROW/HP1c to the regulation of telomere function is direct and independent of their contribution to the regulation of gene expression (Font-Burgada, 2008).

Drosophila HP1c is regulated by an auto-regulatory feedback loop through its binding partner Woc

HP1 is a major component of chromatin and regulates gene expression through its binding to methylated histone H3. Most eukaryotes express at least three isoforms of HP1 with similar domain architecture. However, despite the common specificity for methylated histone H3, the three HP1 isoforms bind to different regions of the genome. Most of the studies so far focused on the HP1a isoform and its role in transcriptional regulation. Since HP1a requires additional factors to bind methylated chromatin in vitro, it was asked whether another isoform might also require additional targeting factors. Indeed, HP1c interacts with the DNA binding factors Woc and Row and requires Woc to become targeted to chromatin in vivo. Moreover, the interaction between HP1c and Woc constitutes a transcriptional feedback loop that operates to balance the concentration of HP1c within the cell. This regulation may prevent HP1c from binding to methylated heterochromatin (Abel, 2009).

HP1a requires additional factors to get targeted to H3K9methylated chromatin. It was thus of interest to determine which factors interact with the highly related HP1c protein. FLAG tagged HP1c (fHP1c) was immunoprecipitated from Drosophila SL2 cells and a protein complex containing fHP1c and two cofactors Woc and Row was purified, in agreement of the results from Font-Burgada (2008). All three proteins were indentified by GeLC MS/MS mass spectrometry and LC MS/MS analysis of the eluted protein complex in at least two independent protein purifications. Besides the three major stoichiometric components HP1c, Row and Woc that were identified with MOWSE scores of (274, 335 and 119), varying amounts of Ubiquilin, HP1b and eIF4A were found, the functional significance of which has not been further studied. Both major HP1c associated proteins (Woc and Row) contain several Zn-finger domains and two or three HMG-I/Y domains, respectiwvely. In the case of Woc three mammalian orthologues (ZNF198, ZNF261 and ZNF 262) have been reported that are mutated in myeloproliferative diseases (ZNF198) or X-linked mental retardation (ZNF 261) (Kulkarni, 1999; Reiter, 1998; Smedley, 1999). In Drosophila, Woc is involved in regulating genes that are crucial for ecdysone biosynthesis (Warren, 2001; Wismar, 2000) and prevents telomeric fusions (Raffa, 2005). The other Zn-finger protein, Row, is poorly characterized but has recently been shown to co-regulate certain neuronal genes together with Woc and HP1c (Font-Burgada, 2008; Abel, 2009).

Interestingly, the HP1c complex that was purified does not contain either Su(var)3-9 or ACF1, two factors that mediate HP1a recruitment to chromatin, suggesting that the HP1c isoform might require a different set of interaction partners for its function. To determine whether HP1a and HP1c form two different complexes with exclusive partners, HP1a and HP1c were exapressed as GST fusion proteins and GST-pull down experiments were performed using in vitro translated Su(var)3-9 or ACF1. Whereas HP1a efficiently precipitated these proteins, HP1c did not. In order to test whether the binding of Row or Woc to HP1c is as exclusive as the binding of ACF1 and Su(var)3-9 to HP1a, the in vitro translated Woc and Row proteins was tested in a pull down assay. The pull down assay demonstrates that Row specifically interacts with HP1c but not with HP1a, suggesting a possible role for Row and/or Woc for the specific targeting of HP1c to eukaryotic regions. Interestingly an interaction between Woc and HP1c was not observed in vitro neither when it was expressed separately or together with Row. This may be due to an improper folding of in vitro translated Woc or a requirement for specific posttranslational modifications that do not occur during in vitro translation and bacterial expression. Alternatively, Woc may require a specific structural arrangement of the complex similarly to the human orthologue of Woc (ZNF198), which has recently been shown to interact with more stably with a trimeric CoRest complex (Gocke, 2008) than with the individual subunits (Abel, 2009).

In order to confirm the specificity of the Woc/HP1c interaction in vivo, an HP1c specific monoclonal antibody was developed. This antibody recognizes a protein of the expected molecular weight in extracts from wild-type flies, that is absent in extracts prepared from HP1c-/- strains and does not recognize any of the other HP1 isoforms. Using this antibody Woc could be coprecipitated from a nuclear extract prepared from 0-12 hr old Drosophila embryos. An anti-Woc antibody was used for immunoprecipitation, which resulted in the co-purification of HP1c. Based on these experiments it is concluded that most HP1c is associated with two Zn-finger proteins Woc and Row, which do not interact with HP1a (Abel, 2009).

Using the highly specific antibody the distribution of HP1c was investigated within chromatin. In agreement with previous reports for the mammalian isoforms and for Drosophila Kc cells (Smothers, 2001), it was found that HP1c is excluded from DAPI dense regions within the nuclei of SL2 cells, To map the sites of HP1c binding more precisely polytene chromosomes prepared from Drosophila third instar larvae were used. Staining of polytenes showed a strong localization of HP1c to interbands, which are considered to be sites of actively transcribed chromatin. This is in marked contrast to known heterochromatic proteins such as HP1a or HP2, which are highly enriched in pericentric heterochromatin. This is of particular interest as Woc has also been shown to bind to interbands of polytene chromosomes (Raffa, 2005). Indeed, when a co staining of HP1c and Woc was performed an almost perfect overlap of the two signals was found, suggesting that the two proteins indeed form a complex on chromatin. Next whether the binding of HP1c to chromatin is dependent on the presence of Woc and vice versa was tested. In order to do this, polytene chromosomes were prepared from HP1c-/- third instar larvae and from a fly strain carrying a heteroallelic combination of woc alleles that result in greatly reduced Woc levels. Whereas HP1c mutations did not have a strong effect on Woc binding, mutations in woc almost completely abolished HP1c binding. However, this is only in part due to a lack of targeting as the reduction of Woc levels also results in decreased HP1c (but not HP1a) levels. As it has been observed that the reduction of one component of a multi-protein complex destabilizes the other, it was wondered whether the reduction is indeed due to a decrease in HP1c stability or if the transcription of HP1c is reduced. Thus RT-PCR analysis was performed using total RNA isolated from salivary glands of 3^rd instar larvae. A strong reduction of HP1c mRNA was observed in two different woc heteroallelic mutant backgrounds, suggesting that besides being a binding partner for Woc, HP1c is also transcriptionally regulated by Woc. The possibility cannot be excluded that the observed effect is indirect but based on the extensive co-localization of HP1c and Woc and the mapping of HP1c to its own genomic locus (Greil, 2003) this seems to be very unlikely (Abel, 2009).

To further investigate the dynamics of the regulation of the HP1c transcript, SL2 cells were treated either with a woc specific dsRNA that efficiently depletes Woc protein or an unrelated gene (GST) as a negative control. Whereas the levels of HP1c did not decrease on the negative control, a considerable drop in HP1c levels were observed in the cells treated with dsRNA against Woc. This was dependent on the endogenous HP1c promoter as the removal of Woc did not lower the amount of exogenous, FLAG tagged HP1c transcribed from an actin promoter. HP1c expression from an exogenous actin promoter on the contrary completely abolishes expression of the endogenous non-tagged HP1c whereas the expression of the HP1a isoform has no effect on HP1c expression. This repression can also be observed on the transcriptional level as exogenous HP1c expression leads to a considerable reduction of the levels of endogenous HP1c mRNA. Based on these observations, it is argued that woc and HP1c can act as antagonistic factors regulating transcription, leading to a simple way of regulating HP1c levels within a cell by a negative feedback loop. Unfortunately the anti Woc antiserum did not allow performing of ChIp experiments to show a direct binding of woc to the HP1c promoter. Therefore it is not known whether the effect that was seen is direct or indirect. However, as Greil (2003) showed a binding of HP1c to the HP1c locus by Dam-ID and this study observed an almost full overlap of Woc and HP1 binding in polytene chromosomes, it is suggested that HP1c as well as its binding partner Woc play a direct role on this locus (Abel, 2009).

The mechanism by which HP1c inhibits the ability of Woc to activate transcription from the HP1c promoter is unclear. In theory, it could either interfere with the interaction between Woc and DNA or between Woc and transcriptional co-activators. As HP1c and Woc co-localize on polytene chromosomes and no effect of HP1c deletion on Woc localization was observed, the first model as considered improbable. Since the human orthologue of Woc, ZNF198, interacts with a series of transcriptional regulators (Gocke, 2008), it is suggested that the Drosophila Woc protein can also bind to such transcriptional cofactors in an HP1c regulated manner. However, additional experiments will be required to dissect the precise molecular function of transcriptional regulation mediated by Woc and HP1c (Abel, 2009).

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).

Distinct HP1 and Su(var)3-9 complexes bind to sets of developmentally coexpressed genes depending on chromosomal location

Heterochromatin proteins are thought to play key roles in chromatin structure and gene regulation, yet very few genes have been identified that are regulated by these proteins. Large-scale mapping and analysis was performed of in vivo target loci of the proteins HP1, HP1c, and Su(var)3-9 in Drosophila Kc cells, which are of embryonic origin. For each protein, ~100-200 target genes were identified among >6000 probed loci. HP1 and Su(var)3-9 bind together to transposable elements and genes that are predominantly pericentric. In addition, Su(var)3-9 binds without HP1 to a distinct set of nonpericentric genes. On chromosome 4, HP1 binds to many genes, mostly independent of Su(var)3-9. The binding pattern of HP1c is largely different from that of HP1 and Su(var)3-9. Target genes of HP1 and Su(var)3-9 show lower expression levels in Kc cells than do nontarget genes, but not if they are located in pericentric regions. Strikingly, in pericentric regions, target genes of Su(var)3-9 and HP1 are predominantly embryo-specific genes, whereas on the chromosome arms Su(var)3-9 is preferentially associated with a set of male-specific genes. These results demonstrate that, depending on chromosomal location, the HP1 and Su(var)3-9 proteins form different complexes that associate with specific sets of developmentally coexpressed genes (Greil, 2003).

The hinge and chromo shadow domain impart distinct targeting of HP1-like proteins

Drosophila heterochromatin-associated protein 1 (HP1) is an abundant component of heterochromatin, a highly condensed compartment of the nucleus that comprises a major fraction of complex genomes. Some organisms have been shown to harbor multiple HP1-like proteins, each exhibiting spatially distinct localization patterns within interphase nuclei. The subnuclear localization patterns of two newly discovered Drosophila HP1-like proteins (HP1b and HP1c) have been characterized, comparing them with that of the originally described fly HP1 protein (here designated HP1a). While HP1a targets heterochromatin, HP1b localizes to both heterochromatin and euchromatin and HP1c is restricted exclusively to euchromatin. All HP1-like proteins contain an amino-terminal chromo domain, a connecting hinge, and a carboxyl-terminal chromo shadow domain. Truncated and chimeric HP1 proteins were expressed in vivo to determine which of these segments might be responsible for heterochromatin-specific and euchromatin-specific localization. Both the HP1a hinge and chromo shadow domain independently target heterochromatin, while the HP1c chromo shadow domain is implicated solely in euchromatin localization. Comparative sequence analyses of HP1 homologs reveal a conserved sequence block within the hinge that contains an invariant sequence (KRK) and a nuclear localization motif. This block is not conserved in the HP1c hinge, possibly accounting for its failure to function as an independent targeting segment. It is concluded that sequence variations within the hinge and shadow account for HP1 targeting distinctions. It is proposed that these targeting features allow different HP1 complexes to be distinctly sequestered in organisms that harbor multiple HP1-like proteins (Smothers, 2001).

Search PubMed for articles about Drosophila Hp1c

Abel, J., Eskeland, R., Raffa, G. D., Kremmer, E. and Imhof, A. (2009). Drosophila HP1c is regulated by an auto-regulatory feedback loop through its binding partner Woc. PLoS One 4(4): e5089. PubMed ID: 19352434

Coustham, V., Bedet, C., Monier, K., Schott, S., Karali, M. and Palladino, F. (2006). The C. elegans HP1 homologue HPL-2 and the LIN-13 zinc finger protein form a complex implicated in vulval development. Dev. Biol. 297: 308-322. PubMed ID: 16890929

Dialynas, G. K., Terjung, S., Brown, J. P., Aucott, R. L., Baron-Luhr, B., Singh, P. B. and Georgatos, S. D. (2007). Plasticity of HP1 proteins in mammalian cells. J. Cell Sci. 120: 3415-3424. PubMed ID: 17855382

Font-Burgada, J., Rossell, D., Auer, H. and Azorín, F. (2008). Drosophila HP1c isoform interacts with the zinc-finger proteins WOC and Relative-of-WOC to regulate gene expression. Genes Dev. 22(21): 3007-23. PubMed ID: 18981478

Greil, F., et al. (2003). Distinct HP1 and Su(var)3-9 complexes bind to sets of developmentally coexpressed genes depending on chromosomal location. Genes Dev 17: 2825-2838. PubMed ID: 14630943

Gocke, C. B. and Yu, H. (2008). ZNF198 stabilizes the LSD1-CoREST-HDAC1 complex on chromatin through its MYM-type zinc fingers. PLoS ONE 3: e3255. PubMed ID: 18806873

Hayakawa, T., Haraguchi, T., Masumoto, H. and Hiraoka, Y. (2003). Cell cycle behavior of human HP1 subtypes: Distinct domains of HP1 are required for their centromeric localization during interphase and metaphase. J. Cell Sci. 116: 3327-3338. PubMed ID: 12840071

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

Kulkarni, S., Reiter, A., Smedley, D., Goldman, J. M. and Cross, N. C. (1999). The genomic structure of ZNF198 and location of breakpoints in the t(8;13) myeloproliferative syndrome. Genomics 55: 118-121. PubMed ID: 9889006

Lechner, M. S., Begg, G. E., Speicher, D. W. and Rauscher, F. J. (2000). Molecular determinants for targeting heterochromatin protein 1-mediated gene silencing: Direct chromoshadow domain-KAP-1 corepressor interactions is essential. Mol. Cell. Biol. 20: 6449-6465. PubMed ID: 10938122

Minc, E., Allory, Y., Wormann, H. J., Courvalin, J. C. and Buendia, B. (1999). Localization and phosphorylation of HP1 proteins during the cell cycle in mammalian cells. Chromosoma 108: 220-23. PubMed ID: 10460410

Raffa, G. D., Cenci, G., Siriaco, G., Goldberg, M. L. and Gatti, M. (2005). The putative Drosophila transcription factor Woc is required to prevent telomeric fusions. Mol. Cell 20: 821-831. PubMed ID: 16364909

Reiter, A., et al. (1998). Consistent fusion of ZNF198 to the fibroblast growth factor receptor-1 in the t(8;13)(p11;q12) myeloproliferative syndrome. Blood 92: 1735-1742. PubMed ID: 9716603

Schott, S., Coustham, V., Simonet, T., Bedet, C. and Palladino, F. (2006). Unique and redundant functions of C. elegans HP1 proteins in post-embryonic development. Dev. Biol. 298: 176-187. PubMed ID: 16905130

Smedley, D., Hamoudi, R., Lu, Y. J., Cooper, C. and Shipley, J. (1999). Cloning and mapping of members of the MYM family. Genomics 60: 244-247. PubMed ID: 10486218

Smothers, J. F. and Henikoff, S. (2000). The HP1 chromo shadow domain binds a consensus peptide pentamer. Curr. Biol. 10: 27-30. PubMed ID: 10660299

Smothers, J. F. and Henikoff, S. (2001). The hinge and chromo shadow domain impart distinct targeting of HP1-like proteins. Mol. Cell. Biol. 21: 2555-2569. PubMed ID: 11259603

Vakoc, C. R., Mandat, S. A., Olenchock, B. A. and Blobel, G. A. (2005). Histone H3 lysine 9 methylation and HP1γ are associated with transcription elongation through mammalian chromatin. Mol. Cell 19: 381-391. PubMed ID: 16061184

Vermaak, D., Henikoff, S. and Malik, H. S. (2005). Positive selection drives the evolution of rhino, a member of the heterochromatin protein 1 family in Drosophila. PLoS Genet. 1: e9. PubMed ID: 16103923

Warren, J. T., Wismar, J., Subrahmanyam, B. and Gilbert, L. I. (2001). Woc(without children) gene control of ecdysone biosynthesis in Drosophila melanogaster. Mol. Cell. Endocrinol. 181: 1-14. PubMed ID: 11476936

Wismar, J., Habtemichael, N., Warren, J. T., Dai, J.-D., Gilbert, L. I. and Gateff, E. (2000). The mutation without children (rgl) causes ecdysteroid deficiency in third-instar larvae of Drosophila melanogaster. Dev. Biol. 226: 1-17. PubMed ID: 10993670

date revised: 10 June 2023

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