Mi-2, the central component of the nucleosome remodeling and histone deacetylation (NuRD) complex, is known as an SNF2-type ATP-dependent nucleosome remodeling factor. No morphological mutant phenotype of Drosophila Mi-2 (dMi-2) has been reported previously; however, it was found that rare escapers develop into adult flies showing an extra bristle phenotype. dMi-2 enhances the phenotype of acHw49c, which is a dominant gain-of-function allele of achaete (ac) and produces extra bristles. Consistent with these observations, the ac-expressing proneural clusters are expanded, and extra sensory organ precursors (SOP) are formed in the dMi-2 mutant wing discs. Immunostaining of polytene chromosomes showed that dMi-2 binds to the ac locus, and dMi-2 and acetylated histones distribute on polytene chromosomes in a mutually exclusive manner. Chromatin immunoprecipitation assay of the wing imaginal disc also demonstrated a binding of dMi-2 on the ac locus. These results suggest that the Drosophila Mi-2/NuRD complex functions in neuronal differentiation through the repression of proneural gene expression by chromatin remodeling and histone deacetylation (Yamasaki, 2006).
Eukaryotic cells respond to genomic and environmental stresses, such as DNA damage and heat shock (HS), with the synthesis of poly-[ADP-ribose] (PAR) at specific chromatin regions, such as DNA breaks or HS genes, by PAR polymerases (PARP). Little is known about the role of this modification during cellular stress responses. This study shows that the nucleosome remodeler dMi-2 is recruited to active HS genes in a PARP-dependent manner. dMi-2 binds PAR suggesting that this physical interaction is important for recruitment. Indeed, a dMi-2 mutant unable to bind PAR does not localise to active HS loci in vivo. Several dMi-2 regions have been identified that bind PAR independently in vitro, including the chromodomains and regions near the N-terminus containing motifs rich in K and R residues. Moreover, upon HS gene activation, dMi-2 associates with nascent HS gene transcripts, and its catalytic activity is required for efficient transcription and co-transcriptional RNA processing. RNA and PAR compete for dMi-2 binding in vitro, suggesting a two step process for dMi-2 association with active HS genes: initial recruitment to the locus via PAR interaction, followed by binding to nascent RNA transcripts. It is suggested that stress-induced chromatin PARylation serves to rapidly attract factors that are required for an efficient and timely transcriptional response (Murawaska, 2011).
Mi-2 is strongly linked to transcriptional repression in both vertebrate and invertebrate organisms. Within NuRD and dMec complexes it contributes to the repression of cell type-specific genes. Therefore, the widespread colocalisation of dMi-2 with active Pol II and elongation factors at many chromosomal sites is surprising and suggests that dMi-2 might play an unappreciated role during active transcription, at least (or specifically) during environmental stresses such as HS. Indeed, dMi-2 is recruited to HS genes within minutes of HS. This property is not shared by other chromatin remodelers: Brahma (BRM) is not enriched at HS puffs and HS gene activation is independent of BRM function. Moreover, although imitation switch (ISWI) containing complexes are important for HS gene transcription, ISWI does not accumulate to high levels at active HS loci. Recruitment to HS puffs has previously been reported for Drosophila CHD1. Thus, accumulation at active HS genes is shared by at least two members of the CHD family of nucleosome remodelers but not by SWI/SNF and ISWI proteins (Murawaska, 2011).
Depletion of dMi-2 or a reduction of dMi-2 recruitment does not significantly perturb hsp70 transcription in Kc cells and, therefore, dMi-2 is dispensable for HS gene activation in this system. By contrast, depletion of dMi-2 in larvae strongly decreases hsp70, hsp26 and hsp83 activation. It is possible, that the RNAi-mediated depletion of dMi-2 is more efficient in transgenic flies compared to cell lines. In addition, it is believed that several factors contributing to HS gene activation are highly abundant or redundant in Kc cells but more limiting in other contexts. Accordingly, FACT and Spt6 are required for a HS gene activation in flies but are not essential in Kc cells (Murawaska, 2011).
The strong decrease of HS gene activation in dMi-2 RNAi larvae indicates a positive contribution of dMi-2 to transcription in vivo. Overexpression of inactive dMi-2 also results in reduced HS gene transcription implying that its enzymatic activity is critical. It is presently unclear whether this reflects a requirement for dMi-2 catalysed nucleosome remodeling or whether its activity is directed towards different substrates (Murawaska, 2011).
While dMi-2 could indirectly influence transcription by remodeling nucleosomes within the transcribed part of hsp70, its physical association with nascent HS gene transcripts argues for a more direct effect. Indeed, dMi-2 is not only required for high HS gene mRNA levels, but also affects the efficiency of co-transcriptional 3' end formation and splicing. A role of chromatin remodelers in splicing has been suggested before: Both CHD1 and BRG1 bind components of the splicing apparatus. CHD1 associates with Pol II and binds nucleosomes containing H3K4me3, which are enriched near the 5' end of active genes . BRG1 is present at the coding region of genes and influences splice site choice. It has been proposed that CHD1 and BRG1 physically recruit splicing factors but it is unclear if their ATPase activities play a role. Indeed, inactive BRG1 retains the ability to affect exon choice. Inefficient processing of the hsp70 and hsp83 transcripts is not only observed in larvae expressing reduced levels of dMi-2. Importantly, even stronger processing defects are generated by overexpression of inactive dMi-2. This strongly suggests, for the first time, that the catalytic activity of a chromatin remodeler is required for correct co-transcriptional RNA processing. It remains to be determined whether dMi-2 nucleosome remodeling activity influences RNA processing indirectly, e.g. by altering Pol II elongation rates, or whether it has a more direct role (Murawaska, 2011).
A series of complementary results support the hypothesis that dMi-2 interacts with PAR polymers that are rapidly synthesized at activated HS loci. First, the broad distribution of dMi-2 over the entire transcribed region correlates with the distribution of PAR polymer. Second, pharmacological inhibition of PARP greatly decreases dMi-2 binding to activated hsp70. Third, dMi-2 directly binds PAR polymers in vitro. Fourth, a dMi-2 mutant unable to bind PAR also fails to localise to active HS loci. dMi-2 physically associates with nascent HS gene transcripts and binds RNA in vitro. While this interaction is potentially important for the efficiency of transcription and processing, it likely plays a minor role in dMi-2 targeting. Accordingly, inhibition of transcriptional elongation has no significant effect on dMi-2 recruitment (Murawaska, 2011).
It is important to note, that while the results argue for an important role of PAR binding in the recruitment of dMi-2 to HS loci, it cannot be excluded that protein-protein interactions with histone or non-histone proteins also play a role (Murawaska, 2011).
This analysis indicates that dMi-2 harbours several PAR binding motifs in its N-terminal region. It has been demonstrated that human CHD4 is recruited to double stranded DNA breaks in a PARP-dependent manner (Polo, 2010). That study demonstrated PAR binding activity to the region N-terminal of the ATPase domain of CHD4. This agrees well with the current data and suggests that the PAR binding function of CHD4/dMi-2 has been conserved in evolution (Murawaska, 2011).
Two structural protein modules directly interact with PAR, the macrodomain and the PBZ domain; however, these domains are not present in dMi-2. In addition, several shorter PAR binding motifs have been identified. These motifs bear little sequence similarity but share the presence of several K/R residues which are interspersed by hydrophobic residues. The current results have uncovered three K/R-rich regions with PAR binding activity near the N-terminus of dMi-2. Two of these three K/R-rich regions (K/R III and K/R IV) consist of interspersed basic and hydrophobic residues and are therefore reminiscent of the previously described PAR binding motifs, and the third (K/R I) lacks hydrophobic residues completely. None of the three K/R regions matches the consensus PAR binding motifs. It is possible that a consensus motif should generally be chosen less stringently and that a high content of K and R-residues in these regions is sufficient to provide PAR binding activity in vitro. Further characterisation of these regions will be required to resolve this issue. In addition to the K/R regions, the tandem chromodomains of dMi-2 bind PAR in vitro. Previous studies have shown that the chromodomains are required for interacting with nucleosomal DNA in vitro. The new data suggests that these domains can interact with different nucleic acids (Murawaska, 2011).
Several potential molecular functions of PARylation at HS genes have been suggested. First, PARP activity is required for the rapid loss of nucleosomes at hsp70 within the first two minutes after HS (Petesch, 2008). It has been suggested that PARylation of histones aids rapid nucleosome disassembly (Petesch, 2008). Second, at later stages of the HS response (20-60 minutes after HS), PARP activity is required to establish a compartment which restricts the diffusion of factors such as Pol II and Spt6 and promotes efficient factor recycling. The current results suggest that PARylation carries out a third task, namely, to recruit factors via their direct interaction with PAR. The earliest time point when dMi-2 binding to hsp70 can be detected is between 2 and 5 minutes after HS. This places dMi-2 recruitment between the early PARP-dependent nucleosome removal (0-2 minutes after HS) and effects of the transcription compartment (20-60 minutes after HS) (Murawaska, 2011).
The ability of dMi-2 to bind both PAR and RNA and the finding that RNA can compete for PAR binding to dMi-2 is consistent with the hypothesis that dMi-2 association with active HS genes is a two step process. It is proposed that dMi-2 is initially recruited via interaction with PAR polymers. Synthesis of these starts prior to the onset of hsp70 transcription (Petesch, 2008). This results in a rapid local increase of the dMi-2 concentration. In the second step, when hsp70 transcripts are produced by elongating RNA polymerase II at high rates, dMi-2 can switch from binding PAR to interacting with nascent transcripts (Murawaska, 2011).
Severe cellular stresses, such as DNA strand breaks and acute HS, must be dealt with quickly and efficiently. In both cases, a multitude of factors are rapidly recruited to orchestrate the repair of DNA and the massive transcriptional activation of HS genes, respectively. It is postulated that rapid synthesis of PAR polymers at both DNA damage sites and HS genes affords an efficient mechanism to recruit chromatin remodelers and other factors. It has recently been shown that PARylation of DNA breaks is instrumental in recruiting chromatin remodelers, including mammalian dMi-2 homologs, to damaged sites. This study shows that dMi-2?s recruitment to activated HS genes requires PARP activity and that dMi-2 binds PAR directly. The high local concentration of PAR polymers at DNA breaks and HS genes might exploit the general affinity of dMi-2 for nucleic acids. Indeed, dMi-2 binds both DNA and RNA as well as PAR in vitro. In this manner, PAR polymers might act as a scaffold to redirect dMi-2 to chromatin regions where high levels of dMi-2 activity are required, thus acting as a stress-dependent, transient affinity site for chromatin remodeling and possibly RNA processing activities. The results highlight a signaling and scaffolding function for PARP activity during transient environmental stresses other than DNA damage, suggesting that PARylation carries out important modulatory functions in the stress-dependent reprogramming of nuclear activities (Murawaska, 2011).
The ATP-dependent chromatin remodeler dMi-2 can play both positive and negative roles in gene transcription. dMi-2 is recruited to the hsp70 gene in a heat shock-dependent manner and is required to achieve high transcript levels. This study used chromatin immunoprecipitation sequencing (ChIP-Seq) to identify other chromatin regions displaying increased dMi-2 binding upon heat shock and to characterize the distribution of dMi-2 over heat shock genes. dMi-2 is shown to be recruited to the body of at least seven heat shock genes. Interestingly, dMi-2 binding extends several hundred base pairs beyond the polyadenylation site into the region where transcriptional termination occurs. dMi-2 does not associate with the entire nucleosome-depleted hsp70 locus 87A. Rather, dMi-2 binding is restricted to transcribed regions. These results suggest that dMi-2 distribution over active heat shock genes are determined by transcriptional activity (Mathieu, 2012).
hsp70 heat shock genes has been used as a model system to study by what parameters chromatin association of dMi-2 is governed. dMi-2 is recruited to heat shock-activated hsp70 genes, and is required for their full activation in flies. dMi-2 appears to occupy several regions within the body of the hsp70 gene. However, it is not known if dMi-2 covers the hsp70 gene completely, if it is evenly distributed or displays preferences for the 5'- or 3'-ends (Mathieu, 2012).
Actively transcribed hsp70 loci are extensively poly-ADP-ribosylated. Binding of dMi-2 to hsp70 in S2 cells is reduced in the presence of a small molecule poly ADP ribose polymerase (PARP) inhibitor. In addition, dMi-2 binds to PAR in vitro and possesses several PAR-binding motifs suggesting that dMi-2 recruitment to hsp70 involves a direct interaction with the PAR polymer (Murawska, 2011). Moreover, dMi-2 binds nascent hsp70 transcripts and can interact both with DNA and RNA in vitro. Based on these results, it is proposed that dMi-2 is initially recruited to the hsp70 locus when this becomes PARylated shortly after heat shock (HS). Once transcription has been activated, dMi-2 engages with nascent transcripts. However, the relative contributions of PAR, DNA and RNA binding to dMi-2 chromatin association and distribution across genes are not well defined (Mathieu, 2012).
Histone PARylation within the hsp70 locus is believed to contribute to the rapid nucleosome loss that occurs within the first 2min of heat shock. Interestingly, nucleosome loss at hsp70 loci is not restricted to the hsp70 transcription units but extends several kilobases up- and down-stream. It is limited on either side by silencer elements (scs and scs'). Nucleosome depletion across the hsp70 locus increases the access of RNAP II and transcription factors for DNA and their concerted action results in the production of thousands of hsp70 RNA molecules per nucleus. It is currently not known if dMi-2 binding is elevated across the entire PARylated hsp70 locus or if dMi-2 binding is restricted to those regions that are actively transcribed (Mathieu, 2012).
In addition to hsp70, the expression of two other HS genes (hsp26 and hsp83) is affected in transgenic flies expressing reduced levels of dMi-2. This raises the possibility that all HS genes require dMi-2 for full activation and that dMi-2 physically associates with other HS genes during the HS response (Mathieu, 2012).
This study extends an analysis of HS-regulated dMi-2-chromatin interaction by addressing several key questions. First, chromatin immunoprecipitation sequencing (ChIP-Seq) has been used to obtain a high resolution, genome-wide dMi-2 binding profile in both untreated and heat-shocked S2 cells. Through this global approach, seven regions were identified which exhibit strong, HS-induced enrichment of dMi-2 binding. In addition to hsp70 genes, these regions harbour six additional HS genes. Inspection of ChIP-Seq profiles revealed that dMi-2 associates with the body of these HS genes. A more detailed analysis of dMi-2 distribution showed that dMi-2 binding closely follows nascent RNA production. Importantly, dMi-2 binding extends several hundred base pairs beyond polyadenylation sites into regions where transcriptional termination occurs. dMi-2 binding within the PARylated hsp70 locus 87A was analyzed and it was found that dMi-2 recruitment is restricted to actively transcribed regions. These results suggest that RNA synthesis, rather than a general increase in DNA accessibility by PARylation and nucleosome depletion, determines the distribution of dMi-2 at active HS loci. However, ChIP-Seq and RT-qPCR analysis of dMi-2 binding to genes that are constitutively transcribed at high levels, and are induced by other forms of stress indicates that strong transcriptional activity is not sufficient to accumulate dMi-2. Together, these results allow identification of transcription as the key parameter that determines the distribution of dMi-2 over active HS genes (Mathieu, 2012).
Transcription by RNAP II continues past the polyadenylation site until transcription is terminated at one of multiple positions downstream. Termination sites for the hsp26 gene have been mapped by KMnO4 hypersensitive site mapping. The furthest detectable hypersensitive site was located at a distance of 526bp from the polyadenylation site. Increased dMi-2 binding to the active hsp26 gene can be detected ~300-bp downstream of the polyadenylation site. Thus, the dMi-2 bound region lies within the region that is transcribed by RNAP II (Mathieu, 2012).
dMi-2 binds nascent hsp70 and hsp83 transcripts in vivo. It is hypothesized that this interaction of dMi-2 with nascent transcripts governs the distribution of dMi-2 over active heat shock genes. This hypothesis predicts that dMi-2 levels should be lower within the 5' halves of HS genes, where RNA transcripts are still short, higher within the 3' halves of HS genes, where transcripts reach their maximum length, and decline again past the polyadenylation signal, where the message has been cleaved off and only short transcripts are produced prior to their termination. Indeed, the relative enrichment of dMi-2 binding that was observed across the six heat shock genes analysed supports this hypothesis. In all cases, dMi-2 enrichment is higher in the 3' half compared to the 5' half of genes and declined again in the region beyond the polyadenylation site (Mathieu, 2012).
Several HS genes exhibit a 'dip' in dMi-2 binding around the polyadenylation site. This is also consistent with the hypothesis that dMi-2 binding is mediated by an interaction with nascent RNA. It is proposed that the decline in dMi-2 binding near the polyadenylation site is a consequence of RNA cleavage there. The downstream dMi-2 peak might reflect dMi-2 interacting with the RNA produced by terminating RNA polymerase II (Mathieu, 2012).
While the interaction of dMi-2 with nascent RNA appears to contribute to its association with chromatin, it is not sufficient for recruiting dMi-2 to active gene loci. This view is supported by several findings. First, two genes that have been shown to be activated more than 10-fold upon heat shock in a HSF-dependent manner do not display a significant increase in dMi-2 binding. Secondly, genes that are strongly transcribed in a constitutive fashion, such as the genes encoding ribosomal protein subunits, do not bind more dMi-2 than neighbouring, untranscribed regions. Thirdly, strong activation of metallothionein A by Cd treatment does not result in increased association of dMi-2 with the promoter or the transcribed part of the gene. Fourthly, dMi-2 is not recruited to a reporter gene under control of the hsp70 promoter that is upregulated 200-fold following heat shock. Fifthly, inhibition of transcriptional elongation does not affect the recruitment of dMi-2 to several regions within the activated hsp70 gene. These findings suggest that the initial recruitment of dMi-2 to heat shock genes requires additional signals. In case of the hsp70 gene in Kc cells, one signal appears to be provided by poly-ADP-ribosylation of the locus. No consistent effects of treatment with the PARP inhibitor PJ34 on dMi-2 recruitment was observed in the S2 cells used for this study. The relative contribution of poly-ADP ribosylation to dMi-2 recruitment in different biological contexts is therefore unclear. It is also not known, whether poly-ADP-ribosylation does also occur during the activation of other HS genes (Mathieu, 2012).
A rapid loss of nucleosomes from the 87A locus after HS has been described. Interestingly, nucleosome loss is not restricted to the two hsp70 genes residing within 87A. Instead, it includes the entire region flanked by the insulator elements scs and scs'. This property of the 87A locus has allowed addressing the question if dMi-2 chromatin association correlates with nucleosome depletion. Interrogation of the ChIP-Seq data revealed that dMi-2 recruitment was restricted to the transcribed part of the hsp70 genes even within the larger nucleosome-depleted locus. This underscores the importance of transcription for governing dMi-2 chromatin distribution (Mathieu, 2012).
Taken together, these results support a two-step recruitment model of dMi-2. Initial recruitment does not depend on RNA synthesis. Rather, it is likely to be facilitated by other signals that are specific for HS gene activation, one of which might be poly-ADP-ribosylation in certain contexts. Other potential recruitment signals might include binding to PARP itself, which is located near the 5'-end of the hsp70 transcription unit and migrates across the gene following heat shock, the interaction with histone variants deposited at hsp70 or particular histone modifications that are generated during the heat shock response (Mathieu, 2012).
Once dMi-2 is brought to activate HS genes by one or more of these mechanisms, it interacts with nascent RNA and by doing so associates with the transcribed body of the gene. It is tempting to speculate that this association with nascent RNA influences transcription and co-transcriptional processes. Indeed, quantitative changes are detected in levels and processing of hsp70 gene transcripts in transgenic flies with compromised dMi-2 activity. The ChIP-Seq study suggests that dMi-2 associates with and regulates an entire suite of heat shock genes and provides the basis for a more systematic analysis of dMi-2's role in the heat-shock response (Mathieu, 2012).
In order to study the chromatin remodelling activities of a putative Drosophila Mi-2 complex two antibodies directed against the N-terminus (alphadMi-2-N) and the C-terminus of Mi-2 (alphadMi-2-C), respectively, were raised as well as an antibody directed against the RPD3 (alphaRPD3) HDAC. These antibodies were used for immunoprecipitation from a crude nuclear extract prepared from Drosophila embryos and for detection by Western blot analysis of Mi-2 and RPD3 in the immunoprecipitates. The alphadMi-2-N antibody recognizes a protein of the expected molecular weight (220 kDa) in the extract. Both Mi-2 antisera as well as the alphaRPD3 antiserum immunoprecipitate the 220 kDa alphadMi-2-N-reactive protein. It is concluded that the 220 kDa protein is Mi-2. The alphaRPD3 antiserum recognizes a protein of ~60 kDa in nuclear extract, which will be referred to as RPD3. alphaRPD3 as well as both alphadMi-2 antisera immunoprecipitate RPD3. These results argue that Mi-2 and RPD3 form a stable complex in Drosophila embryos. The immunoprecipitates were shown to be active in an in vitro HDAC assay. and the immunoprecipitated Mi-2 complex possesses ATPase activity that is stimulated by nucleosomes but not by naked DNA. Superose 6 gel filtration chromatography suggests that, like its vertebrate counterparts, Mi-2 resides in a high molecular weight complex that possesses HDAC and nucleosome-stimulated ATPase activity (Brehm, 2000).
Although it has not yet been formally demonstrated, it is widely assumed that Mi-2 and its vertebrate homologs confer ATPase activity to Mi-2-containing complexes. It has been proposed that the non-enzymatic subunits of chromatin remodelling complexes regulate the activity and specificity of the ATPases (Kingston, 1999). For example, it is conceivable that nucleosome stimulation of the ATPase requires a non-enzymatic subunit of the Mi-2 complex. To establish the intrinsic properties of Mi-2, flag epitope-tagged full length Mi-2 (dMi-2-F) was expressed using the baculovirus system and the recombinant protein was purified to apparent homogeneity by immunoaffinity chromatography. The purified recombinant protein gave rise to two closely migrating bands on SDS–PAGE gels. Both bands are recognized by alpha-Flag antibody in a Western blot analysis. It is not know at present whether these bands represent degradation products or post-translationally modified forms of Mi-2. As expected, recombinant Mi-2 does not display HDAC activity. Recombinant Mi-2 has weak ATPase activity in the absence of DNA or nucleosomes, which is not significantly stimulated by the addition of DNA or core histones. However, addition of nucleosomal arrays isolated from Drosophila embryos or nucleosomes reconstituted from plasmid DNA and purified Drosophila histones greatly stimulate the ATPase activity of the recombinant protein. These results verify that the nucleosome-stimulated ATPase activity is an intrinsic feature of Mi-2 and is not conferred by other subunits of the Mi-2 complex (Brehm, 2000).
The Drosophila histones used for reconstitution of nucleosomes represent a mixture of the different post-translationally modified forms that exist in vivo. It is conceivable that the Mi-2 ATPase does not respond to the nucleosome structure as such but rather to a particular histone isoform. To address this issue nucleosomes were reconstituted from recombinant histones expressed in Escherichia coli, which lack post-translational modifications. Nucleosomes assembled on plasmid DNA either with purified or recombinant histones activate Mi-2 to a similar extent. Thus, nucleosomal arrays consisting of fully defined components (DNA and four recombinant core histones) suffice to stimulate the Mi-2 ATPase (Brehm, 2000).
Drosophila Mi-2 protein binds to a domain in the gap protein Hunchback, which is specifically required for the repression of HOX genes. Using LexA-Hb as bait, cDNAs were isolated representing six different genes. In interaction tests with various unrelated LexA baits, proteins encoded by three of the six cDNAs interacted exclusively with Hb. Among these proteins, the hip76 clone product exhibits the strongest interaction with Hb. Multiple cDNA clones were isolated that span a complete open reading frame (ORF) encoding a 1982-amino acid protein with high sequence similarity to the human autoantigen (Kehle, 1998).
To map the Mi-2-interacting domain in Hb, Hb fragments were generated and tested for Mi-2 interaction in yeast two-hybrid assays. Mi-2 interacts very strongly with sequences overlapping the D domain, a stretch of amino acids that is conserved between Hb proteins of different insect species. Mutations in the D box cause extensive derepression of HOX genes of the Bithorax complex (BXC). Both D box alleles are premature termination codons, suggesting that the D domain and its COOH-terminal flanking sequences are critical for repression of BXC genes. The interaction tests show that this protein portion of Hb interacts with Mi-2. In vitro binding assays with bacterially expressed Mi-2 and Hb proteins confirm that these proteins bind directly to each other. Thus, Mi-2 binds to a portion of Hb that appears to be critical for repression of BXC genes (Kehle, 1998).
Mi-2 homozygotes survive until the first or second larval instar. Mutant embryos and larvae show no obvious mutant phenotypes. Specifically, expression of BXC genes such as Ultrabithorax (Ubx) and Abdominal-B (Abd-B) is completely normal in these mutant embryos. This normal expression may be due to maternally deposited Mi-2 RNAs or proteins that persist through subsequent development. Consistent with this, all early embryos from a Mi-2 deletion stock (including those lacking the gene) show the same high levels of Mi-2 RNA. An attempt was made to generate embryos from mutant Mi-2 germ cells. However, germ cells that are mutant for any of the seven tested Mi-2 alleles fail to develop. This failure can be rescued by a Mi-2 transgene, demonstrating that Mi-2 is essential for the development of germ cells (Kehle, 1998).
An attempt was made to detect a genetic interaction between Mi-2 and hb. hb9Q mutants (carrying a premature stop codon upstream of the first finger domain) show only slight anterior derepression of Ubx in embryos because of perdurance of maternal hb products. hb9K57 mutants (carrying a D box lesion) show more extensive anterior derepression of Ubx; this mutant protein is thought to have dominant-negative effects on the persisting maternal wild-type product. Mi-24;hb9K57 double mutants show much more extensive derepression of Ubx than hb9K57 mutants. Similarly, Mi-24;hb9Q double mutants show more extensive derepression than hb9Q mutants alone. These results demonstrate a synergy between hb and Mi-2 that is consistent with the finding that Mi-2 binds to Hb. Furthermore, it provides strong evidence that Mi-2 functions in the repression of BXC genes (Kehle, 1998).
Mi-2 protein was tested to see if it participates in PcG repression. As in the case of Mi-2, maternally deposited PcG product often rescues homozygous mutant PcG embryos to a considerable extent. Extensive derepression of HOX genes can be observed if such homozygous embryos are also mutant for another PcG gene. Thus embryos homozygous for the PcG gene Posterior sex combs (Psc) and Mi-2 were examined and it was found that Ubx and Abd-B are derepressed more extensively in this double mutant than in Psc homozygotes alone. A similar result was found if Mi-2 is combined with other PcG mutations; these double mutants consistently lead to much enhanced homeotic transformations compared with the single PcG mutants. Thus, there is a synergy between Mi-2 and PcG genes. Mi-2 behaves like the PcG mutations Enhancer of Polycomb and Suppressor 2 of zeste, neither of which on their own cause a homeotic phenotype but do so in combination with other PcG mutations. This suggests that Mi-2 functions in PcG repression (Kehle, 1998).
Imaginal discs were examined for derepression of HOX genes as well as the phenotypes of their adult derivatives. Clonal analysis suggests that Mi-2 is required for the survival of somatic cells. Do Mi-2 mutations exhibit gene-dosage interactions with PcG mutations? While larvae heterozygous for Polycomb (Pc) mutations show slight derepression of Ubx, larvae transheterozygous for both Pc and Mi-2 mutations show more extensive derepression. Furthermore, derepression of the HOX gene Sex combs reduced (Scr) in the second and third leg discs of Pc heterozygotes results in the formation of a first leg structure, the sex comb, on the second and third legs. The extent of this homeotic transformation reflects the number of cells that misexpress Scr protein. This homeotic transformation is far stronger in Mi-2/Pc transheterozygotes than in adults heterozygous for Pc alone, which is consistent with more extensive derepression of Scr in the double mutant. These results are further evidence that Mi-2 acts together with PcG proteins to repress HOX genes (Kehle, 1998).
It has been proposed that Hb directly or indirectly recruits PcG proteins to DNA to establish PcG silencing of homeotic genes. The present data suggest that Mi-2 might function as a link between Hb and PcG repressors. Although Mi-2 contains two motifs with similarity to DNA-binding domains (the myb and HMG domains), Mi-2 does not seem to bind to DNA on its own. Therefore, Hb may recruit Mi-2 to DNA. Xenopus Mi-2 has recently been purified as a subunit of a histone deacetylase complex with nucleosome remodeling activity. In yeast and in vertebrates, several transcription factors repress transcription by recruiting histone deacetylases. It is possible that in Drosophila, nucleosome remodeling and deacetylase activities of a Mi-2 complex, recruited to homeotic genes by Hb, may result in local chromatin changes that allow binding of PcG proteins to the nucleosomal template. Alternatively, the proposed Hb-Mi-2 complex might directly bind a PcG protein and recruit it to DNA. Finally, the involvement of Mi-2 in PcG silencing suggests that this process may involve deacetylation of histones (Kehle, 1998 and references).
dMi-2, the ATPase subunit of the Drosophila nucleosome remodelling and histone deacetylation (dNuRD) complex, has been identified in a two-hybrid screen as an interacting partner of the transcriptional repressor, Tramtrack69 (Ttk69). A short region of Ttk69 is sufficient to mediate this interaction. Ttk69, but not the Ttk88 isoform, co-purifies with the dNuRD complex isolated from embryo extracts. dMi-2 and Ttk69 co-immunoprecipitate from embryonic extracts, indicating that they can associate in vivo. Both dMi-2 and Ttk69 co-localize at a number of discrete sites on polytene chromosomes, showing that they bind common target loci. dMi-2 and Ttk interact genetically, indicating a functional interaction in vivo. It is proposed that Ttk69 represses some target genes by remodelling chromatin structure through the recruitment of the dNuRD complex (Murawsky, 2001).
Fractionation of 0-24 h embryonic extracts by gel-filtration chromatography and subsequent Western blotting shows that the majority of Ttk69 elutes in a broad peak with a molecular weight of ~1 MDa, much larger than the predicted monomer size of 69 kDa. Antibodies directed against the C-terminal region of dMi-2 show that dMi-2 and Ttk69 are present in an overlapping set of high-molecular-weight fractions. In a separate purification procedure developed to isolate the dMi-2-containing dNuRD complex, Ttk69 consistently co-purified with dMi-2 through multiple fractionation steps. Western analysis has confirmed that Ttk69 is present in the purest dMi-2 fractions. Ttk69 also physically associates with dMi-2 in partially purified nuclear extracts, as shown by co-immunoprecipitation of dMi-2 from these fractions using alpha-Ttk69 antibodies. alpha-Ttk69 antibodies co-precipitate dMi-2. dMi-2 is also precipitated by alpha-dMi-2 and alpha-dRpd3 (HDAC subunit of dNuRD) antibodies (Murawsky, 2001).
To establish if dMi-2 is required for Ttk69-mediated repression in vivo, an examination was made to see whether simultaneous loss of dMi-2 would increase neuron over-production seen in the peripheral nervous system (PNS) of embryos mutant for the hypomorphic ttkrM730 allele. Normally, Ttk is expressed in all non-neuronal cells of PNS sensory organs and prevents these cells from becoming neurons. In ttk mutants, a variable number of these cells are transformed into neurons, causing excess neurons in the PNS. An antibody against the neuronal antigen 22C10 was used to detect neurons of the lateral pentascolopidial sensory organ of the embryonic PNS. In wild-type embryos, five neurons are present. This number is approximately doubled in ttkrM730 mutants. Although homozygous mutant dMi-24 embryos show no defects in PNS development, loss of dMi-2 in a ttkrM730 mutant background significantly increases neuron number. Moreover, a number of tissues that are not stained by mAb 22C10 in wild-type and ttkrM730 mutants now express the antigen. Epidermal staining is increased and body-wall muscles stain strongly. mAb 22C10 staining of the somatic musculature has been reported for strong ttk loss-of-function alleles such as ttkD2-50, providing further evidence that loss of dMi-2 increases the strength of ttk mutant phenotypes (Murawsky, 2001).
ttk also shows dominant interactions with mutations affecting the components of the dNuRD complex. Titration of Ttk function can increase the number of precursor cells that are recruited to initiate sensory-organ development. One manifestation is the production of ectopic bristles along veins of the adult wing. ttkrM730 interacts dominantly with mutants affecting rpd3 to cause bristle de-repression on the adult wing. Further reduction of dMi-2 levels in the presence of one copy of the null allele dMi-24 increases bristle number synergistically, reflecting functional in vivo interactions between Ttk and both Rpd3 and dMi-2 (Murawsky, 2001).
Interestingly, the interaction of Ttk69 with the dNuRD complex parallels that of another BTB/POZ-containing protein, GAGA, with the ISWI-containing remodelling complex NURF. A second Drosophila protein, Hb, has also been shown to interact with dMi-2, both genetically and by two-hybrid analysis. Hb binds to the same C-terminal region of dMi-2 as does Ttk69, suggesting that this region may be a docking platform for proteins that recruit the dNuRD complex. However, thus far Hb has not been detected in the dNuRD complex. This failure may simply reflect the relative abundance of Ttk69 and Hb in Drosophila embryos. Nevertheless, the association of dMi-2 with more than one Drosophila transcriptional repressor implies that it can be recruited by a number of different proteins. Such recruitment may be a general mechanism by which specific repressors silence their targets. Indeed, MBD2, Ikaros and Aiolos, also known repressors, have been shown to purify with the NuRD complex from mammalian cells (Murawsky, 2001 and references therein).
The association of Ttk69 with the dNuRD complex implies that one mechanism by which Ttk69 may repress its targets is to direct the histone deacetylation and chromatin remodelling activities of the dNuRD complex. Such a function would be consistent with the developmental expression pattern of Ttk69. High levels of Ttk69 expression are detected toward the end of embryogenesis, when most cell types have already been specified and the chosen fate needs to be stabilized. These results imply in particular that dMi-2 is involved in ttk-mediated neuronal repression. It is noteworthy that all known transcription factors that interact with Mi-2-containing complexes are deployed when particular cell fates or expression patterns need to be maintained. Thus, Hb mediates stable homeotic repression, Ikaros/Aiolos maintains B and T cell fates and MBD2 is required for DNA-methylation-dependent silencing. It is speculated that Ttk69 also uses this mechanism stably to repress targets incompatible with determined cell fates (Murawsky, 2001).
These results suggest a model by which Ttk69 interacts with the dMi-2 component of the dNuRD complex and subsequently recruits its repressive activities to target genes. It is speculated that in vivo the association of Ttk69 with dMi-2 is probably not the only route by which Ttk69 may repress transcription. It is known that Ttk69 interacts genetically with dCtBP, another transcriptional co-repressor. Moreover, Ttk69 and dMi-2 do not completely co-localize on polytene chromosomes, suggesting that Ttk69 binds a subset of its targets in the absence of dMi-2. Finally, it is noted that only a small fraction of the total amount of Ttk69 present in embryonic extracts co-purifies with the dNuRD complex. Taken together, it is surmised that Ttk69 is a component of more than one repressive complex and that different target genes may be regulated by different mechanisms (Murawsky, 2001).
DREF is a transcriptional regulatory factor required for the expression of genes carrying the 5'-TATCGATA DRE. DREF has been reported to bind to a sequence in the chromatin boundary element, and thus may play a part in regulating insulator activity. To generate further insights into DREF function, a Saccharomyces cerevisiae two-hybrid screening was screened with DREF polypeptide as bait, and Mi-2 was identified as a DREF-interacting protein. Biochemical analyses revealed that the C-terminal region of Drosophila Mi-2 (dMi-2) specifically binds to the DNA-binding domain of DREF. Electrophoretic mobility shift assays showed that dMi-2 thereby inhibits the DNA-binding activity of DREF. Ectopic expression of DREF and dMi-2 in eye imaginal discs resulted in severe and mild rough-eye phenotypes, respectively, whereas flies simultaneously expressing both proteins exhibited almost-normal eye phenotypes. Half-dose reduction of the dMi-2 gene enhanced the DREF-induced rough-eye phenotype. Immunostaining of polytene chromosomes of salivary glands showed that DREF and dMi-2 bind in mutually exclusive ways. These lines of evidence define a novel function of dMi-2 in the negative regulation of DREF by its DNA-binding activity. Finally, it is postulated that DREF and dMi-2 may demonstrate reciprocal regulation of their functions (Hirose, 2002).
This report proposes a novel mechanism whereby dMi-2 is involved in repressing transcription of DRE-containing genes by inhibiting the DNA binding of DREF. The observations point to a first example of a member of the SWI/SNF2 family of DNA-stimulated ATPases directly interacting with a transcription factor to attenuate its activity. Although the present biochemical and genetic analyses clearly indicated direct interaction between DREF and dMi-2, it is uncertain whether the dMi-2 polypeptide alone or in association with another subunit of the chromatin-remodeling complex, such as HDAC, binds to DREF in vivo. It is worth noting that treatment of Drosophila cultured cells with trichostatin A, a microbial metabolite generally used as an inhibitor of HAT, did not affect the PCNA promoter activity, whereas cotransfection of a dMi-2-expressing plasmid with reporter plasmid significantly decreased PCNA promoter activity depending on the presence of the DRE sequence. This indicates that accompanying histone acetyltransferase activity might not be involved in repression by dMi-2 (or the dMi-2 complex). However, a requirement for other subunits cannot be ruled out. Although previous studies on mammalian Mi-2 (Mi-2 ß, CHD4) complexes characterized the Mi-2 polypeptide as a major component, biological functions of separate components have not been examined. Importantly, several different strategies resulted in the purification of slightly different Mi-2 complexes. In the case of the original NRD complex, the purification was performed by pursuing HDAC activity by conventional chromatography, followed by affinity chromatography for Mi-2 ß (CHD4). With this purification method, the bulk of the tightly associated NRD core complex does not contain sequence-specific DNA-binding protein. Recently, MeCP1 complexes have been purified to homogeneity and the presence of the core polypeptide of the known NRD complex was demonstrated, indicating that several kinds of complexes, including the Mi-2 polypeptide, might exist in cells. Considering that only 5% of the total dMi-2 polypeptide was estimated to be associated with DREF by immunoprecipitation experiments, it is hypothesized that binding with DREF in vivo may also be limited. Furthermore, it is interesting to note that the amino-terminal region of dMi-2 exhibits inhibitory effects on its binding to DREF, suggesting a possible regulation by change in the structure of the molecule. To assess this possibility, a challenge for the future will be the determination of the three-dimensional structure of Mi-2 (or the Mi-2 complex) that binds and modulates DREF activity. Transgenic fly lines have been established expressing HA epitope-tagged dMi-2 and Flag epitope-tagged DREF by using the GAL4-UAS system. These flies should be powerful tools for the purification of DREF/dMi-2 complexes (Hirose, 2002).
dMi-2 protein is localized at several hundred loci of the polytene chromosomes of salivary glands. A model of dMi-2 protein function has been proposed featuring repression of transcription by binding to a Polycomb group protein in the form of a Hunchback-dMi-2 complex, with consequent recruitment to DNA. However, this study observed dMi-2 in interbands and regions associated with high transcriptional activity (puffs), suggesting an ability to enhance as well as to repress gene expression. To address this question, it is important that genes that are positively regulated by dMi-2 be identified (Hirose, 2002).
Another important finding of the immunostaining is that DREF and dMi-2 bind to polytene chromosomes in a mutually exclusive manner. This seems contrary to the results of immunoprecipitation and in vitro binding experiments but can be explained as follows. Since the DNA-binding domain and the Mi-2-binding domain of DREF overlap, dMi-2 cannot interact with DREF bound to DNA. In contrast, dMi-2 presumably has access to free DREF. If dMi-2 cannot disrupt DREF/DNA complexes, genes adjacent to DREF binding sites will be kept in a transcriptionally active state. Furthermore, overexpression of DREF or dMi-2 in eye imaginal discs induces a rough-eye phenotype although the eyes of transgenic flies simultaneously expressing DREF and dMi-2 appear normal. These results suggest that DREF and dMi-2 negatively regulate each other's functions. To date, although there is no evidence that molecules recruit dMi-2 to specific loci of polytene chromosomes, it can be speculated that DREF could be involved in the regulation of such dMi-2 recruitment. If this is the case, an important mechanism for the maintenance of epigenetic activation (or silencing) of genes can be envisaged. This idea is not contradictory to the model in which DREF contributes to the cancellation of chromatin boundary function by displacing BEAF from its binding sites (Hirose, 2002).
In summary, evidence has been provided for a novel function of dMi-2 in repressing transcription of DRE-containing genes by attenuating the DRE-binding activity of DREF. In addition, it is hypothesized that DREF and dMi-2 may demonstrate reciprocal regulation of their functions. To probe this possibility, efforts to isolate DREF mutant flies and determine the dMi-2 (complex) structure in association with DREF are necessary in the future (Hirose, 2002).
Attempts were made to confirm the association between MBD2/3 and the MI-2 complex at the functional level. It has been shown previously that MBD2/3 and MI-2 interact in vitro (Tweedie, 1999). Similarly, both proteins have been co-fractionated in protein extracts from Drosophila SL-2 cells (Ballestar, 2001). In order to look for a genetic interaction between MBD2/3 and Mi-2, homozygous MBD1 flies were crossed with flies carrying a heterozygous mutant allele for Mi-2 (Mi-24). Compound heterozygotes for both mutations had significantly rougher and smaller eyes in about 25% of the progeny, while both homozygous MBD1 flies and heterozygous Mi-24 flies had completely normal eyes. This result strongly suggests a functional interaction between MBD2/3 and MI-2. The interaction between MBD2/3 and MI-2 was examined by determining the subcellular distribution of MI-2 protein in MBD1 mutants. Wild-type and mutant embryos were immunostained with a specific antiserum against MI-2 and the subnuclear distribution of the protein was examined by confocal microscopy. This revealed a homogeneous distribution of MI-2 in wild type embryos. However, the protein appeared to be absent from about 10-15 nuclear foci in the mutant. These results are consistent with a functional interaction between MBD2/3 and MI-2, and suggest that the MI-2 complex might be absent from a subset of target loci in MBD2/3 mutants (Marhold, 2004).
In order to analyze the relationship between MBD2/3 and MI-2 in greater detail, double immunostaining was performed. MBD2/3 has been shown to form nuclear foci at the cellular blastoderm stage that remain detectable until after gastrulation (Marhold, 2002). However, the precise nature of these foci could not be determined further because of the lack of suitable antibodies. As a prerequisite to double immunostaining experiments a monoclonal MBD2/3-specific antibody (MBD 8E7) was raised by immunizing rats with an MBD2/3 peptide. The peptide was selected from the exon 2 region of MBD2/3 that is not present in the short MBD2/3 isoform, and the antibody recognized a single band in Western blots from embryonic nuclear extracts that corresponds to the long isoform of MBD2/3. The specificity of the antibody was confirmed by the absence of detectable signals in Western blots of protein extracts from homozygous MBD1 embryos. Similarly, immunostaining of homozygous MBD1 embryos with 8E7 antibody failed to detect any signals above the background level. Double immunostaining of wild-type embryos with the 8E7 antibody and an MI-2-specific antiserum revealed a speckled nuclear pattern for MBD2/3, which is in agreement with previous observations (Marhold, 2002). By contrast, MI-2 is found in a rather ubiquitous distribution in embryonic nuclei. This result argues against MBD2/3 being an integral component of all MI-2 complexes and suggested a more peripheral association with only a subset of MI-2 complexes (Marhold, 2004).
Drosophila Mi-2 (dMi-2) is the ATPase subunit of a complex combining ATP-dependent nucleosome remodelling and histone deacetylase activities. dMi-2 contains an HMG box-like region, two PHD fingers, two chromodomains and a SNF2-type ATPase domain. It is not known which of these domains contribute to nucleosome remodelling. A panel of dMi-2 deletion mutants was tested in ATPase, nucleosome mobilization and nucleosome binding assays. Deletion of the chromodomains impairs all three activities. A dMi-2 mutant lacking the chromodomains is incorporated into a functional histone deacetylase complex in vivo but has lost nucleosome-stimulated ATPase activity. In contrast to Drosophila HP1, dMi-2 does not bind methylated histone H3 tails and does not require histone tails for nucleosome binding. Instead, the dMi-2 chromodomains display DNA binding activity that is not shared by other chromodomains. These results suggest that the chromodomains act at an early step of the remodelling process to bind the nucleosome substrate predominantly via protein-DNA interactions. Furthermore, this study identifies DNA binding as a novel chromodomain-associated activity (Bouazoune, 2002).
Fusion of the chromodomain region and the ATPase domain is sufficient for DNA and nucleosome binding, but not for nucleosome-stimulated ATPase and nucleosome mobilization activities. It follows that additional regions outside these domains make critical contributions to nucleosome remodelling. Conversion of the chromo domain-ATPase domain fusion (dMi-2 484-1271) to an active nucleosome remodeller can be achieved by addition of the remainder of the N-terminal region (NTR) or by addition of the C-terminal region (CTR). This implies that both NTR and CTR provide activities that are redundant in the assay. The CTR binds the repression domains of Hunchback and Tramtrack 69. The C-terminus of mammalian Mi2ß interacts with the KAP-1 co-repressor. The results suggest that CTR function is not restricted to transcription factor binding. Instead, it plays an active role in ATPase regulation: although deletion of the CTR does not affect nucleosome mobilization it makes the ATPase responsive to DNA. In this respect, the dMi-2 1-1271 mutant resembles ATPases of the SWI/SNF subgroup. This observation suggests that the CTR is directly involved in regulation of the ATPase domain: it is required to suppress activity in presence of the 'wrong' effector (DNA), when no remodelling substrate (nucleosome) is available. It is conceivable that the CTR might undergo a change in conformation following nucleosome recognition, which then allows the ATPase domain to function (Bouazoune, 2002).
The Drosophila trithorax group gene kismet (kis) was identified in a screen for extragenic suppressors of Polycomb (Pc) and subsequently shown to play important roles in both segmentation and the determination of body segment identities. One of the two major proteins encoded by kis (Kis-L) is related to members of the SWI2/SNF2 and CHD families of ATP-dependent chromatin-remodeling factors. To clarify the role of Kis-L in gene expression, its distribution on larval salivary gland polytene chromosomes was examined. Kis-L is associated with virtually all sites of transcriptionally active chromatin in a pattern that largely overlaps that of RNA Polymerase II (Pol II). The levels of elongating Pol II and the elongation factors SPT6 and CHD1 are dramatically reduced on polytene chromosomes from kis mutant larvae. By contrast, the loss of Kis-L function does not affect the binding of PC to chromatin or the recruitment of Pol II to promoters. These data suggest that Kis-L facilitates an early step in transcriptional elongation by Pol II (Srinivasan, 2005).
To clarify the functional relationship between Kis-L and other chromatin-remodeling factors, their distributions on polytene chromosomes were compared. The distributions of Kis-L and Brm were compared, since previous studies have suggested that the two proteins have similar functions. For example, brm and kis were both identified in genetic screens for dominant suppressors of Pc and mutations in the two genes cause similar homeotic transformations. In addition, Brm plays an extremely general role in transcription by Pol II and, like Kis-L, is associated with almost all transcriptionally active regions of polytene chromosomes. Consistent with a close functional relationship between the two proteins, it was found that the distributions of Brm and Kis-L on polytene chromosomes are virtually identical. In addition, the relative levels of the two proteins do not vary from site to site. The striking similarities between the chromosomal distributions of Brm and Kis-L strongly suggest that the functions of the two trxG proteins are intimately related (Srinivasan, 2005).
Based on its association with histone deacetylases and transcriptional repressors, Mi-2 is thought to be involved in transcriptional repression. Genetic studies in Drosophila also suggest that Mi-2 acts in concert with PcG proteins to repress Hox transcription. It was therefore anticipated that the chromosomal distributions of Kis-L and Mi-2 would be very different, if not mutually exclusive. Surprisingly, it was found that the patterns of Kis-L and Mi-2 are actually very similar. Although the relative levels of Kis-L and Mi-2 vary from site to site, only 1 to 2% of the binding sites of the two proteins fail to overlap. These data suggest that Mi-2 plays an unanticipated and relatively general role in transcription by Pol II (Srinivasan, 2005).
The NURD and Sin3 histone deacetylase complexes are involved in transcriptional repression through global deacetylation of chromatin. Both complexes contain many different components that may control how histone deacetylase complexes are regulated and interact with other transcription factors. In a genetic screen for modifiers of wingless signaling in the Drosophila eye, mutations were isolated in the Drosophila homolog of p66, a protein previously purified as part of the Xenopus NURD/Mi-2 complex. p66 encodes a highly conserved nuclear zinc-finger protein that is required for development and it is proposed that the p66 protein acts as a regulatory component of the NURD complex. Animals homozygous mutant for p66 display defects during metamorphosis possibly caused by misregulation of ecdysone-regulated expression. Although heterozygosity for p66 enhances a wingless phenotype in the eye, loss-of-function clones in the wing and the eye discs do not have any detectable phenotype, possibly due to redundancy with the Sin3 complex. Overexpression of p66, in contrast, can repress wingless-dependent phenotypes. Furthermore, p66 expression can repress multiple reporters in a cell culture assay, including a Wnt-responsive TCF reporter construct, implicating the NURD complex in repression of Wnt target genes. By co-immunoprecipitation, p66 associates with dMi-2, a known NURD complex member (Kon, 2005).
Thus, loss of p66 enhances the sev-wgts phenotype in the eye; i.e., the resulting phenotype resembles that of increased wingless signaling. Since loss of p66 does not affect other transgenes expressed using the same promoter as sev-wgts, it is believed that removal of p66 does not affect wingless expression through the sevenless promoter, but instead represses wingless target genes involved in bristle formation. The results are in agreement with experiments that have implicated the NURD complex in Wnt signaling. It has been reported that Lef-1 repression involves HDAC-1 function. This interaction takes place in the absence of mSin3A, leading to the hypothesis that Lef-1 repression involves recruitment of the NURD complex. Three pieces of evidence are presented to further support this hypothesis: (1) loss of p66 enhances a wg overexpression phenotype; (2) overexpression of p66 can repress activation of a TCF reporter by ß-catenin in tissue culture cells; (3) overexpression of p66 in vivo represses the formation of wingless-dependent scutellar bristles without repressing wingless expression. Together, these experiments provide additional evidence that the NURD complex is involved in repression of Wnt target genes (Kon, 2005).
The lethality of p66 mutants is caused by misregulation of ecdysone-regulated genes during larval stages. If p66 was involved in repression of ecdysone-induced targets such as E74 and DHR3, then loss of p66 should lead to ectopic gene expression. In contrast, it was found that ecdysone-induced genes are not activated, suggesting that the role of p66 is more complex and indirect. In microarray studies of ecdysone response, 44% of genes that changed expression were repressed. It is speculated that repression of one or more of these genes is required for ecdysone-induced expression and that p66 is required for this process (Kon, 2005).
An additional connection between the NURD complex and ecydsone response is made through Bonus (bon), the Drosophila homolog of TIF1. TIF1 was identified as a protein that interacts with HP1, a heterochromatin-associated protein. The NURD complex may be involved in histone modification to allow HP1 binding. Similar to p66 mutants, bon mutants die during pupal stages due to misregulation of ecdysone-induced genes. Furthermore, E74 expression is also reduced in bon mutants. Thus it is speculated that p66 and the NURD complex may be involved in regulation of ecdysone response through HP1-mediated repression (Kon, 2005).
p66 mutations can affect both wg and ecdysone-induced gene expression. Furthermore, in cell culture reporter assays, it was found that expression of p66 inhibits activation of a TCF reporter by ß-catenin, of an SRE reporter by the M1 receptor, and of an NF-AT reporter by high levels of intracellular calcium. In addition, two human p66 homologs, hp66alpha and hp66ß, function as transcriptional repressors when tethered to a promoter, suggesting that transcriptional repression is a shared activity of p66 proteins. However, p66 did not repress an albumin luciferase reporter, implying that expression of p66 does not cause a defect in general transcription. Therefore, it is concluded that p66 can repress multiple signaling pathways, and it is hypothesized that this repression is mediated by recruitment of the NURD histone deacetylase complex. This conclusion is also supported by previous reports that expression of p66 can change the localization of MBD3, a component of the NURD complex (Kon, 2005).
Although overexpression of p66 can repress multiple signaling pathways, no loss-of-function phenotypes were detected that would be consistent with this hypothesis. In Drosophila, relatively mild and tissue-specific phenotypes for repressors have also been found for Pangolin/dTCF and naked cuticle. With respect to Pangolin/dTCF, this could be due to its dual role as both a repressor and an activator, but in other cases, a lack of phenotypes is possibly due to redundancy among parallel pathways (not necessarily among related genes). This suggestion is in analogy to the SynMuv genes in C. elegans. Animals mutant in either a synMuvA or a synMuvB gene alone have a normal vulva; animals mutant for both a synMuvA gene and a synMuvB gene have a multi-vulval (Muv) phenotype. Indeed, in the C. elegans vulva, p66 functions in a redundant pathway (Kon, 2005).
P66 is present when NURD complex is purified from Xenopus oocytes and is found to be associated with the NURD complex in mammalian cells in association with the methyl DNA-binding protein MBD2, as part of the MeCP1 complex. Is p66 a component of the NURD complex or an accessory factor? WdMi-2 and p66 co-purify, suggesting that they form a complex. However, if all of the complex members participate in the same processes, then the corresponding mutants should also have the same phenotypes. To ascertain whether p66 and rpd3 (HDAC homolog) function together is difficult since rpd3 also participates in the Sin3 complex. Thus, it is likely that rpd3 will display a wider range of phenotypes than a mutant of the NURD complex alone (Kon, 2005).
However, the p66 mutant can be compared to the other NURD complex mutant characterized in Drosophila, dMi-2. dMi-2 is required for oogenesis and for cell viability. rpd3 is also likely important for oogenesis; a hypomorphic rpd3 allele produces very few eggs. In contrast, p66 mutant germlines produce normal embryos, and p66 mutant clones survive in third instar imaginal discs. dMi-2 zygotic mutants die during larval stages, which coincides with the lethality of strong allele combination of p66 mutants. It is possible that dMi-2 may also be required for ecdysone response in larval stages. Since the p66 mutant phenotype does not completely mimic Mi-2 mutant phenotypes, it is suggested that p66 is not a core component of the NURD complex, but could have a regulatory function. However, it is equally possible that differences in allelic strength or perdurance of maternal contributions obscures the full range of phenotypes on the several components (Kon, 2005).
These results are further supported by experiments demonstrating that dMi-2 and the Drosophila MBD2/3 protein do not colocalize in nuclei. dMi-2 is distributed ubiquitously in embryonic nuclei, while Drosophila MBD2/3 is localized in a speckled pattern. This result suggests that Drosophila MBD2/3 is not an integral component of all dMi-2 complexes (Kon, 2005).
How might p66 function? In mammalian cells, human p66 protein is associated only with the NURD complex as part of the MeCP1 complex, which additionally contains methylated DNA-binding activity through the MBD2 protein. Similarly, the Xenopus NURD complex, which copurifies with p66, in contrast to mammalian NURD complexes, also has methylated DNA-binding activity. The NURD complex, through Mi-2, interacts with the zinc-finger proteins Hunchback, Tramtrak, and Ikaros. It is hypothesized that p66, also a zinc-finger-containing protein, functions similarly to these transcription factors to recruit the NURD complex to methylated DNA (Kon, 2005).
This hypothesis is supported by experiments that demonstrate that human p66 interacts with MBD2 and MBD3 in vitro. Furthermore, overexpression of p66 can change localization of MBD3. On the basis of these results, it is hypothesized that p66 may function as a link between the NURD complex and the methylated DNA-binding proteins MBD2 and MBD3. It is proposed that p66 recruits the NURD complex to mediate methylation-mediated silencing. Since both DNA methyl transferases and DNA-methylated binding proteins exist in Drosophila, it is likely that there is some vestige of a methylation system in Drosophila, although the function is unknown at this time (Kon, 2005).
dMi-2 is a highly conserved ATP-dependent chromatin-remodeling factor that regulates transcription and cell fates by altering the structure or positioning of nucleosomes. dMi-2 plays an unanticipated role in the regulation of higher-order chromatin structure in Drosophila. Loss of dMi-2 function causes salivary gland polytene chromosomes to lose their characteristic banding pattern and appear more condensed than normal. Conversely, increased expression of dMi-2 triggers decondensation of polytene chromosomes accompanied by a significant increase in nuclear volume; this effect is relatively rapid and is dependent on the ATPase activity of dMi-2. Live analysis revealed that dMi-2 disrupts interactions between the aligned chromatids of salivary gland polytene chromosomes. dMi-2 and the cohesin complex are enriched at sites of active transcription; fluorescence-recovery after photobleaching (FRAP) assays showed that dMi-2 decreases stable association of cohesin with polytene chromosomes. These findings demonstrate that dMi-2 is an important regulator of both chromosome condensation and cohesin binding in interphase cells (Fasulo, 2012).
Cohesin has been the topic of intensive study due to its critical role in sister chromatid cohesion during mitosis, and its roles in gene regulation and DNA repair. The complex forms a ring-like structure that encircles chromosomes beginning in telophase, and mediates sister chromatid cohesion upon DNA replication. Cohesin binding is dynamic, but unusually stable compared to most DNA-binding proteins. Interphase cohesin is continuously loaded by the kollerin complex containing Nipped-B and released from chromosomes by the releasin complex containing Pds5 and Wap. The current studies revealed an intriguing connection between dMi-2 and the cohesin complex, and argue that dMi-2 facilitates removal of cohesin from chromosomes during interphase. This activity is not restricted to situations in which dMi-2 is expressed at unusually high levels, since a twofold reduction in dMi-2 dosage counteracts the developmental consequences of reduced dosage of Nipped-B. These findings add dMi-2 to the list of factors that regulate cohesin binding (Fasulo, 2012).
Cohesin regulates transcription by multiple mechanisms, including long-range interactions between insulators, enhancers and promoters via the formation of DNA loops, repression in collaboration with Polycomb proteins, and controlling transition of paused polymerase to elongation. The observed suppression of a dominant Nipped-B mutant phenotype by reduced dMi-2 gene dosage suggests that regulation of cohesin chromosome binding may be one mechanism by which dMi-2 controls gene expression (Fasulo, 2012).
The live analysis of a LacO array tagged with GFP in living cells is consistent with a potential role for dMi-2 in chromosome cohesion. The array is organized in a compact disc due to cohesion between precisely aligned chromatids. The over-expression of dMi-2 caused the LacO array to disperse into hundreds of discrete foci, presumably due to the disruption of interactions between sister chromatids. The over-expression of dMi-2 also disrupted the organization of mitotic chromosomes along their longitudinal axes, possibly by interfering with chromosomal interactions in cis that contribute to the organization of chromosome shape (Fasulo, 2012).
The findings of this study show that dMi-2 plays unanticipated roles in both the regulation of higher-order chromosome structure and cohesin dynamics. Is there a causal relationship between the two activities? The sudden removal of cohesin in late larval development by targeted proteolysis does not dramatically alter polytene structure and thus cohesin may not be critical for maintenance of polytene structure once fully established. However, genetic studies of pds5 have revealed a role for both cohesin binding and sister chromatid cohesion in forming the normal structure of polytene chromosomes. A pds5 null allele and an allele encoding an N-terminally truncated protein alter polytene chromosome structure in distinctive ways, but in both cases the size and normal banding pattern are disrupted. Taken together, the above considerations prevent the conclusion that dMi-2 promotes chromosome decondensation by destabilizing cohesin binding. However, because dMi-2 over-expression causes a large reduction in both the amount of stable cohesin and its chromosomal residence time, it can be concluded that cohesin binding has been reduced, and that it is also likely that cohesion is affected (Fasulo, 2012).
The internal diameter of cohesin is ~35 by 50 nm; it can therefore encircle only one 30 nm or two 10 nm chromatin fibers. Interactions between cohesin complexes are thought to contribute to chromatid cohesion and presumably anchor chromatin loops to form 'hubs' of high transcriptional activity . The destabilization of cohesin binding therefore may be a secondary consequence of changes in chromatin structure catalyzed by dMi-2. Further work will be necessary to test this possibility and clarify the causal relationship, if any, between changes in chromosome structure and cohesin binding catalyzed by dMi-2 (Fasulo, 2012).
It is intriguing that dMi-2, an antagonist of cohesin binding and well-characterized transcriptional repressor, co-localizes with cohesin at sites of active transcription. Although cohesin subunits and Nipped-B were not identified as stable subunits of dMi-2 containing complexes in cultured cells, the extensive overlap between their chromosomal distributions suggests that chromatin structure and gene activity may be dependent on a fine balance of opposing dMi-2 and cohesin activities. Cohesin selectively binds and regulates active genes that have paused RNA polymerase, and can both positively and negatively regulate these genes by multiple mechanisms, including controlling the transition of paused polymerase to elongation. It is possible that dMi-2 may also influence this transition by regulating cohesin binding and the chromatin structure at the pause sites. Intriguingly, mouse Mi-2ß and the NuRD complex bind active and poised gene promoters in thymocytes, and have both negative and positive effects on expression of these genes. The Mi-2/NuRD complex regulates the expression of genes involved in lymphocyte differentiation [and is also involved in stem cell renewal and determination. As in Drosophila, mammalian cohesin also regulates many genes critical for growth and development. These findings raise the interesting possibility that Mi-2 may regulate cellular differentiation in vertebrates by modulating chromosome condensation and cohesin activity (Fasulo, 2012).
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