Targets of Activity

Evidence in this study suggests that a strong hypomorphic mutation in the Drosophila Rpd3 gene causes embryonic lethality and a specific pair-rule segmentation phenotype. The analysis of a number of segmentation genes suggests that the repressor function of Even-skipped (Eve) may be diminished, causing an indirect loss of Ftz-mediated activation of engrailed. Thus Rpd3 may be involved as a cofactor in Eve function. The possibility is discussed that Eve mediates multiple mechanisms of repression, so that Rpd3 mutants disrupt the regulation of just a subset of Eve target genes (Mannervik, 1999).

During segmentation of the Drosophila embryo, even skipped is required to activate engrailed stripes and to organize odd-numbered parasegments. A 16 kb transgene containing the even skipped coding region can rescue normal engrailed expression, as well as all other aspects of segmentation, in even skipped null mutants. To better understand its mechanism of action, the Even-skipped protein was functionally dissected in the context of this transgene. Even-skipped utilizes two repressor domains to carry out its function. Each of these domains can function autonomously in embryos when fused with the Gal4 DNA-binding domain. A chimeric protein consisting only of the Engrailed repressor domain and the Even-skipped homeodomain, but not the homeodomain alone, is able to restore function, indicating that the repression of target genes is sufficient for even skipped function at the blastoderm stage, while the homeodomain is sufficient to recognize those target genes. When Drosophila Even skipped is replaced by its homologs from other species, including a mouse homolog, these homologs could provide substantial function, indicating that these proteins can recognize similar target sites and also provide repressor activity. Using this rescue system, it has been shown that broad, early even skipped stripes are sufficient for activation of both odd- and even-numbered engrailed stripes. Furthermore, these 'unrefined' stripes organize odd-numbered parasegments in a dose-dependent manner, while the refined, late stripes, which coincide cell-for-cell with parasegment boundaries, are required to ensure the stability of the boundaries (Fujioka, 2002).

The histone deacetylase Rpd3 affects eve function. In Rpd3 mutant embryos, although the expression pattern of eve is not changed, even-numbered en stripes are very weak or missing owing to a lack of repression of odd. However, odd-numbered en stripes are expressed with only minor alterations. This is in contrast to the relative effects on odd- versus even-numbered en stripes when the eve dose is reduced, or in hypomorphic mutants, suggesting that Rpd3 may affect the repression of odd more than that of slp and prd. The Rpd3 effect similarly contrasts with the effects of removing either of the Eve repressor domains, suggesting that Rpd3 specificity cannot be explained by a selective effect on one of the Eve corepressors. This is true despite the fact that Rpd3 has been shown to mediate Gro repressor activity. Therefore, the apparent specificity of action of Rpd3 during segmentation is not easily explained solely through an effect on Eve activity. Conceivably, Rpd3 might affect the target specificity of the Eve HD, perhaps through selective effects on chromatin structure at different target sites. Another possibility is that it might affect the activities of other pair-rule gene products in addition to Eve. For example, it has been shown that Slp interacts with Gro in vitro. If Rpd3 reduces slp activity, then the effect of Rpd3 on Eve repressor function might be partially antagonized at the odd-numbered parasegment boundaries by its effect on slp (Fujioka, 2002).

Low-level ectopic expression of the Runt transcription factor blocks activation of the Drosophila melanogaster segmentation gene engrailed (en) in odd-numbered parasegments and is associated with a lethal phenotype. By using a genetic screen for maternal factors that contribute in a dose-dependent fashion to Runt-mediated repression, it is shown that there are two distinct steps in the repression of en by Runt. The initial establishment of repression is sensitive to the dosage of the zinc-finger transcription factor Tramtrack. By contrast, the co-repressor proteins Groucho and dCtBP, and the histone deacetylase Rpd3, do not affect establishment but instead maintain repression after the blastoderm stage. The distinction between establishment and maintenance is confirmed by experiments with Runt derivatives that are impaired specifically for either co-repressor interaction or DNA binding. Other transcription factors can also establish repression in Rpd3-deficient embryos: this indicates that the distinction between establishment and maintenance may be a general feature of eukaryotic transcriptional repression (Wheeler, 2002).

IkappaB kinase (IKK) and Jun N-terminal kinase (Jnk) signaling modules are important in the synthesis of immune effector molecules during innate immune responses against lipopolysaccharide and peptidoglycan. However, the regulatory mechanisms required for specificity and termination of these immune responses are unclear. Crosstalk occurs between the Drosophila Jnk and IKK pathways; this leads to downregulation of each other's activity. The inhibitory action of Jnk is mediated by binding of Drosophila activator protein 1 (AP1) to promoters activated by the transcription factor NF-kappaB. This binding leads to recruitment of the histone deacetylase dHDAC1 to the promoter of the gene encoding the antibacterial protein Attacin-A and to local modification of histone acetylation content. Thus, AP1 acts as a repressor by recruiting the deacetylase complex to terminate activation of a group of NF-kappaB target genes (Kim, 2005).

dSAP18 and dHDAC1 contribute to the functional regulation of the Drosophila Fab-7 element

The Drosophila GAGA factor [Trithorax-like (Trl)] interacts with dSAP18, which, in mammals, is a component of the Sin3-HDAC co-repressor complex. GAGA-dSAP18 interaction has been proposed to contribute to the functional regulation of the bithorax complex (BX-C). Mutant alleles of Trl, dsap18 and drpd3/hdac1 enhance A6-to-A5 transformation indicating a contribution to the regulation of Abd-B expression at A6. In A6, expression of Abd-B is driven by the iab-6 enhancer, which is insulated from iab-7 by the Fab-7 element. GAGA, dSAP18 and dRPD3/HDAC1 co-localize to ectopic Fab-7 sites in polytene chromosomes, and mutant Trl, dsap18 and drpd3/hdac1 alleles affect Fab-7-dependent silencing. Consistent with these findings, chromatin immunoprecipitation analysis shows that, in Drosophila embryos, the endogenous Fab-7 element is hypoacetylated at histones H3 and H4. These results indicate a contribution of GAGA, dSAP18 and dRPD3/HDAC1 to the regulation of Fab-7 function (Canudas, 2005).

The conclusion that GAGA, dSAP18 and dRPD3/HDAC1 contribute to the function of the Fab-7 element of BX-C is based on the following observations:

  1. the localization of GAGA, dSAP18 and dRPD3/HDAC1 at ectopic Fab-7 elements (Canudas, 2005).
  2. the effects of Trl, dsap18 and drpd3/hdac1 mutations on Fab-7-dependent silencing. Ectopic Fab-7 constructs are known to mediate silencing of flanking reporter genes both in cis, as in heterozygous GCD6 flies, as well as in trans, as in 5F24 flies, where silencing is pairing-sensitive being observed only when the transgene is in a homozygous state. This study shows that Trl, dsap18 and drpd3/hdac1 mutations affect both cis- and trans-silencing mediated by Fab-7 (Canudas, 2005).
  3. the homeotic A6-to-A5 transformation observed in flies heterozygous for various Trl, dsap18 and drpd3/hdac1 mutant alleles and hemizygous for Df(3R)sbd45, which uncovers dsap18. This homeotic transformation results from the ectopic repression of the iab-6 enhancer at A6 that is insulated from the repressed iab-7 enhancer by the Fab-7 element. The fact that this homeotic transformation is very infrequent in hemizygous Df(3R)sbd45 flies, as well as in the heterozygous mutants, demonstrates that it is directly associated to the Trl, dsap18 and drpd3/hdac1 mutations. Moreover, a single copy of a transgene expressing dsap18 significantly rescues this phenotype. The results also indicate that an unidentified element(s) contained within Df(3R)sbd45 is also contributing to the establishment of the phenotype. In addition to sap18, Df(3R)sbd45 uncovers at least 11 other genes including the trithorax gene, taranis. However, the homeotic transformation described in this study does not appear to be associated to a loss of taranis function since no transformation is observed in flies trans-heterozygous for a null taranis allele and Trl, dsap18 or drpd3/hdac1 mutations (Canudas, 2005).

Together, these results indicate a contribution of GAGA, dSAP18 and dRPD3/HDAC1 to the structural and functional properties of Fab-7. What could this contribution be? Several models might account for these results. Fab-7 is known to contain two functional elements: a PRE, which is required for Pc-dependent silencing, and an adjacent boundary element that insulates iab-6 from iab-7. The finding that, in heterozygous GCD6 flies, mutant Trl, dsap18 and drpd3/hdac1 alleles enhance cis-silencing imposed by Fab-7 suggests that their functions might antagonize Pc-dependent silencing. Several observations, however, make this hypothesis unlikely: (1) at some PREs, GAGA helps recruitment of PcG complexes and contributes to silencing; (2) dRPD3/HDAC1 was shown to be a component of several PcG complexes, and genetic analysis indicates a contribution to homeotic silencing; (3) in mammals, SAP18 acts as a repressor when targeted to an active promoter (Canudas, 2005).

An alternative possibility is that GAGA, dSAP18 and dRPD3/HDAC1 contribute to the function of the Fab-7 boundary element. In fact, the Fab-7 boundary contains several GAGA-binding sites that are required for its enhancer blocking activity and, it is hypoacetylated at histones H3 and H4. In GCD-6 flies, the Fab-7 boundary element is located proximal to the reporter mini-white gene with respect to the PRE so that it might help to insulate the reporter gene from repression by the PRE. In this context, mutations that affect boundary function would result in a less efficient insulation and, therefore, would enhance silencing (Canudas, 2005).

In contrast to the enhancer effect observed in heterozygous GCD6 flies, mutations in Trl, dsap18 and drpd3/hdac1 suppress pairing-dependent trans-silencing in transgenic 5F24(25,2) flies. A contribution to boundary-functions might also account for this effect. Pairing-sensitive trans-silencing results from long-distance chromosomal interactions that involve the association of the transgenes with each other and with the endogenous Fab-7 element, even when located in different chromosomes. These long-distance interactions that require the contribution of PcG proteins might be facilitated by a functional boundary element as has been described for the gypsy insulator (Canudas, 2005).

The incomplete A6-to-A5 homeotic transformation observed in the presence of Trl, dsap18 and drpd3/hdac1 mutations might also reflect a contribution to the boundary function of Fab-7 as, in the mutant conditions, it might not properly insulate the iab-6 enhancer from the repressing activity of the Fab-7 PRE, thereby becoming partially inactivated. Interestingly, mutations that delete the Fab-7 boundary but not the PRE produce, in addition to strong A6-to-A7 transformation, incomplete A6-to-A5 transformation. Moreover, replacement of the Fab-7 boundary by the gypsy or the scs insulator (both of which are not functional in the context of BX-C) results in complete A6-to-A5 transformation (Canudas, 2005).

The results indicate that GAGA, dSAP18 and dRPD3/HDAC1 have similar effects on the functional properties of Fab-7 suggesting a functional link. A physical interaction between GAGA and dSAP18 has been reported. Moreover, in mammals, SAP18 is associated with the Sin3-HDAC co-repressor complex and, in Drosophila, dSAP18 modulates bicoid activity through the recruitment of dRPD3/HDAC1 and it is required to suppress bicoid activity in the anterior tip of the embryo. In this context, it is tempting to speculate that GAGA helps in the recruitment of dSAP18 and dRPD3/HDAC1 to Fab-7 resulting in a concerted contribution to its boundary function (Canudas, 2005).

In mammals, SAP18 is also associated with ASAP, a protein complex involved in RNA processing. In Drosophila, dSAP18 may also participate in RNA processing; in cultured S2 cells, a large proportion of dSAP18 co-immunoprecipitates with factors that participate in RNA processing. It is possible that, in response to cellular signals, the association of dSAP18 to different protein complexes would be regulated during development and/or cell cycle progression (Canudas, 2005).

Histone deacetylase Rpd3 regulates olfactory projection neuron dendrite targeting via the transcription factor Prospero

Compared to the mechanisms of axon guidance, relatively little is known about the transcriptional control of dendrite guidance. The Drosophila olfactory system with its stereotyped organization provides an excellent model to study the transcriptional control of dendrite wiring specificity. Each projection neuron (PN) targets its dendrites to a specific glomerulus in the antennal lobe and its axon stereotypically to higher brain centers. Using a forward genetic screen, a mutation in Rpd3 was identified that disrupts PN targeting specificity. Rpd3 encodes a class I histone deacetylase (HDAC) homologous to mammalian HDAC1 and HDAC2. Rpd3−/− PN dendrites that normally target to a dorsolateral glomerulus mistarget to medial glomeruli in the antennal lobe, and axons exhibit a severe overbranching phenotype. These phenotypes can be rescued by postmitotic expression of Rpd3 but not HDAC3, the only other class I HDAC in Drosophila. Furthermore, disruption of the atypical homeodomain transcription factor Prospero (Pros) yields similar phenotypes, which can be rescued by Pros expression in postmitotic neurons. Strikingly, overexpression of Pros can suppress Rpd3−/− phenotypes. This study suggests a specific function for the general chromatin remodeling factor Rpd3 in regulating dendrite targeting in neurons, largely through the postmitotic action of the Pros transcription factor (Tea, 2010).

To identify genes that are essential for dendrite wiring specificity in Drosophila olfactory projection neurons (PNs), a MARCM-based forward genetic screen was performed using ethyl methanesulfonate as mutagen. MARCM allows visualization and genetic manipulation of single cell or neuroblast clones in an otherwise heterozygous animal, allowing the study of essential genes in mosaic animals. GH146-GAL4 was used to label a single PN born at newly hatched larva, which in wild-type animals always projects its dendrites to the dorsolateral glomerulus DL1 in the antennal lobe. A mutant, called 12-37, was identified in which DL1 PNs mistargeted towards dorsomedial or ventromedial regions of the antennal lobe. SNP and deletion mapping identified the causal gene to be Rpd3, encoding a homolog of mammalian HDAC1 and HDAC2. The mutant 12-37 causes a G136R missense mutation in the catalytic domain; the glycine at this position is conserved from yeast to humans (Tea, 2010).

The mistargeting phenotype was conserved to be caused by the mutation in Rpd3 using the following two criteria. First, MARCM single cell clones of two previously existing Rpd3 mutants gave similar mistargeting phenotypes. Second, MARCM expression of UAS-Rpd3 in DL1 single cell clones using GH146-GAL4 significantly rescued the phenotype for all three Rpd3 alleles, especially at higher temperatures where GAL4 activity increases. Because GH146-GAL4 expresses UAS-Rpd3 only in postmitotic PNs, it was conclude that Rpd3 plays an essential role in postmitotic PNs to regulate dendrite targeting (Tea, 2010).

Three lines of evidence indicate that Pros expression and function in the PN lineage differs from its function in the embryonic lineage. First, Pros is clearly present in postmitotic PNs. Immunostaining for Pros at 24hAPF shows that wild-type postmitotic PNs express varying amounts of Pros protein; this variable expression persists throughout development and adulthood. Wild-type DL1 PNs express an intermediate level of Pros protein. pros−/− DL1 or neuroblast clone PNs have an undetectable level of Pros protein in the cell body, confirming both antibody specificity and the nature of the loss-of-function mutation. Pros immunoreactivity can be restored by MARCM expression of UAS-pros in postmitotic PNs (Tea, 2010).

Second, the pros−/− single cell clone phenotype indicates that pros mRNA must be transcribed in postmitotic neurons and/or ganglion mother cells; it cannot be transcribed only in neuroblasts. There are two scenarios in which a single cell MARCM clone can be produced. The mitotic recombination could occur right before the ganglion mother cell divides to give rise to two postmitotic cells, and therefore only the postmitotic DL1 PN is pros−/−. Alternatively, because the sibling of the DL1 PN dies during development, the mitotic recombination could also occur in the neuroblast; in this case, the ganglion mother cell giving rise to the DL1 PN is pros−/−. In either case, the neuroblast remains pros+/−. Therefore pros must be transcribed either in the postmitotic PN or the ganglion mother cell to account for a single cell phenotype (Tea, 2010).

Third, postmitotic UAS-pros expression can rescue the mistargeting defect of pros−/− DL1 dendrites as well as overbranching of DL1 axons, indicating that Pros functions in postmitotic neurons to regulate PN dendrite targeting and axon branching (Tea, 2010).

It has long been thought that histone deacetylases play a general role in chromatin remodeling and transcriptional control, and many studies have examined genome-wide patterns of histone modifications. Yet recent studies have also suggested that 'general' chromatin remodeling factors can have very specific roles. This study has shown a new function for Rpd3, a ubiquitously expressed protein that is the major histone deacetylase in Drosophila olfactory projection neurons. Rpd3 plays a specific role in controlling dendrite targeting and axon terminal branching, and this function cannot be replaced by the only other class I HDAC. Furthermore, this study has shown that the majority of its function in regulating dendrite targeting and a portion of its function in regulating axon branching are likely carried out through the downstream transcription factor Prospero (Tea, 2010).

Although the possibility cannot be ruled that Rpd3 and Pros act in parallel pathways to regulate dendrite targeting and axon branching, several lines of evidence support the notion that Rpd3 acts via Pros to regulate these events. First, loss-of-function mutations in single cell clones of Rpd3 and pros give similar dendrite targeting and axon branching phenotypes. Second, overexpression of Pros can largely suppress the dendrite mistargeting phenotype of Rpd3−/− and can partially suppress the axon overbranching phenotype of Rpd3−/−. This suppression is specific, as overexpressing Pros does not cause defects in wild-type cells nor does it suppress mistargeting phenotypes due to a few other mutations. Conversely, overexpression of Rpd3 does not suppress pros mutant phenotypes. Because the suppression is more robust for dendrite mistargeting defects compared to axon overbranching defects, it is possible that Pros function accounts for more of Rpd3’s activity in regulating dendrite targeting than axon branching (Tea, 2010).

To address the mechanism by which Rpd3 and Pros function together in regulating PN development, several models of their interactions were tested. One model is that Rpd3 functions to upregulate the expression of Pros, which predicts that Rpd3−/− would lead to a decrease in Pros protein. However, anti-Pros immunoreactivity in Rpd3−/− DL1 PN clones was not decreased compared to wild-type DL1 PN clones. A second model is that Rpd3 and Pros directly bind and work together to regulate the transcription of Pros target genes. Yet in wild-type embryos or in S2 cell culture, no complex between Rpd3 and Pros could be detected via immunoprecipitation. A third model is that Rpd3 directly deacetylates the Pros protein to affect its function, but Pros acetylation could not be detected in the presence of HDAC inhibitors by immunostaining or mass spectrometry. Together, these data suggest that Rpd3 indirectly affects the function of Pros. This effect may be through posttranslational modification of Pros to modify its activity. For example, Pros has previously been shown to be phosphorylated. If posttranslational modification increases Pros activity, then Rpd3−/− would result in reduced Pros activity. Overexpression of Pros in Rpd3−/− may compensate for the reduced activity of unmodified Pros, and therefore suppress the Rpd3−/− phenotype (Tea, 2010).

Future studies will determine how Rpd3 regulates Prospero, how these factors act together with other transcription factors, and what transcriptional target genes they regulate in order to orchestrate the developmental program for precise wiring of the olfactory circuit (Tea, 2010).

dLin52 is crucial for dE2F and dRBF mediated transcriptional regulation of pro-apoptotic gene hid

Drosophila lin52 (dlin52) is a member of Myb transcription regulator complex and it shows a dynamic pattern of expression in all Drosophila tissues. Myb complex functions to activate or repress transcription in a site-specific manner; however, the detailed mechanism is yet to be clearly understood. Members of the Drosophila melanogaster Myb-MuvB/dREAM complex have been known to regulate expression of a wide range of genes including those involved in regulating apoptosis. E2F and its corepressor RBF also belong to this complex and together they regulate expression of genes involved in cell cycle progression, apoptosis, differentiation, and development. This study examined whether the depletion of dlin52 in developing photoreceptor neurons results in enhanced apoptosis and disorganisation of the ommatidia. Strikingly, it was found that dLin52 is essential for transcriptional repression of the pro-apoptotic gene, hid; decrease in dlin52 levels led to dramatic induction of hid and apoptosis in eye-antennal discs. Reduction of Rpd3 (HDAC1), another member of the dREAM complex, also led to marginal upregulation of Hid. In addition, it was demonstrated that an optimum level of dLin52 is needed for dE2F1/2 activity on the hid promoter. dlin52 cooperates with dRBF and dE2F1/2 for recruitment of repressor complex on the hid promoter. Preliminary data indicates that Rpd3/HDAC1 also contributes to hid repression. Based on the findings, it is concluded that dLin52 functions as a co-factor and modulates activity of members of dMyb/dREAM complex at hid promoter, thus, regulating apoptosis by repressing this pro-apoptotic gene in the developing Drosophila eye (Bhaskar, 2014).

Regulated progression through the cell cycle is essential for ordered cell proliferation. Changes in the balance between cell cycle-driving proto-oncogene-dependent pathways and inhibiting signals from tumour suppressors are a common cause for cancer. One of the best characterised tumour suppressors is the retinoblastoma protein pRB, the first cloned tumour suppressor. This gene was first described as a susceptibility gene for retinoblastoma, an eye tumour in children; it is now known to be mutated in many cancers. Currently, more than 120 proteins have been reported to be associated with pRb, and a wide assortment of chromatin-modifying and binding complexes have been implicated in pRB mediated repression. The association of these complexes with pRb has broadened its range of functions. In humans, one of the complexes known as the DREAM complex is required to arrest expression of genes essential for cell cycle progression. A similar complex is also found in C.elegans (synMuvB complementation group or DRM complex) as well as in Drosophila (dREAM complex). The function of this complex seems to be conserved across taxa and has definitive control over normal cell development. In the past few years, the function and composition of this complex has been unravelled. The role of the dREAM complex has now been extended to development, differentiation, and apoptosis. Its composition has also been reported to vary according to the function and site of the action (Bhaskar, 2014).

The smallest member of dREAM/MMB complex, dLin52 is found in Drosophila. The dynamic pattern of expression of dLin52 in various tissues has been reported and a strong conservation was found of this protein across taxa (Bhaskar, 2012). Hence, this research was extended to explore the functional characterisation of dlin52. GMR-GAL4 and UAS-dlin52-RNAi were used to deplete dLin52 in the developing eye and examine its role in the developing photoreceptors (Bhaskar, 2014).

A recent study, Lewis (2012), has shown that dLin52 is needed for viability, adult eye development and embryogenesis via its maternal effect. The mutant phenotypes could be rescued by heterozygous deletion of mip120 or loss of function allele of mip130. It was concluded that Lin-52 and Myb proteins counteract against the repressive activities of the other members of the MMB/dREAM complex at specific genomic loci in a developmentally controlled manner (Bhaskar, 2014).

The findings of the current study showed that down regulation of dlin52 leads to rough eye phenotype, which is primarily caused by enhanced apoptosis. this observation is in accordance with a study of mammalian cells where depletion of LIN52 sensitised gastrointestinal stromal cells to imatinib induced apoptosis, suggesting a similar mechanism for regulation of apoptosis by LIN52 in higher organisms (Boichuk, 2013). A connection between E2Fs, regulation of hid, and apoptosis has also been found in Drosophila. While dE2F1 activates, dE2F2 has been found to represses hid in wing discs. However, it has been reported that the depletion of dE2F1 in S2 cells leads to activation of hid, showing for the first time the ability of dE2F1 to repress or negatively regulate transcription (Bhaskar, 2014).

The present study established that apoptosis due to dlin52 down regulation is cell autonomous. The components of Drosophila cell death regulatory pathway are conserved in higher organisms. This includes inhibitor of apoptosis proteins (IAPs) which bind to caspases and pro-apoptotic proteins. This study observed that over-expression of DIAPI rescued dlin52-RNAi phenotype. Further, only loss of function alleles of hid suppressed dlin52 rough eye phenotype but not rpr or grim alleles. Similarly, depletion of dLin52 also led to increase in hid transcript levels but not that of rpr or grim. The pattern of hid activation caused by dlin52-RNAi is in agreement with the earlier studies which reported the loss of dRBF and dE2F1; similar to those of previous studies, the cells immediately posterior to the MF were more sensitised to dLin52 downregulation in the eye-antennal discs. Therefore, these findings, supported by the previous observations with loss of RBF and E2F1, suggest that the reduction of dLin52 results in apoptosis is mediated via upregulation of Hid. Additionally, dlin52 also showed robust genetic interaction with Rbf, E2f, and E2f2alleles. It is evident that dLin52 works synergistically with dRBF and dE2F1/2 to mediate transcriptional repression of hid. Previous studies revealed that loss of dRBF and dE2F1/2 activates hid and increases apoptosis, while this tudy identified a third important component, dLin52, vital for hid regulation (Bhaskar, 2014).

Although Lin52 itself is not DNA binding, in humans, Lin52 has been shown to be essential for the formation of transcription repressor complex. Thiss tudy demonstrated that ablated dLin52 levels diminish both dE2F1 and dE2F2 recruitment on the hid promoter. As mutated dE2F binding site on the hid promoter fails to enhance dlin52-RNAi phenotype, it indicates that dE2F1 mediates repression of hid, aided by dLin52 and dRBF1. It was observed that recruitment of dE2F2 was also affected with loss of dLin52; it is likely that both E2F1 and E2F2 function to repress hid, and the stoichiometry of both E2F1 and E2F2 are important for their regulatory function. Taken together these data suggest dLin52 as the essential factor for dRBF, dE2F1/2 mediated repression of hid expression. It was also found that transcriptional regulation of hid is mediated via the dE2F binding site present in the 5' UTR of hid. Previous studies showed that dE2F1 not only binds to a unique site in promoter region of hid but also regulates its transcription in presence of dRBF. Therefore, it is concluded that optimum level of dLin52 is needed for recruitment of dE2F1/2 to the hid promoter; reducing dLin52 dramatically compromises with the dE2F1/2 binding to the cis-acting sequences on the hid promoter (Bhaskar, 2014).

Histone deacetylase1 (HDAC1) has been the most thoroughly studied HDAC at the biochemical and functional levels. HDACs are known to promote heterochromatin silencing by deacetylating H3. They target hundreds of genes in the genome and play a major role as a direct transcriptional repressor. HDAC1 controls segmentation genes through interaction with the Groucho corepressor and has also been linked to silencing by Polycomb repressors. HDAC3 and HDAC1 mutants together can suppress position-effect variegation (PEV), indicating the ability of both the HDACs in mediating heterochromatin silencing while working together. Recently, Zhu, found Drosophila HDAC3 regulating the wing imaginal disc size through suppression of apoptosis, while HDAC3 mutants shows ectopic Hid levels in wing discs. Mammalian HDAC1 mutant also shows increased apoptosis. Rpd3, the Drosophila homologue of HDAC1/2, has been purified along with dMyb/MMB/dREAM complex. Function of both HDAC3 and Rpd3 appears to overlap in regulating silencing and chromatin modification. In this study, it was observed that down-regulation of Rpd3 alone in eye-antennal discs led to a rough eye phenotype and induced marginal increase of Hid; however, ectopic expression was not limited to a few rows of cells posterior to the MF but traversed the differentiating photoreceptors, implying that Rpd3 mediated hid regulation is not limiting to cells specifically regulated by dLin52, dRbF and dE2F1/2 but expands to a much broader area in the developing photoreceptors. Similarly, lowering of both dlin52 and Rpd3 at the same time led to substantial increase in Hid levels with enhanced disorganisation of the ommatidia. Although Rpd3 in Drosophila lacks LXCXE motif, it might be recruited at the repressed sites by other members of the complex. Hence, it may be concluded that both dLin52 and Rpd3 (HDAC1) have a major role to play in negative regulation of hid expression. However, this is only a preliminary report suggesting that dLin52 and Rpd3 can work in parallel to regulate hid expression. Further experiments like biochemical purification of dREAM complex in the presence and absence of dLin52 would help in elaborating function of Rpd3 in regulating apoptosis (Bhaskar, 2014).

This study is in agreement with the earlier findings that Lin52 plays an important role in forming dREAM complex. Human Lin52 phosphorylation is needed for assembling of the dREAM complex. Earlier, it was reported that the Serine-28 residue in the human Lin52 is conserved in dLin52. This study proposes that limiting dLin52 might actually be equivalent to un-phosphorylated dLin52 which is non-functional, resulting in assembling defects of the dREAM complex. Therefore, it is hypothesised that dLin52 is a vital survival signal, needed for suppressing hid transcription and apoptosis and conclude that dLin52 is a crucial cofactor essential for assembling members of dREAM/MMB complex (dRBF, dE2F1/2). In addition, this study has also presented preliminary data indicating that Rpd3 functions together with dLin52, dRBF, and dE2F1/2 for mediating transcriptional repression of hid (Bhaskar, 2014).

A proposed model shows that dLin52 and Rpd3 (HDAC1) together with dRBF and dE2Fs are part of a repressor complex, repressing hid activation. When dLin52 becomes limiting, E2F1/2 cannot be recruited to the consensus E2F1/2 binding site on the hid promoter, resulting in the inability of the repressor complex to assemble. This leads to derepression and activation of hid, followed by increased apoptosis (Bhaskar, 2014).

Chemical inhibitors that block HDAC activity are of considerable interest in cancer research because of their ability to induce tumour cell killing by activating cell death pathway leading to apoptosis. Hence, it is proposed that Lin52 may also be selectively inhibited in inducing apoptosis in tumour cells (Bhaskar, 2014).

This study established the important role of dLin52 in repressing apoptosis. This leads to the belief that dLin52 is needed for maintenance of proper development, differentiation, normal physiology, and homeostasis in Drosophila (Bhaskar, 2014).

This study has found down regulation of dlin52 resulting in a rough eye phenotype. Based on these findings, it is suggested that the rough eye phenotype is due to increase in apoptosis. This study established through genetic analysis that downregulation of dlin52 increases hid expression. As dLin52 by itself is not DNA binding, it is predicted that it may be regulating hid along with the members of dREAM complex. Based on the observations, it can be concluded that dRBF, dE2F1, dE2F2, and Rpd3 work together with dLin52 for repressing hid activation, because the loss of function alleles of these genes led to strong enhancement of dlin52-RNAi eye phenotype. ChIP experiment demonstrated that reduced dlin52 levels also affect dE2F1 and dE2F2 binding to its consensus binding site on hid promoter (Bhaskar, 2014).

Furthermore, it can be concluded that dLin52 regulates apoptosis in eye-antennal discs by repressing hid transcription; loss of dLin52 induces hid expression. This suggests that dLin52 is a co-factor, needed for repression of hid along with dE2Fs and dRBF. Additionally, loss of dLin52 also affected binding of dE2F1 and dE2F2 to their consensus sequence, which means like DP, dLin52 can also affect DNA binding capacity of RBF and E2Fs. Taken together these data suggest that dLin52, dRBF, dE2F1, dE2F2, and dRpd3 cooperate to negatively regulate hid transcription and apoptosis. Further studies in understanding the role of Drosophila lin52 in apoptosis will shed light on the role of LIN52 in higher organisms (Bhaskar, 2014).

Protein Interactions

Protein structural evidence suggests that Drosophila Sin3, a widely distributed transcription factor essential for embryonic viability, may interact with Rpd3. Expression of many mammalian genes is activated by the binding of heterodimers of the Myc (see Drosophila Myc) and Max proteins to specific DNA sequences called the E-boxes. Transcription of the same genes is repressed upon binding to the same DNA sequences of complexes composed of Max, Mad/Mxi1, the co-repressors Sin3 and N-CoR, and the histone deacetylase Rpd3. Max-Mad/Mxi1 heterodimers, which bind to E-boxes in the absence of co-repressors, do not inhibit gene expression simply by competition with Myc-Max heterodimers, but require Sin3 and Rpd3 for efficient repression of transcription. The Drosophila homolog of Sin3 (dSin3) has been cloned and found to be ubiquitously expressed during embryonic development. Yeast, mouse and Drosophila Sin3 proteins share six blocks of strong homologies, including four potential paired amphipathic helix domains. In addition, the domain of binding to the histone deacetylase Rpd3 is strongly conserved. Null mutations of Sin3 cause recessive embryonic lethality. It is likely that Drosophia Rpd3 protein interacts with dSin3 via the histone deacetylase domain (Pennetta, 1998).

Since Rpd3 mutation is known to dominantly enhance position effect variegation (PEV) (De Rubertis, 1996) it was asked whether dSin3 mutations likewise affect PEV. Surprisingly, no enhancement of PEV was observed. By contrast, sin3 and rpd3 mutations have been shown to have a similar effect on gene silencing at yeast telomeres (Vannier, 1996). One trivial explanation would be that the dSin3 gene is expressed at such a high level that reducing its dose by half (the combination that was tested) is not rate-limiting in the control of heterochromatin. This hypothesis is not favored, considering the sensitivity of PEV to the dose of many modifiers, including enzymes such as the histone deacetylase Rpd3 (De Rubertis, 1996). A more interesting possibility would be that the function of the Rpd3 histone deacetylase in counteracting gene silencing is mediated by Sin3 in yeast but depends on interaction with other partners in Drosophila (Pennetta, 1998).

Ras1 plays a critical role in receptor tyrosine kinase (RTK) signal transduction pathways that function during Drosophila development. Mis-expression of constitutively active forms of Ras1 (Ras1V12) and the Sevenless (Sev) RTK (SevS11) during embryogenesis causes lethality due to inappropriate activation of RTK/Ras1 signaling pathways. Genetic and molecular data indicate that the rate of SevS11/sev-Ras1V12 lethality is sensitive to the expression level of both transgenes. To identify genes that encode components of RTK/Ras1 signaling pathways or modulators of RNA polymerase II transcription, advantage was taken of the dose-sensitivity of the system and a screen was carried out for second site mutations that would dominantly suppress the lethality. The collection of identified suppressors includes the PR55 subunit of Protein Phosphatase 2A, indicating that downstream of Sev and Ras1 this subunit acts as a negative regulator of phosphatase activity. The isolation of mutations in the histone deacetylase RPD3 suggests that it functions as a positive regulator of sev enhancer-driven transcription. Finally, the isolation of mutations in the Trithorax group gene devenir and the characterized allelism with the Breathless RTK encoding gene provides evidence for Ras1-mediated regulation of homeotic genes (Maixner, 1998).

The Drosophila gene groucho encodes a transcriptional corepressor that has critical roles in many development processes. In an effort to illuminate the mechanism of Gro-mediated repression, Gro was employed as an affinity reagent to purify Gro-binding proteins from embryonic nuclear extracts. One of these proteins was found to be the histone deacetylase Rpd3. Protein-protein interaction assays suggest that Gro and Rpd3 form a complex in vivo and that they interact directly via the glycine/proline rich (GP) domain in Gro. Cell culture assays demonstrate that Rpd3 potentiates repression by the GP domain. Furthermore, experiments employing a histone deacetylase inhibitor, as well as a catalytically inactive form of Rpd3, imply that histone deacetylase activity is required for efficient Gro-mediated repression. Finally, mutations in gro and rpd3 have synergistic effects on embryonic lethality and pattern formation. These findings support the view that Gro mediates repression, at least in part, by the direct recruitment of the histone deacetylase Rpd3 to the template, where it can modulate local chromatin structure. They also provide evidence for a specific role of Rpd3 in early development (Chen, 1999).

Immunoprecipitation assays suggest that endogenous Gro and Rpd3 are associated in Drosophila nuclei, as antibodies against Gro coprecipitate Gro and Rpd3 from both Drosophila embryonic and S2 cell nuclear extracts. In each case, ~10%-20% of Rpd3 in the nuclear extracts has been found to precipitate with Gro. In vitro histone deacetylase assays using 3H-acetyl-labeled histones as the substrate indicates that ~20% of the histone deacetylase activity in the embryonic extracts coprecipitates with Gro. The histone deacetylase activity in the crude extracts and in the Gro immunoprecipitate is largely inhibited by the histone deacetylase inhibitor trichostatin A (TSA), as is the activity of purified recombinant Rpd3 (Chen, 1999).

To map the domain(s) of Gro responsible for the interaction with Rpd3, a series of truncated forms of Gro was constructed. When epitope tagged Groucho (M2-Gro) lacking the carboxy-terminal WD repeat domain (M2GroN420) is coexpressed with epitope tagged Rpd3 (H6Rpd3) in insect cells, M2GroN420 and H6Rpd3 copurify on both Ni2+-NTA-agarose and anti-Flag affinity beads. Therefore, the amino-terminal region of Gro is necessary, whereas the WD repeat domain is dispensable for the Rpd3 interaction (Chen, 1999).

The domain(s) in the amino-terminal region of Gro required for the Rpd3 interaction were further mapped by incubating in vitro-translated 35S-labeled Gro deletions with anti-Flag affinity beads containing purified M2Rpd3. Those 35S-labeled Gro variants that contain the glycine/proline-rich (GP) domain bind to beads containing purified M2Rpd3 but not to beads alone. Conversely, those 35S-labeled Gro variants lacking the GP domain fail to associate with M2Rpd3. In addition, a GST pull-down assay using purified GST-Gro fusion proteins confirms that the GP domain of Gro is required for the interaction with Rpd3. Furthermore, the deletion of the amino-terminal glutamine-rich (Q) domain of Gro severely reduces the affinity of the interaction. In conclusion, these findings suggest that the GP domain is required for the interaction, whereas the Q domain, which is required for Gro tetramerization, significantly stimulates the interaction (Chen, 1999).

To address whether the interaction between Gro and Rpd3 is functional in Gro-mediated repression, it was first determined if histone deacetylase activity is important for transcriptional repression by Gro in cultured cells. Gro strongly represses activated transcription in S2 cells when directly targeted to a promoter by fusion to the DNA binding domain (DBD) of the yeast transcription factor Gal4. The Gal4-Gro fusion strongly represses the transcriptional activation promoted by the combination of Dorsal and Twist, when expression vectors encoding these factors were cotransfected with a luciferase reporter (G5DE5tkLuc) driven by a herpes simplex virus thymidine core promoter, an artificial enhancer element containing multimerized Dorsal and Twist binding sites (Dl-Ebox), and multimerized Gal4-binding sites (USAG) (Chen, 1999).

Transfected S2 cells were treated with the histone deacetylase inhibitor TSA to determine whether deacetylase activity is important for Gal4-Gro-mediated repression. TSA treatment dramatically reduces Gal4-Gro-mediated repression, suggesting that histone deacetylation does contribute to this repression. Introduction of TSA results in a small (less than 2-fold) increase in the level of reporter activity in the absence of Gal4-Gro, but a much larger (up to 20-fold) increase in the level of reporter activity in the presence of Gal4-Gro. As a result, the calculated repression by Gal4-Gro decreases from 25-fold in the absence of TSA to ~3-fold in the presence of 300 nM TSA. The residual repression observed at high concentrations of TSA suggests that although full repression of transcription by Gro requires histone deacetylase activity, Gro may also utilize histone deacetylase-independent mechanisms for transcriptional repression (Chen, 1999).

In agreement with the finding that the GP domain of Gro is required for the interaction with Rpd3, this GP domain functions as a repression domain, when fused to the Gal4 DBD and the tetramerization domain (TD) of p53 (construct G4TDGP). The p53 TD, which itself does not repress transcription when fused to the Gal4 DBD, was utilized in place of the Gro TD to avoid the repression activity that is believed to result from the association of Gal4-Gro TD fusion with endogenous full-length Gro. In the absence of the p53 TD, the Gal4-GP domain fusion failed to repress transcription. These findings suggest that efficient repression by the GP domain of Gro requires tetramerization. This agrees with the finding that the Q domain is required for efficient binding between Gro and Rpd3 and with results showing that efficient repression by Gro requires a functional tetramerization domain (Chen, 1999 and references therein).

To further determine whether histone deacetylase activity is critical for the function of the Gro GP domain, a single point mutation in Rpd3 was generated in which a highly conserved histidine residue (H196) is replaced with a phenylalanine (H196F). Consistent with studies on mammalian histone deacetylases, the mutation decreases the specific activity of the enzyme by about sevenfold. Additionally, using a GST pull-down assay, both Rpd3WT and Rpd3H196F were found to bind to Gro with comparable affinity. Unlike Rpd3WT, which represses transcription three- to fourfold when fused to the Gal4 DBD, the Gal4-Rpd3H196F fusion fails to repress transcription in a similar assay. Therefore, Rpd3H196F represents an enzymatically inactive form of Drosophila histone deacetylase that binds to Gro with the same affinity as wild-type Rpd3 (Chen, 1999).

Although the Gal4-GP domain fusion protein does not repress activate transcription on its own due to the lack of a tetramerization domain, the Gal4-GP fusion is able to synergize with cotransfected wild-type Rpd3 to repress transcription but does not synergize with the mutant catalytically inactive form of Rpd3. These results strongly suggest that the GP domain contributes to Gro-mediated repression by interacting directly with the histone deacetylase Rpd3 and that the histone deacetylase activity of Rpd3 is essential for its ability to contribute to Gro-mediated repression (Chen, 1999).

In addition to Rpd3, Drosophila histone H1 has also been found to associate with Gro. H1 is a linker histone that stabilizes higher-order chromatin structure. Several previous findings suggest that the interaction between Gro and H1 may be functionally relevant to Gro-mediated repression. Drosophila H1 was purified and identified as a general inhibitor of transcription by RNA polymerase II in vitro . Genetic analysis has indicated that H1 does not appear to affect global transcription; instead, H1 functions as a gene-specific transcriptional repressor. In addition, the expression of genes encoding various H1 subtypes is developmentally regulated. Thus, it is possible that the interaction between Gro and H1 functions in a developmentally regulated manner to facilitate chromatin condensation and/or establish a repressive chromatin enviroment for transcription (Chen, 1999 and references therein).

The findings reported here, as well as those from previous studies showing that Gro family proteins make specific contacts with the amino-terminal tails of core histones (Palaparti, 1997), suggest that Gro represses transcription by inducing a silenced chromatin conformation. Thus it is proposed that after a direct interaction with DNA-binding transcription factors brings Gro to the template, the known ability of Gro to oligomerize, together with the known favorable interactions between Gro and core histones and/or histone H1, results in the nucleation of a Gro polymer that spreads along the template. Template-bound Gro may then provide an interface for recruitment to the template of key chromatin-modifying enzymes including histone deacetylases. These enzymes may then serve to modulate local higher order chromatin structure to establish a transcriptionally silenced domain. It is known that the interaction between Tup1 (a possible yeast homolog of Gro) and core histones is actually enhanced by histone deacetylation (Edmondson, 1996), so it is possible that histone deacetylation also serves to facilitate the further recruitment of the corepressor to the template, thereby reenforcing the transcriptionally repressed state (Chen, 1999).

Although histone deacetylation contributes to Gro-mediated repression, there are likely to be additional mechanisms by which Gro represses transcription. For example, cotransfection experiments show that Gro possesses some repression activity that is resistant to the deacetylase inhibitor TSA. In addition, the WD repeat domain of Gro functions as a weak repression domain (Fisher, 1996), and this repression activity appears to be independent of Rpd3. Consistent with the idea that corepressors can modulate transcription by multiple mechanisms, histone deacetylase-independent repression has also been observed for the Sin3 corepressor. It will thus be interesting to determine the identities of additional Gro-interacting proteins to see if they provide any clues to these possible alternative mechanisms of repression (Chen, 1999).

To study the mechanisms by which deacetylases regulate transcription by RNA polymerase II, the biochemical properties of purified recombinant Drosophila histone deacetylase 1 (dHDAC1, also known as dRPD3) were investigated. dHDAC1 and Gal4-dHDAC1 polypeptides possess substantial deacetylase activity. Thus, deacetylation by dHDAC1 does not require any additional cofactors. Gal4-dHDAC1, but not dHDAC1, was observed to repress transcription in vitro by about 2-3-fold from chromatin templates, but not from naked DNA templates, in a Gal4 site-dependent manner. This magnitude of repression is similar to that commonly seen by deacetylases in vivo, as assessed by treatment of cells with deacetylase inhibitors. Transcriptional repression by Gal4-dHDAC1 is blocked by the deacetylase inhibitor, FR901228, and thus, deacetylase activity correlates with repression. Single round transcription analyses have shown that Gal4-dHDAC1 reduces the absolute number of productive initiation complexes with chromatin templates. Moreover, with chromatin templates that were assembled with completely purified components, Gal4-dHDAC1 was found to deacetylate nucleosomal histones as well as to repress transcription. These experiments provide biochemical evidence for the requirement of chromatin for transcriptional repression by dHDAC1 and further show that dHDAC1 acts to repress the transcription initiation process (Huang, 2001).

A Drosophila corepressor mediates transcriptional silencing of the Ecdysone receptor:Ultraspiracle heterodimer. SMRT-related ecdysone receptor-interacting factor (Smrter), formally known as SANT domain protein , is a large nuclear protein that, surprisingly, shows only limited homology to the vertebrate corepressors SMRT and N-CoR. Nevertheless, the fact that EcR:USP associates with Smrter and Smrter associates with murine Sin3A and Drosophila Sin3A, co-repressors known to form a complex with the histone deacetylase Rpd3/HDAC, indicates a conserved mechanism underlying transcriptional repression by vertebrate and invertebrate nuclear receptors. Given the genetic and biochemical evidence that Sin3A associates with Rpd3/HDAC in both yeast and mammalian cells, and the likelyhood for a similar association in Drosophila, it is expected that Smrter also recruits a histone deacetylase complex to EcR. The linkage of EcR to Rpd3 is a potential explaination for the role of histone deacetylase in triggering the regression of chromosome puffs. Yet the presence of Smrter in puffed loci of polytene chromosomes indicates that a complete dissociation of Smrter complex may not be a prerequisite step for the formation of chromosomal puffs. Rather, other factors, such as coactivators with histone acetyltransferase activity, may play a significant role in triggering the formation of chromosome puffs (Tsai, 1999 and references).

The Drosophila Polycomb Group (PcG) proteins are required for stable long term transcriptional silencing of the homeotic genes. Among PcG genes, esc is unique in being critically required for establishment of PcG-mediated silencing during early embryogenesis, but not for its subsequent maintenance throughout development. Esc has been shown to be physically associated with the PcG protein E(Z). Esc, together with E(z), is present in a 600 kDa complex that is distinct from complexes containing other PcG proteins. This Esc complex has been purified and it also contains the histone deacetylase Rpd3 and the histone-binding protein p55 (Chromatin assembly factor 1 subunit), which is also a component of the chromatin remodeling complex NURF and the chromatin assembly complex CAF-1. The association of Esc and E(z) with p55 and Rpd3 is conserved in mammals. Rpd3 is required for silencing mediated by a Polycomb response element (PRE) in vivo and E(z) and Rpd3 are bound to the Ubx PRE in vivo, suggesting that they act directly at the PRE. It is proposed that histone deacetylation by this complex is a prerequisite for establishment of stable long-term silencing by other continuously required PcG complexes (Tie, 2001).

To test whether the association of Esc and E(z) with p55/Caf1 and Rpd3 has been conserved in mammals, the human complex containing the Esc homolog (EED) was examined for the presence of Rpd3 and p55 homologs. Database searches reveal that Drosophila Rpd3 is most closely related to two human histone deacetylases, HDAC1 and HDAC2 (77% and 75% identical to Rpd3). Similarly, there are two closely related p55 homologs in mammals, RbAp48 and RbAp46 (91% and 86% identical to p55). RbAp48 and RbAp46 have also been found together in the SIN3 and Mi-2 deacetylase complexes, as have HDAC1 and HDAC2. A test was performed to see whether all four proteins are associated with the human EED complex. A GST-ESC fusion protein encoding full-length Esc can pull down full-length in vitro translated Esc and a GST-ESC1-60 fusion protein encoding just the N-terminal 60 residues of Esc is sufficient to pull down full-length in vitro translated Esc. Similarly, GST-EED1-81, which contains the corresponding N-terminal region of EED, binds directly to in vitro translated EED. In addition to FLAG-Esc, GST-ESC1-60 also pulls down p55 and Rpd3 from Drosophila embryo nuclear extract. This strongly suggests that GST-ESC1- 60 specifically pulls down the Esc complex. GST-EED1-81 pulls down HDAC1, HDAC2 and RbAp48 from HeLa cell nuclear extract. RbAp46 has also been detected. Thus, the association of ESC with p55 and Rpd3 is mirrored in the conserved association of mammalian EED with RbAp48, RbAp46 and HDAC1 and HDAC2. These results confirm the previously reported association of EED with HDAC1 and HDAC2 (Tie, 2001).

The presence of p55 in the ESC complex provides a direct molecular link to chromatin. The highly conserved mammalian p55 homologs, RbAp48 and RbAp46, have been shown to bind directly to histone H4 and possibly H2A, but not H2B or H3. The N- and C-terminal regions of RbAp48 that mediate binding to histone H4 are virtually identical to the corresponding regions of Drosophila p55, strongly suggesting that p55 has the same histone-binding specificity (Tie, 2001).

What, then, is the role of p55 in the Esc complex? It is unlikely that p55 is responsible for the selective recruitment or targeting of Esc and E(Z) to the ~100 specific chromosomal sites at which they co-localize. The histone-binding activity of p55 does not, by itself, suggest a mechanism for such specificity and p55 binds to many more sites on the polytene chromosomes than Esc and E(Z), presumably reflecting its distribution in other complexes, such as CAF1 and NURF. It seems more likely that p55 acts after the Esc complex is recruited and serves to direct the deacetylase activity of Rpd3 to local histone substrates. This is analogous to the role proposed for RbAp46 in the heterodimeric HAT1 complex. RbAp46 greatly stimulates the acetyltransferase activity of the non-histone-binding HAT1 catalytic subunit, presumably by tethering it to its substrate via its histone-binding activity. Similarly, although recombinant Rpd3 can deacetylate histone H4 in vitro, free Rpd3 does not bind to H4 when the two are co-expressed in vivo and is unlikely to be able to deacetylate nucleosomal histones. This suggests that p55 may play a similar essential role in the Esc complex by targeting Rpd3 to histone substrates for deacetylation (Tie, 2001).

The presence of Rpd3 in the Esc complex suggests that histone deacetylation is an intrinsic activity of the Esc complex and that Rpd3 is required for PRE-mediated silencing. The related mammalian EED complex has been shown to contain the Rpd3 homologs HDAC1 and HDAC2, and immunoprecipitates containing this complex can deacetylate a histone H4 tail-peptide in vitro. In yeast, Rpd3-dependent repression in vivo has been shown to be associated with deacetylation of histones H4 and H3. Which nucleosomes would be deacetylated by the Esc complex? Histone deacetylation by yeast Rpd3 appears to be highly localized, extending only one or two nucleosomes from a site to which it is recruited. Since components of the Esc complex are physically associated with the Ubx PRE in vivo, Esc-mediated deacetylation may be restricted to nucleosomes comprising and immediately adjacent to PREs. Nucleosomes outside the PRE might also be targeted if the PRE has long-range interactions with the promoter or if the Esc complex itself also binds to the promoter or other regions outside the Ubx PRE, a possibility that the data presented here do not rule out. Although an effect is observed of several Rpd3 mutations on silencing of a PRE-mini-white reporter, which is an extremely sensitive assay, PcG phenotypes have not been reported for Rpd3 mutants. A hypomorphic Rpd3 allele associated with the insertion of a P-element transposon in the noncoding 5' untranslated region has been analyzed in the most detail. Homozygous mutant embryos derived from germline clones of this allele do not exhibit PcG phenotypes, but have a pair-rule phenotype similar to that of ftz mutants. Abundant ubiquitously distributed Rpd3 RNA and protein of maternal origin are detectable in early (0-2 hour) wild-type embryos, but are reduced no more than fivefold in these Rpd3 mutant embryos derived from germline clones. By stage 9-10, the level of maternally derived Rpd3 RNA and protein is greatly diminished. Localized zygotic expression of Rpd3 becomes detectable in the brain and ventral nervous system of wild-type embryos, but is not detectable in these mutant embryos, suggesting that this Rpd3 allele may have a stronger effect on zygotic expression than maternal expression. If Rpd3 protein derived from maternally synthesized RNA is sufficient to promote development of a normal cuticular phenotype, then it remains possible these mutant embryos may contain sufficient maternally derived protein to do so and that germline clones of a true null Rpd3 allele would display PcG phenotypes. Alternatively, it is possible that the function of Rpd3 in the Esc complex is not absolutely essential for Esc-dependent silencing or is redundant, i.e. when eliminated, it can be compensated by another histone deacetylase, either one normally associated with the Esc complex or a related one that can associate with the complex in the absence of Rpd3. A number of other histone deacetylases have been identified in Drosophila and at least two are reported to be ubiquitously distributed in the early embryo (Tie, 2001).

However, unlike mammals, which have two very closely related Rpd3 orthologs (HDAC1 and HDAC2), both of which are associated with mouse EED, the Drosophila genome contains no equally closely related homolog of Rpd3. The next most closely related Drosophila HDAC is an unequivocal ortholog of mammalian HDAC3, which is a class I HDAC like Rpd3. Interestingly, mouse HDAC3 has been reported to interact with the mouse Esc homolog EED in a yeast two-hybrid assay, consistent with the possibility that Rpd3 function in the Esc complex might be at least partially redundant. Further genetic analysis of Rpd3 should help to clarify its role in the Esc complex (Tie, 2001).

The 600 kDa Esc complex is distinct from complexes containing PC and other PcG proteins. This suggests that the Esc complex and other PcG complexes are likely to have separate functions. Furthermore, in embryos lacking any functional Esc protein, some weak residual Pc-dependent silencing activity is still detected, also supporting separate, if interdependent, functions. Similar conclusions have been drawn for the homologous mammalian PcG complexes, which have been reported to be expressed in temporally distinct stages of B cell differentiation, further suggesting they have distinct functions. In Drosophila, derepression of homeotic genes is detected slightly earlier in Esc mutants than in other PcG mutants, raising the possibility that Esc complex function might be required earlier than other PcG complexes. However, unlike the apparent temporal separation of the homologous complexes during mammalian B cell development, both Esc- and PC-containing complexes are present together throughout most of embryogenesis, before Esc disappears, and E(z), like other PcG proteins, is required continuously throughout development. The phenotypic similarities between Esc, E(z) and other PcG mutants, the genetic interactions among them and their common association with PREs, suggests that their functions, however distinct at the biochemical level, are interdependent (Tie, 2001).

What role might Esc-mediated histone deacetylation play in PcG silencing? Given the critical early requirement for Esc, Esc-mediated deacetylation of PRE-associated nucleosomes might be an essential prerequisite for the initial binding of one or more components of PRC1 or other PcG complexes to PREs. A schematic model is presented for such a function of the Esc complex in which Esc complex-mediated deacetylation of PRE associated histones is a critical step in establishing stable long-term PcG silencing. Alternatively, the Esc complex may be required for events subsequent to the initial binding of other PcG proteins to a PRE, perhaps for their assembly into active silencing complexes or for interaction of PRE-bound PcG complexes with the promoter. Indeed, repression of a reporter gene by a tethered GAL4-Pc fusion protein remains dependent on endogenous Esc and E(z) as well as other PcG proteins. This indicates that, at least for PC, constitutive binding to DNA does not bypass the requirement for Esc and E(z). This also suggests that while the biochemical evidence reveals no stable direct association of the Esc complex with other PcG complexes, it remains possible that there is a transient or less stable association in vivo that is essential for establishing PcG silencing (Tie, 2001).

The association of mammalian EED with the two closely related HDACs and two histone-binding proteins could reflect the existence of two separate EED complexes or some different functionality of the EED complex compared with the Esc complex. Consistent with this latter possibility, EED has recently been shown to be required after embryogenesis for aspects of adult hematopoietic development. Interestingly, analysis of the complete Drosophila genome sequence using the BLASTP and TBLASTN algorithms reveals that p55 has no other closely related Drosophila homologs, strongly suggesting that it is the functional counterpart of both RbAp48 and RbAp46 in Drosophila. Likewise, Rpd3 is the only Drosophila counterpart of mammalian HDAC1 and HDAC2. Given the remarkably high degree of similarity between RbAp48 and RbAp46 and HDAC1 and HDAC2, it is not yet clear whether each of these proteins has a distinct or redundant role in the EED complex. Perhaps this situation reflects a greater degree of functional specialization or versatility within the mammalian EED complexes. Since HDAC1 and HDAC2 have also been found together with RbAp48 and RbAp46 in other co-repressor complexes, it is also possible that the EED and Esc complexes represent specialized relatives of these complexes, perhaps more dedicated to a specific subset of genes (Tie, 2001).

The Wingless signaling pathway directs many developmental processes in Drosophila by regulating the expression of specific downstream target genes. The product of the trithorax group gene osa is required to repress such genes in the absence of the Wingless signal. The Wingless-regulated genes nubbin, Distal-less, and decapentaplegic and a minimal enhancer from the Ultrabithorax gene are misexpressed in osa mutants and repressed by ectopic Osa. Osa-mediated repression occurs downstream of the up-regulation of Armadillo but is sensitive both to the relative levels of activating Armadillo/Pangolin and repressing Groucho/Pangolin complexes that are present, and to the responsiveness of the promoter to Wingless. Osa functions as a component of the Brahma chromatin-remodeling complex; other components of this complex are likewise required to repress Wingless target genes. These results suggest that altering the conformation of chromatin is an important mechanism by which Wingless signaling activates gene expression (Collins, 2000).

In addition to transducing the wg signal in a complex with Arm, Pan is also required for the active repression of Wg target genes in the absence of the Wg signal. This repression requires the association of Pan with the corepressor Groucho (Gro). Gro functionally interacts with the histone deacetylase Rpd3, and this interaction is important for at least some of the repressive activity of Gro. Thus, both Osa-containing Brm complexes and Pan/Gro/Rpd3 complexes repress the expression of Wg target genes and probably mediate this repression by altering the local chromatin architecture at the promoters of these genes. Consistent with this, reduction of gro or rpd3 dosage reduces the ability of Osa to repress nub. The loss of nub expression caused by expression of UAS-Osa with ap-Gal4 is significantly rescued in wing discs homozygous for a hypomorphic allele of rpd3. Also, larvae transheterozygous for osaeld308 and groE48 often ectopically express nub in the wing disc, and 40% of transheterozygous adults have notum-to-wing transformations. These phenotypes are not seen when osa or gro single mutants are crossed to wild-type flies (Collins, 2000).

Interestingly, Gro has been shown to interact with the N-terminal tail of histone H3 and with the histone deacetylase Rpd3, and it has therefore been proposed that Gro mediates repression by altering chromatin structure. Consistent with this, a strong genetic interaction exists between osa and gro that suggests that their activities in repressing Wg target genes are closely related. Although it has not previously been reported that Rpd3 functions in the repression of wg target genes, reducing the function of rpd3 can partly rescue the loss of nub expression caused by the overexpression of Osa. Rpd3 is therefore important for the repression of Wg target genes; testing whether it is essential awaits the isolation of null alleles (Collins, 2000).

The loss of either osa or gro leads to ectopic expression of Wg target genes; thus, the activity of one is not sufficient to repress the expression of these genes without the activity of the other. Osa and Gro may, therefore, be mediating the same repressive event rather than acting in parallel. Interestingly, human SWI/SNF forms a repressor complex with Rb and the histone deacetylase HDAC. This complex interacts with the cyclin E promoter through the binding of Rb to E2F-1 and represses E2F-1 activation of cyclin E expression. This suggests the intriguing possibility that Osa and the Brm complex function in a larger repressor complex containing Gro and Rpd3 and that this complex is recruited to Wg target genes though the binding of Gro to Pan. However, Gro acts as a corepressor for a large number of transcription factors, and Osa cannot be required for all repression mediated by Gro because loss of osa does not result in neurogenic phenotypes like those caused by the loss of gro. Further research is needed to determine if Gro and/or Rpd3 can directly interact with components of the Brm complex and, if so, what determines the specificity of this interaction (Collins, 2000 and references therein).

A model for the regulation of gene expression by components of the Wg pathway is presented. The chromatin remodeling activity of the OsaBrm complex is required to maintain the chromatin at the promoters of wg-responsive genes in a repressive conformation. This would prohibit the association of other transcription factors with their binding sites and prevent the recruitment of components of the basal transcription machinery. Osa/Brm complexes may be recruited to Wg-responsive genes through an association with Pan/Gro/Rpd3 complexes. In response to the Wg signal, Arm is stabilized and accumulates in the cytosol. This accumulation of cytosolic Arm permits Arm to translocate to the nucleus and displace Gro from Pan and, in so doing, relieve the repression mediated by Gro, Rpd3, and Osa/Brm complexes. Arm may also promote a more open chromatin conformation by recruiting the HAT activity of dCBP, thus permitting the association of other transcription factors with their binding sites. Also, the stimulation of the DNA-bending activity of Pan by Arm may bring distantly spaced transcription factors into juxtaposition to promote the activation of gene expression. In the absence of osa, the chromatin is maintained in a more open and less repressive conformation. This would permit other transcription factors to interact with their binding sites at lower concentrations than would otherwise be possible. Under these conditions, the low levels of Arm that are always present in the cell may be sufficient to promote the activation of gene expression without the Wg signal (Collins, 2000).

Bicoid directs anterior development in Drosophila embryos by activating different genes along the anterior-posterior axis. However, its activity is down-regulated at the anterior tip of the embryo, in a process known as retraction. Retraction is under the control of the terminal polarity system, and results in localized repression of Bicoid target genes. A Drosophila homolog of human SAP18 (Sin3A-associated polypeptide p18), a member of the Sin3A/Rpd3 histone deacetylase complex (HDAC), is described. Termed Bicoid interacting protein 1 (Bip1), the SAP18 homolog interacts with Bicoid in yeast and in vitro, and is expressed early in development coincident with Bicoid. In tissue culture cells, Bip1 inhibits the ability of Bicoid to activate reporter genes. These results suggest a model in which Bip1 interacts with Bicoid to silence expression of Bicoid target genes in the anterior tip of the embryo (Zhu, 2001).

A cDNA encoding Bin1 was identified using a custom two-hybrid selection in which Bicoid was bound to DNA via its homeodomain. The 5' end of the bin1 cDNA was cloned by RACE and a full-length cDNA sequence was assembled. The bin1 cDNA encodes a 150-amino-acid protein with a predicted molecular weight of 17.3 kDa. The protein is 58% identical to the human and murine SAP18 proteins and 42% identical to a C. elegans ORF. The Drosophila genome sequence does not predict any other homologs. A search of the Berkeley Drosophila Genome Project database revealed an EP insertion line EP(3)3462 in which an EP-transposon is inserted 259 bp upstream of the bin1 start codon, and 151 bp upstream of the transcription initiation site. This insertion is within the 5' UTR of nebula, an ORF oriented opposite to that of bin1 (Zhu, 2001).

LexA-Bicoid fusion proteins were used to map the regions within Bicoid that are important for the Bin1-Bicoid interaction. In these experiments, Bin1 was fused to the B42 activation domain. Results from these assays show that interaction with Bin1 does not require the Bicoid acidic activation domain (AD), or the polyglutamine (Q) or polyalanine (A) domains. The homeodomain is not sufficient for interaction, but seems to be required along with flanking regions, each of which contributes modestly to the interaction. Thus, the interaction requires two distinct regions of Bicoid, aa 1-95 and aa 163-246. To test whether Bin1 interacts directly with Bicoid in vitro, the full-length Bin1 was expressed as a GST fusion protein in Escherichia coli. GST-Bin1 was attached to glutathione beads and used in pull-down experiments with 35 S-methionine-labeled, full-length Bicoid generated by in vitro translation. GST-Bin1 interacts with Bicoid in this system. Thus, Bin1 interacts directly with Bicoid in vitro (Zhu, 2001).

If Bin1 is required for Bicoid function, then its protein expression pattern should overlap with that of Bicoid temporally and spatially. Bicoid is translated from maternally deposited mRNA shortly after egg laying. After 3 h of development, the protein level begins to diminish, and after 4 h, Bicoid is undetectable. To determine when the Bin1 gene is expressed, Northern analysis was carried out using mRNA isolated from unfertilized eggs and from 0- to 2-h, 2- to 4-h, and 4- to 24-h developing embryos. A Bin1 mRNA of about 500 nt is detected in unfertilized eggs and in early embryos. The mRNA levels peak around the cellularization to early gastrulation stages (2-4 h). These results indicate that Bin1 is transcribed both maternally and zygotically, and the Bin1 mRNA is present at the time that Bicoid is present (Zhu, 2001).

To determine the spatial distribution of Bin1 mRNA within the early embryo, whole-mount in situ hybridization was carried out using anti-sense. In contrast to the highly localized BCD mRNA, Bin1 mRNAs are distributed throughout the early embryo and in the unfertilized egg. Thus, the expression pattern of Bin1 overlaps spatially with that of Bicoid protein, which is detectable over the anterior two-thirds of the embryo. The temporal and spatial expression pattern of Bin1 mRNA suggests that Bin1 protein is present throughout the embryo, although proof of protein localization will require anti-Bin1 immunostaining (Zhu, 2001).

Based on the role of human SAP18 in transcription repression by HDAC complexes, tests were performed to see whether over-expression of Bin1 inhibits Bicoid-dependent transcription in Drosophila S2 cells. In this assay, plasmids expressing Bicoid and Bin1 were co-transfected along with a Bicoid binding site-CAT reporter construct. The results indicate a dose-sensitive inhibition of Bicoid-dependent transcription by Bin1. The effect is greater at lower Bicoid concentrations, suggesting that the ratio of Bin1 to Bicoid is important for the effect (Zhu, 2001).

The Sin3A Rpd3 histone deacetylase complex is conserved in Drosophila. Both Sin3 and an Rpd3 homolog (HDAC1) have been identified in Drosophila and are required for embryogenesis. By analogy with mammalian systems, Bin1 is likely to function in co-repression as part of a Drosophila Sin3/HDAC1 complex. It is proposed that interaction with Bin1 recruits the HDAC complex to DNA, converting Bicoid from an activator into a repressor, or at least neutralizing its ability to stimulate expression of its target genes. In this model, interaction of Bicoid with Bin1 would be stimulated by the action of the terminal polarity-system kinases. For example, phosphorylation of either Bicoid or Bin1 might trigger a conformational change that strengthens their interaction. The Bin1-Bicoid complex would then recruit Sin3/HDAC1 to down-regulate Bicoid's transcription activity beginning at late cellularization stages. In this way, Bicoid-dependent gene expression could be down-regulated exclusively at the anterior tip of the embryo, where the Bicoid concentration is high and the terminal system is active, resulting in the observed retraction (Zhu, 2001).

Human SAP18 has been found to interact with a cAMP-GEF protein. cAMP-GEF proteins function in MAPK signal transduction pathways to activate the GTPases Rap1 and Ras, which in turn leads to activation of Raf kinases (MAPKKK). Members of this pathway are present in Drosophila, including two putative proteins similar to human cAMP-GEFs, CG3427, located at 42C4-5, and CG9494, located at 26C3, as well as dRap1 (Roughened) and Raf kinase (Pole hole protein), which is the kinase downstream of the Torso receptor in the terminal system. By analogy with human SAP18, Drosophila Bin1 might interact with a cAMP-GEF, and thereby be linked directly to the terminal system MAP-kinase pathway. For example, interaction of Bin1 with cAMP-GEF might result in phosphorylation of Bin1 by Raf upon stimulation of the Torso receptor tyrosine kinase. This, in turn, might stimulate Bin1 to interact with Bicoid and trigger recruitment of the HDAC complex to Bicoid-regulated promoters (Zhu, 2001).

Bin1 has also been identified as a protein that interacts with Enhancer of Zeste, E(z), (L. Ding and R. Jones, personal communication to Zhu, 2001), which is a Polycomb group protein important for maintenance of repression of homeotic genes, and with GAGA factor, the trithorax-like gene product required for activation of homeotic genes. These and other examples suggest that control of expression of homeobox genes by histone deacetylases is important for embryogenesis. Histone deacetylases may also alter homeodomain protein activity by direct interaction. Mobilization of the EP-transposon insertion near Bin1 should make it possible to generate mutant alleles, which will be important for studying the role of Bin1 in development (Zhu, 2001).

Modification of histones can have a dramatic impact on chromatin structure and function. Acetylation of lysines within the N-terminal tail of the histone octamer marks transcriptionally active regions of the genome whereas deacetylation seems to play a role in transcriptional silencing. The methylation of the histone tails has also been shown to be important for transcriptional regulation and chromosome structure. It is shown by immunoaffinity purification that two activities important for chromatin-mediated gene silencing, the histone methyltransferase SU(VAR)3-9 and the histone deacetylase HDAC1 (Rpd3), associate in vivo. The two activities cooperate to methylate pre-acetylated histones. Both enzymes are modifiers of position effect variegation and interact genetically in flies. A model is suggested in which the concerted histone deacetylation and methylation by a SU(VAR)3-9/HDAC1-containing complex leads to a permanent silencing of transcription in particular areas of the genome (Czermin, 2001).

This study reports the association of the HIM SU(VAR)3-9 and the deacetylase HDAC1 within Drosophila embryo extracts. This interaction plays an essential role in SU(VAR)3-9's ability to methylate acetylated histone tails, which is probably important during the 'invasion' of euchromatic regions of the genome by heterochromatin, an process observed when a gene is placed closed to heterochromatin (Czermin, 2001).

In addition to their biochemical interaction, a strong genetic interaction is seen between Su(var)3-9 and HDAC1. A point mutation within the HDAC1 gene, which has a strong Su(var) phenotype, very efficiently dominates the 'triplo-enhancer effect' usually seen in flies carrying additional copies of Su(var)3-9. The effect of different HDAC1 mutations on PEV has been described as enhancing, suppressing or neutral depending on the experimental setup. These controversial results are probably due to the different nature of the mutants used in the different laboratories. A functional redundancy of HDAC1 with other known HDACs could, for example, obscure the contribution of HDAC1 in a hypomorphic strain. It is postulated that in the HDAC1326 strain a mutant protein is made that is able to interact with SU(VAR)3-9 but fails to deacetylate the histone substrate and therefore acts as a dominant-negative suppressor (Czermin, 2001).

Based on these findings a model is proposed in which deacetylation precedes methylation of lysine 9 in the N-terminus of histone H3. Methylated H3 would then serve as a docking site for HP1, which in turn could help in assembling a specialized higher order chromatin structure. Since the turnover of methylated histones is slow in comparison to acetylation, it is suggested that histone methylation serves as a permanent epigenetic mark that freezes a particular chromatin conformation (Czermin, 2001).

The concerted action of deacetylation and methylation of lysine 9 in histone H3 could allow the generation of a permanently repressed chromatin structure within otherwise more accessible, acetylated chromatin of the early embryo. The interaction between SU(VAR)3-9 and HDAC1 is probably especially important at boundaries between eu- and hetero-chromatin and under circumstances when heterochromatin has been shown to 'invade' euchromatic regions like the white gene in the In(1)wm4h strain (Czermin, 2001).

The existence of a complex containing a HDAC and a HIM provides a molecular mechanism for the close connection between histone deacetylation and histone methylation. This link between deacetylation and methylation is also evident in S. pombe, where inhibition of deacetylases by TSA as well as mutations of the Su(var)3-9 ortholog clr4 leads to defects in centromere function. It will be very interesting to see whether the coupling of histone deacetylation and histone methylation is a mechanism commonly used to stably repress gene expression not only around the centromere but also in euchromatic regions of the genome (Czermin, 2001).

Dosage compensation ensures equal expression of X-linked genes in males and females. In Drosophila, equalization is achieved by hypertranscription of the male X chromosome. This process requires an RNA/protein containing dosage compensation complex (DCC). RNA interference of individual DCC components has been used to define the order of complex assembly in Schneider cells. Interaction of MOF with MSL-3 leads to specific acetylation of MSL-3 at a single lysine residue adjacent to one of its chromodomains. Localization of MSL-3 to the X chromosome is RNA dependent and acetylation sensitive. The acetylation status of MSL-3 determines its interaction with roX2 RNA. Furthermore, RPD3 interacts with MSL-3 and MSL-3 can be deacetylated by the RPD3 complex. It is proposed that regulated acetylation of MSL-3 may provide a mechanistic explanation for spreading of the dosage compensation complex along the male X chromosome (Buscaino, 2003).

The Polycomb Group proteins are required for stable long-term maintenance of transcriptionally repressed states. Two distinct Polycomb Group complexes have been identified, a 2-MDa PRC1 complex and a 600-kDa complex containing the ESC and E(Z) proteins together with the histone deacetylase RPD3 and the histone-binding protein p55. There are at least two embryonic ESC/E(Z) complexes that undergo dynamic changes during development and a third larval E(Z) complex that forms after disappearance of ESC. A larger embryonic ESC complex has been identified containing RPD3 and p55, along with E(Z), that is present only until mid-embryogenesis, while the previously identified 600-kDa ESC/E(Z) complex persists until the end of embryogenesis. Constitutive overexpression of ESC does not promote abnormal persistence of the larger or smaller embryonic complexes and does not delay a dissociation of E(Z) from the smaller ESC complex or delay appearance of the larval E(Z) complex, indicating that these changes are developmentally programmed and not regulated by the temporal profile of ESC itself. Genetic removal of ESC prevents appearance of E(Z) in the smaller embryonic complex, but does not appear to affect formation of the large embryonic ESC complex or the PRC1 complex. The ESC complex is already bound to chromosomes in preblastoderm embryos and genetic evidence is presented that ESC is required during this very early period (Furuyama, 2003).

Polycomb group (PcG) proteins are required to maintain stable repression of the homeotic genes and others throughout development. The PcG proteins ESC and E(Z) are present in a prominent 600-kDa complex as well as in a number of higher-molecular-mass complexes. A 1-MDa ESC/E(Z) complex has been identified and characterized that is distinguished from the 600-kDa complex by the presence of the PcG protein Polycomblike (PCL) and the histone deacetylase RPD3. In addition, the 1-MDa complex shares with the 600-kDa complex the histone binding protein p55 and the PcG protein Su(z)12. Coimmunoprecipitation assays performed on embryo extracts and gel filtration column fractions indicate that, during embryogenesis E(Z), SU(Z)12, and p55 are present in all ESC complexes, while PCL and RPD3 are associated with ESC, E(Z), SU(Z)12, and p55 only in the 1-MDa complex. Glutathione transferase pulldown assays demonstrate that RPD3 binds directly to PCL via the conserved PHD fingers of PCL and the N terminus of RPD3. PCL and E(Z) colocalize virtually completely on polytene chromosomes and are associated with a subset of RPD3 sites. As shown for E(Z) and RPD3, PCL and SU(Z)12 are also recruited to the insertion site of a minimal Ubx Polycomb response element transgene in vivo. Consistent with these biochemical and cytological results, Rpd3 mutations enhance the phenotypes of Pcl mutants, further indicating that RPD3 is required for PcG silencing and possibly for PCL function. These results suggest that there may be multiple ESC/E(Z) complexes with distinct functions in vivo (Tie, 2003).

SIR2 is required for polycomb silencing and is associated with an E(Z) histone methyltransferase complex: RPD3 and SIR2 appear to have similarly broad substrate specificities

SIR2 was originally identified in S. cerevisiae for its role in epigenetic silencing through the creation of specialized chromatin domains. It is the most evolutionarily conserved protein deacetylase, with homologs in all kingdoms. SIR2 orthologs in multicellular eukaryotes have been implicated in lifespan determination and regulation of the activities of transcription factors and other proteins. Although SIR2 has not been widely implicated in epigenetic silencing outside yeast, Drosophila SIR2 mutations were recently shown to perturb position effect variegation, suggesting that the role of SIR2 in epigenetic silencing may not be restricted to yeast. Evidence is presented that Drosophila SIR2 is also involved in epigenetic silencing by the Polycomb group proteins. Sir2 mutations enhance the phenotypes of Polycomb group mutants and disrupt silencing of a mini-white reporter transgene mediated by a Polycomb response element. Consistent with this, SIR2 is physically associated with components of an E(Z) histone methyltransferase complex. SIR2 binds to many euchromatic sites on polytene chromosomes and colocalizes with E(Z) at most sites. It is concluded that SIR2 is involved in the epigenetic inheritance of silent chromatin states mediated by the Drosophila Polycomb group proteins and is physically associated with a complex containing the E(Z) histone methyltransferase (Furuyama, 2004).

The ability of Sir2 mutations to enhance PcG mutant phenotypes and perturb PRE-mediated silencing indicates that SIR2 plays a role in Polycomb silencing. However, like their yeast and C. elegans counterparts, Drosophila Sir2 mutants are viable under standard laboratory conditions, and they do not exhibit obvious PcG phenotypes. Mutations in several other genes that play a role in Polycomb silencing enhance the phenotypes of PcG mutants but do not themselves exhibit Polycomb phenotypes. These include E(Pc), Su(z)2, and the histone deacetylase Rpd3/HDAC1. Uncovering the role of Sir2 in Polycomb silencing required sensitive genetic assays. This could be due to functional redundancy; four other Drosophila genes encode conserved SIR2 paralogs, corresponding respectively to the mammalian SIRT2 (similar to yeast HST2), SIRT4, and the closely related SIRT6 and SIRT7. Although these SIR2 paralogs are likely to have physiological roles distinct from that of SIR2, in the absence of SIR2, one or more of them might at least partially compensate for the function of SIR2 in Polycomb silencing. In S. cerevisiae, the Sir2p paralog Hst1p, which normally functions as a gene-specific repressor, can rescue the silencing defects of Sir2 mutants when it is overexpressed or targeted to the mating-type locus. Another yeast Sir2p paralog, Hst2p, although not required for silencing, improves rDNA silencing when it is overexpressed almost as efficiently as overexpressed Sir2p itself, even though Hst2p remains exclusively cytoplasmic (Furuyama, 2004).

It is also possible that the another deacetylase, e.g., RPD3/HDAC1, which is also present in E(Z) complexes, may be able to at least partially substitute for the SIR2 function in these complexes. Indeed, Drosophila RPD3 and SIR2 appear to have similarly broad substrate specificities, at least in vitro. Alternatively, SIR2 may be more critically required for Polycomb silencing under particular environmental or nutritional conditions that differ from standard laboratory conditions. It was originally suggested that the NAD+ dependence of SIR2 deacetylase activity (or its inhibition by nicotinamide could serve to link SIR2 activity to environmental or nutritional conditions. Indeed, the yeast Sir2p paralog Hst1p has been shown to regulate genes involved in de novo NAD+ biosynthesis by functioning as a direct sensor of cellular NAD+ levels. Various stresses and nutritional conditions appear to regulate the expression or activity of mammalian SIRT1 as well as the association of SIRT1 with its protein substrates. By analogy, the requirement for SIR2 or its activity in Polycomb silencing may be modulated by environmental or nutritional conditions, perhaps so that the fidelity of Polycomb silencing and its epigenetic inheritance is maintained under unfavorable or stressful culture conditions during larval life (Furuyama, 2004).

The physical association of Drosophila SIR2 with E(Z), RPD3, and p55 is the first evidence that SIR2 is associated with proteins known to be involved in epigenetic silencing in multicellular eukaryotes. The association of SIR2 with E(Z) was only detected in post-embryonic extracts, despite the presence of SIR2 and E(Z) in embryos. This suggests that E(Z) complex(es) differ in their composition and possibly their physiological functions at different developmental stages. It also suggests that the role of Drosophila SIR2 in Polycomb silencing may be restricted to post-embryonic stages. The transition from embryonic to larval period upon hatching from the egg marks the onset of active feeding and concomitant exposure to fluctuations in nutrient sources and other environmental variables from which embryonic development may be relatively more insulated. The differential association of SIR2 with E(Z) complexes during the larval stages may serve to increase the fidelity of PcG silencing under stressful conditions, a function that might not be expected to be uncovered without sensitive genetic assays or knowledge of the conditions that would render SIR2 more critical for maintenance of PcG silencing (Furuyama, 2004).

The high degree of protein sequence conservation among SIR2 orthologs from divergent species suggests that their biological functions, including their roles in epigenetic silencing, are also likely to be generally conserved. The conserved NAD+-dependent histone deacetylase activity and chromosomal localization of the Drosophila SIR2 protein is further consistent with this. However, although other components of E(Z) complexes, including RPD3 and the histone binding protein p55, have been highly conserved among all eukaryotes during evolution, an unequivocal E(Z) ortholog is not identifiable in S. cerevisiae or S. pombe, despite the presence of E(Z) orthologs in plants and animals and the presence of SET domain-containing histone methyltransferases in yeast. Conversely, Drosophila and mammals contain no identifiable homologs of S. cerevisiae SIR3 and SIR4, two key proteins that collaborate with SIR2 in the creation of silent chromatin domains at the mating-type loci and telomeres. This suggests that the mechanisms underlying SIR2-dependent silencing in yeast and multicellular eukaryotes, although broadly similar, are likely to differ in additional mechanistic details. On the other hand, the conservation of PcG proteins between Drosophila and mammals suggests that the association of SIR2 with E(Z) complex(es) is also likely to be conserved in mammals (Furuyama, 2004).

At present it is not evident why both NAD+-dependent (SIR2) and NAD+-independent (RPD3) HDACs are associated with E(Z) in larval extracts, but this arrangement is not unique. The S. cerevisiae SET domain protein SET3 is also found in a complex that contains two HDACs, including Hos2p, an RPD3-related class I HDAC, and Hst1p, which is closely related to yeast and Drosophila SIR2. Drosophila Hairy also interacts with both Rpd3 and Sir2. Perhaps in such situations each HDAC functions in different contexts or deacetylates different substrates. Drosophila RPD3 is found in a complex with the SET domain protein SU(VAR)3-9 and appears to be required for SU(VAR)3-9 histone methyltransferase function in vivo. It remains to be determined whether SIR2 is required for or modulates the histone methyltransferase function of E(Z) in vivo. The developmentally regulated association of SIR2 with E(Z) raises the interesting possibility that SIR2 may alter the activity or substrate specificity of E(Z). Although the chromosomal association of Drosophila SIR2 suggests it could target histones, the identification of multiple transcription factors and other proteins as substrates of mammalian SIRT1 suggests that the SIR2 associated with E(Z) may also have other non-histone substrates that regulate transcriptional silencing, perhaps including proteins in the E(Z) complex itself (Furuyama, 2004).

Genetic identification of a network of factors that functionally interact with the nucleosome remodeling ATPase ISWI

Nucleosome remodeling and covalent modifications of histones play fundamental roles in chromatin structure and function. However, much remains to be learned about how the action of ATP-dependent chromatin remodeling factors and histone-modifying enzymes is coordinated to modulate chromatin organization and transcription. The evolutionarily conserved ATP-dependent chromatin-remodeling factor ISWI plays essential roles in chromosome organization, DNA replication, and transcription regulation. To gain insight into regulation and mechanism of action of ISWI, an unbiased genetic screen was conducted to identify factors with which it interacts in vivo. It was found that ISWI interacts with a network of factors that escaped detection in previous biochemical analyses, including the Sin3A gene. The Sin3A protein and the histone deacetylase Rpd3 are part of a conserved histone deacetylase complex involved in transcriptional repression. ISWI and the Sin3A/Rpd3 complex co-localize at specific chromosome domains. Loss of ISWI activity causes a reduction in the binding of the Sin3A/Rpd3 complex to chromatin. Biochemical analysis showed that the ISWI physically interacts with the histone deacetylase activity of the Sin3A/Rpd3 complex. Consistent with these findings, the acetylation of histone H4 is altered when ISWI activity is perturbed in vivo. These findings suggest that ISWI associates with the Sin3A/Rpd3 complex to support its function in vivo (Burgio, 2008).

This study involved an unbiased genetic screen for regulators of ISWI function in Drosophila. A screen produced the first genetic interaction map for the ATP-dependent chromatin remodeler ISWI in higher eukaryotes. Misexpression of dominant-negative alleles of chromatin-remodeling enzymes in the eye-antennal disc can compromise eye development, often causing roughness and/or reduced eye size. A single K159R amino acid substitution in Drosophila ISWI (ISWIK159R) eliminates its ATPase activity, without affecting the ability of the mutant protein to be incorporated into native complexes. The expression of a UAS-ISWIK159R transgene in the developing eye, using an ey-GAL4 driver, has strong effects on cell viability and chromosome organization and results in flies with rough and reduced eyes. It was reasoned that mutations that enhance or suppress phenotypes resulting from the expression of ISWIK159R can be used to define genes involved in the same biological process as ISWI. This approach has been successfully used to conduct a genetic screen for modifiers of phenotypes caused by loss of the chromatin-remodeling factor brm. It was found that ISWI genetically interacts with a network of cellular and nuclear factors that escaped previous biochemical analyses, indicating the participation of ISWI in variety of biological processes (Burgio, 2008).

Interestingly, unbiased genetic screens aimed at the identification of factors involved in the regulation of vulval cell fates in C.elegans and sensory neuron morphogenesis in Drosophila have identified ISWI and some of the ISWIK159R enhancers as key regulators of these biological processes (Burgio, 2008).

GO analysis indicates 'neuron differentiation' and 'cell cycle regulation' as overrepresented categories within the combined strong and medium ISWIK159R enhancers. With hindsight this result is not surprising considering that the screen targeted the eye, an organ whose development is tightly linked to nervous system differentiation and the spatial as well as temporal control of cell division. Therefore, it is likely that some of the ISWIK159R enhancers isolated could work in concert with ISWI to support the differentiation and development of the adult fly eye (Burgio, 2008).

One of the goals of this screen was to isolate factors encoding enzymatic activities that could play a role in the regulation of ISWI in vivo by modifying ISWI or chromatin components with which ISWI interacts. As expected, the screen led to the isolation of a group of genes that includes kinases (e.g. trbl, grp, snf4ag), ATPases (e.g., pont), proteins associated with deacetylases (Sin3A), methyl binding factor (mbf1) and enzymes regulating the metabolism of poly-ADP-ribose (Parp). The variety of chromatin components found in the screen indicates that it is likely that a functional cross talk exists between ISWI and other chromatin-remodeling and modifying activities working in the nucleus (Burgio, 2008).

It was found that Drosophila ISWI genetically interacts with Sin3A and with its associated histone deacetylase subunit Rpd3. This genetic interaction may reflect a physical interaction between ISWI, Sin3A and Rpd3, since the three proteins co-localizes at many, though not all, sites on polytene chomosome. Although the resolution of polytene chromosome staining is limited, these biochemical data are consistent with a physical interaction between ISWI and Sin3A/Rpd3 in embryo and larval stages. Previous biochemical studies in flies have not detected the presence of Sin3A and Rpd3 proteins as integral subunits of Drosophila ISWI complexes. Therefore, the physical interaction that found between ISWI and Sin3A/Rpd3 could be transient or indirect (Burgio, 2008).

The nucleosome stimulated ATPase activity of ISWI co-purifies with a histone deacetylase activity associated with the Sin3A/Rpd3 complex in larvae. Interestingly changes in the levels of ISWI alter the binding of Sin3A/Rpd3 to polytene chromosomes and are correlated with changes in global histone H3 and H4 acetylation. Because ISWI function can be antagonized by the site-specific acetylation of histones, it is possible that the Sin3A/Rpd3 complex positively regulates ISWI activity in vivo. Therefore, ISWI and the Sin3A/Rpd3 complex may facilitate each other's function, forming a positive feedback system for chromatin regulation (Burgio, 2008).

Genetic and biochemical studies in yeast have shown that the nucleosome spacing activity of the Isw2 complex can repress transcription in a parallel pathway with the yeast Sin3/Rpd3 histone deacetylase complex. Although, the functional organization of DNA into chromatin is conserved among eukaryotes, mutations in the two yeast counterparts of ISWI, Isw1 and Isw2, do not show any severe phenotype. Conversely, ISWI is a unique and essential gene in Drosophila highlighting a possible divergent role for ISWI in flies and a distinct mechanism of interaction with the Sin3A/Rpd3 complex in higher eukaryotes. Indeed, interactions between SNF2L, a mouse ISWI homolog, and the Sin3A/Rpd3 complex have been proposed to play a role in repressing ribosomal gene transcription in mammals. Furthermore, studies of the thymocyte-enriched chromatin factor SAT1B indicate that its ability to regulates gene expression and organize chromatin folding into loop domains at the IL-2Ra locus is dependent on the catalytic activities of Sin3A/HDAC1 (the mammalian Rpd3) and the ISWI homolog SNF2H protein (Burgio, 2008).

ISWI can also be a target of site-specific acetylation by the GCN5 histone acetyltransferase. Therefore, the functional association found between ISWI and Sin3A/Rpd3 could help regulate the acetylation state of ISWI and modulate its activity. Interestingly, it has been recently reported that ISWI genetically interact with the histone acetyltransferase GCN5. gcn5 mutations cause chromosome condensation defects very similar to the one observed in ISWI and E(bx) mutants, as well as global loss of histone H4 acetylation on lysine 12. A decrease in ISWI activity as a consequence of loss of GCN5-dependent acetylation could in theory account for the observed defects. An alternative possibility is that specific histone acetylations differently regulate ISWI function. Therefore, further studies will be necessary to clarify the roles of Sin3A, Rpd3 and other histone modifying enzymes in the regulation of ISWI-containing complexes function in vivo (Burgio, 2008).

SAP18 promotes Krüppel-dependent transcriptional repression by enhancer-specific histone deacetylation

Body pattern formation during early embryogenesis of Drosophila relies on a zygotic cascade of spatially restricted transcription factor activities. The gap gene Krüppel ranks at the top level of this cascade. It encodes a C2H2 zinc finger protein that interacts directly with cis-acting stripe enhancer elements of pair rule genes, such as even skipped and hairy, at the next level of the gene hierarchy. Krüppel mediates their transcriptional repression by direct association with the corepressor Drosophila C terminus-binding protein (dCtBP). However, for some Krüppel target genes, deletion of the dCtBP-binding sites does not abolish repression, implying a dCtBP-independent mode of repression. This study identified Krüppel-binding proteins by mass spectrometry and found that SAP18 can both associate with Krüppel and support Krüppel-dependent repression. Genetic interaction studies combined with pharmacological and biochemical approaches suggest a site-specific mechanism of Krüppel-dependent gene silencing. The results suggest that Krüppel tethers the SAP18 bound histone deacetylase complex 1 at distinct enhancer elements, which causes repression via histone H3 deacetylation (Matyash, 2009).

This study provides evidence that Kr exerts transcriptional repression not only by association with the corepressor dCtBP but also by site-specific deacetylation of histones, a mechanism that involves an interaction between Kr and dSAP18. The dual mode of Kr-dependent repression might explain earlier studies showing that Kr represses eve stripe 2 expression, but not h stripe 7 expression, in a dCtBP-dependent manner. Consistent with these observations, a mutant Kr protein that lacks dCtBP-binding sites still associates with dSAP18, which in turn interacts with the Sin3A-HDAC1 repressor complex (Drosophila HDAC1 is Rpd3). dSAP18 was also shown to bind the homeodomain transcription factor Bicoid, causing repression of anterior gap genes such as hunchback in the late Drosophila blastoderm embryo. SAP18-dependent repression involves histone deacetylase both in flies and mammals, and SAP18 that links the HDAC1 complex with sequence-specific transcriptional repressors bound to chromatin is also found in plants. These results are consistent with such a SAP18-dependent mode of Kr-dependent repression that provides target gene-specific repression. Because both dCtBP and SAP18 are uniformly distributed in the embryo, it will be important to learn how the eve stripe 2 and the h stripe 7 enhancer distinguish between the dCtBP- or SAP18-dependent modes of repression. One possibility is that differential packing of the enhancer DNA into nucleosomes might account for the difference in susceptibility to the SAP18/HDAC1-mediated repression (Matyash, 2009).

dSAP18 binds to three distinct regions of Kr, including the 42-amino acid-long repressor region, which is conserved in Kr homologs of all Drosophila species. However, as observed for dCtBP, dSAP18 alone cannot account for Kr-dependent repression of h7-lacZ, because prolonged expression of Kr is able to overcome the lack of dSAP18 activity as observed for the h7 element in dSAP18 mutants. Therefore, it is likely that the full spectrum of Kr-dependent repression is mediated redundantly, employing at least two different corepressors that involve different modes of repression (Matyash, 2009). In vitro, dSAP18 binds to the sequence motif 344RRRHHL349 of Kr and to a similar motif (143RRRRHKI149) of Bicoid; the latter is consistent with the results reported by Zhu (2001). In both proteins, the dSAP18-binding sites are localized in the C-terminal portion of their DNA-binding domains. Thus, when acting from weak binding sites in vivo, transcription factors might be able to form strong complexes with dSAP18. In fact, Bicoid-dependent repression of hunchback, which depends on both SAP18 and HDAC1 (Singh, 2005), occurs only at the very anterior tip of blastoderm embryos where the Bicoid concentration is highest and the target gene enhancers contain multiple weak Bicoid-binding sites (Matyash, 2009).

dSAP18 also interacts with the histone-specific H3K27 methyl-transferase E(z) (Enhancer of zeste) (Wang, 2002), a component of the polycomb group protein complex, and with the GAGA factor, a transcription factor of the trxG (trithorax group) protein complex. Thus, dSAP18 is capable of interacting with two regulatory protein complexes that have antagonistic functions in gene regulation. Whereas the polycomb group complex acts as a repressor of homeotic genes in ectopic locations, the trxG complex is required for activation and maintenance of their transcription. However, this clear-cut distinction between polycomb group and trxG functions has been questioned, because polycomb group and trxG group members were shown to act both as context-dependent repressors and activators of transcription, and factors with such dual functions include both the E(z) and GAGA factor proteins. In fact, interactions between dSAP18 and GAGA factor at the iab-6 element of the bithorax complex, for example, were shown to cause transcriptional activation and not repression (Matyash, 2009).

This study suggests that Kr mediates repression through at least two pathways involving either dCtBP or SAP18. dCtBP-dependent and -independent repression of the transcription factors Knirps and Hairless exert quantitative effects, whereas Kr distinguishes dCtBP and dSAP18 recruitment at different enhancers. It was observed, however, that the loss of SAP18 activity does not affect the pattern of eve stripe expression and that prolonged Kr can suppress h7-lacZ expression in the absence of dSAP18. Thus, although both dSAP18 and dCtBP act independently from each other, the two corepressors, or other yet unknown corepressors, can functionally substitute for each other under forced conditions. However, their mode of repression appears to involve different mechanisms. One mechanism is exemplified by the dCtBP-dependent repression of eve-stripe 2 and not yet established at the molecular level. dCtBP-dependent repression does not act via unleashing local heterochromatization, does not require dHDAC1 activity, and is insensitive to the HDAC inhibitor TSA. Consistently, coimmunoprecipitation studies failed to detect HDAC activity in the dCtBP immunoprecipitates, histone H3 remained acetylated in dCtBP-deficient embryos, and transcription was not repressed. Other studies, however, implied an association of dCtBP with HDACs. Thus, the mechanism of the dCtBP mode of repression is not yet fully understood (Matyash, 2009).

The results of this study showing a lack of H3 deacetylation at the eve stripe 2 enhancer in response to Kr repression are consistent with the argument that eve stripe 2-mediated repression involves the corepressor CtBP. The second, dCtBP-independent mode of Kr-dependent repression, as exemplified by the h stripe 7 element (and possibly also eve stripes 1, 3, and 4) does require both dSAP18 and HDAC1 activities. In support of this mode of repression, the following phenomena were observed in Kr-overexpressing embryos (1) a dSAP18-dependent loss of K9,14H3 acetylation on the h stripe 7 element, (2) an increased resistance of the h7 enhancer DNA to sonication, and (3) SAP18-dependent repression of the h7 reporter gene in response to Kr activity. These Kr-dependent effects were dependent on HDAC1 enzymatic activity as revealed by experiments using the HDAC1 inhibitor, TSA. These results therefore suggest that dSAP18-dependent repression by Kr involves structural changes of chromatin, such as compaction or condensation, likely to be caused by site-specific heterochromatization in response to enhancer-specific HDAC1 activity (Matyash, 2009).

A Drosophila Smyd4 homologue is a muscle-specific transcriptional modulator involved in development

SET and MYND domain (Smyd) proteins are involved in the transcriptional regulation of cellular proliferation and development in vertebrates. However, the in vivo functions and mechanisms by which these proteins act are poorly understood. This study used biochemical and genetic approaches to study the role of a Smyd protein in Drosophila. Eleven Drosophila genes were identified that encode Smyd proteins. CG14122 encodes a Smyd4 homologue that has been named dSmyd4. dSmyd4 repressed transcription and recruited class I histone deacetylases (HDACs). A region of dSmyd4 including the MYND domain interacted directly with approximately 150 amino acids at the N-termini of dHDAC1 (Rpd3) and dHDAC3. dSmyd4 interacts selectively with Ebi, a component of the dHDAC3/SMRTER co-repressor complex. During embryogenesis dSmyd4 was expressed throughout the mesoderm, with highest levels in the somatic musculature. Muscle-specific RNAi against dSmyd4 resulted in depletion of the protein and lead to severe lethality. Eclosion is the final moulting stage of Drosophila development when adult flies escape from the pupal case. 80% of dSmyd4 knockdown flies were not able to eclose, resulting in late pupal lethality. However, many aspects of eclosion were still able to occur normally, indicating that dSmyd4 is likely to be involved in the development or function of adult muscle. Repression of transcription by dSmyd4 and the involvement of this protein in development suggests that aspects of Smyd protein function are conserved between vertebrates and invertebrates (Thompson, 2008).

The large number of Smyd family members in Drosophila may allow these proteins to assume a repertoire of functions, or ensure redundancy between family members during development. Further analysis of vertebrate genomes may also reveal larger numbers of Smyd proteins than had previously been anticipated. Studies in vertebrates show that individual Smyd proteins control gene expression in order to fulfil varied functions during development. The tissue specific expression patterns of Drosophila Smyd family members suggest that these proteins may play equivalent roles in the development of specific tissues in this species (Thompson, 2008).

dSmyd4 represses transcription and recruits HDACs in a manner analogous to vertebrate Smyd1 and Smyd2. This study gives additional insight into the HDAC co-repressors that are involved in repression by dSmyd4. dSmyd4 was shown to interact with both dHDAC3 and Ebi, components of the SMRTER co-repressor complex. This contrasts with mammalian Smyd2 protein, which interacts with the Sin3A-HDAC complex. No interaction could be detected between dSmyd4 and HDAC1-containing NuRD, CoREST and Sin3A co-repressors by immunoprecipitation. Nevertheless, a common feature of dSmyd4 and vertebrate Smyd2 and Smyd1 is the association of a potential methyltransferase with histone deacetylase activity in a single complex. This implies that a primary role of these proteins is to co-ordinate changes in modification status at their target sites (Thompson, 2008).

This paper has described a direct interaction between dSmyd4 and the N-terminal regions of dHDAC1 and dHDAC3. There is a high level of identity between Drosophila and vertebrate class I HDACs, especially at the N-termini where this interaction occurs, therefore this interaction may be relevant to recruitment of HDACs by Smyd family members in other species. The recruitment of HDAC co-repressor complexes by MYND domains is also of clinical importance. AML/MTG8 fusions lead to the aberrant recruitment of HDAC co-repressor complexes in the development of leukaemia. The MTG8 MYND domain interacts with components of these complexes, but the interaction between the MYND domain of MTG8 and HDACs is poorly described. The novel interaction described in this study may also apply to other interactions such as these (Thompson, 2008).

The cytoplasmic over-expression pattern of dSmyd4 resembles that of vertebrate Smyd2, providing another parallel between vertebrate and invertebrate Smyd proteins. However, a more relevant indicator of biological function is the distribution of endogenous protein. Endogenous dSmyd4 is predominantly nuclear in S2 cells. The strong cytoplasmic localisation of dSmyd4 in embryos suggests that in addition to its activity as a transcriptional repressor, dSmyd4 may perform additional cytoplasmic functions, for example the methylation of non-histone substrates. This raises additional parallels with Smyd2, since a cytoplasmic role has been suggested for this protein. The cell-type dependent localisation of endogenous dSmyd4 raises interesting questions about how the localisation of dSmyd4 is regulated. The subcellular localisation of human Smyd3 is regulated in a cell cycle dependent manner and analogous developmental regulation may be required for the function of other Smyd proteins such as dSmyd4 (Thompson, 2008).

Knockdown of dSmyd4 in muscle tissue resulted in reduced rates of survival. dSmyd4 was expressed during embryogenesis, yet the majority of knockdown flies died at the late pupal stage suggesting that there is a greater requirement for dSmyd4 in processes involved in adult myogenesis. This may be due to redundancy between Smyd proteins during embryogenesis since CG8503 and CG18136 are also expressed in muscle tissue at this time. The majority of knockdown flies were not able to escape from the pupal case but performed other eclosion behaviours normally. The neural networks and signalling required for eclosion therefore appear to be intact, indicating that dSmyd4 is likely to play a role in controlling muscle development or function. Identifying the precise nature of the eclosion defect caused by dSmyd4 knockdown will be an important step in understanding the function of this and other Smyd proteins in the development of multicellular organisms. Much is known about the transcription factors involved in Drosophila muscle development but little is understood about how chromatin structure is regulated during this process. dSmyd4 is a good candidate to direct chromatin remodelling during muscle development. Smyd1 is required for cardiac development in vertebrates and a number of other Drosophila Smyd proteins appear to be specifically expressed in muscle. These results suggest that members of the Smyd family play conserved roles in muscle development in both vertebrate and invertebrate species. Drosophila provides a tractable system for the analysis of gene function, for example testing genetic interactions with other genes involved in muscle development. Analysis of mutants in dSmyd4 and other Smyd genes using this approach may also shed light on conserved aspects of Smyd function in vertebrates (Thompson, 2008).

This study presents the first analysis of both Smyd proteins in Drosophila and of a Smyd4 homologue. It appears that aspects of mechanism and function are conserved between Drosophila and vertebrate Smyd proteins. The repression of transcription by SMRTER complex recruitment and the requirement of dSmyd4 for survival highlight the importance of this protein family as transcriptional modulators of developmental processes (Thompson, 2008).

Histone chaperones ASF1 and NAP1 differentially modulate removal of active histone marks by LID-RPD3 complexes during NOTCH silencing

Histone chaperones are involved in a variety of chromatin transactions. By a proteomics survey, the interaction networks of histone chaperones ASF1 (Anti-silencing factor 1), CAF1, HIRA, and NAP1 were identified. This study analyzed the cooperation of H3/H4 chaperone ASF1 and H2A/H2B chaperone NAP1 with two closely related silencing complexes: LAF and RLAF. NAP1 binds RPD3 and LID-associated factors (RLAF) comprising histone deacetylase RPD3, histone H3K4 demethylase LID/KDM5, SIN3A, PF1, EMSY, and MRG15. ASF1 binds LAF, a similar complex lacking RPD3. ASF1 and NAP1 link, respectively, LAF and RLAF to the DNA-binding Su(H)/Hairless complex, which targets the E(spl) Notch-regulated genes. ASF1 facilitates gene-selective removal of the H3K4me3 mark by LAF but has no effect on H3 deacetylation. NAP1 directs high nucleosome density near E(spl) control elements and mediates both H3 deacetylation and H3K4me3 demethylation by RLAF. It is concluded that histone chaperones ASF1 and NAP1 differentially modulate local chromatin structure during gene-selective silencing (Moshkin, 2009).

Regulated modulation of the chromatin structure is essential for the transmission, maintenance, and expression of the eukaryotic genome. The combined actions of ATP-dependent chromatin-remodeling factors (remodelers), histone chaperones, and histone-modifying enzymes drive chromatin dynamics. Histones are subjected to a wide range of reversible posttranslational modifications, including acetylation, phosphorylation, methylation, and ubiquitylation. Histone modifications, in turn, can promote the recruitment of selective regulatory factors and modulate chromatin accessibility. Chromatin remodelers control DNA accessibility by mediating nucleosome mobilization either through sliding or by nucleosome (dis)assembly (Moshkin, 2009).

Whereas originally considered mainly as mere chaperones, it has become clear that histone chaperones play diverse roles during chromatin transactions. Histone chaperones bind selective histones and include the highly conserved H3/H4 chaperones ASF1, CAF1, HIRA, and Spt6 and the H2A/H2B chaperones NAP1, Nucleoplasmin, and FACT. Although their biochemical activity, binding and release of histones, appears rather mundane, in conjunction with other factors, histone chaperones participate in a variety of chromatin transactions and other cellular tasks. For example, yeast NAP1 participates in an extensive interaction network including a diverse set of transcription initiation/elongation factors, chromatin remodelers, RNA-processing factors, cell-cycle regulators, and other proteins (Moshkin, 2009).

ASF1 is one of the major H3/H4 chaperones, and through association with other proteins, it contributes to diverse chromatin transactions. (1) In conjunction with CAF1 and the MCM2-7 DNA helicase, ASF1 participates in replication-coupled chromatin assembly. (2) When associated with HIRA, ASF1 participates in replication-independent chromatin assembly and histone replacement. (3) DNA-repair-associated chromatin assembly requires the cooperation between ASF1 and the H3K56 acetyltransferase Rtt109. (4) ASF1 functionally cooperates with the Drosophila BRM chromatin remodeler, and (5) interaction of ASF1 with transcription activators stimulates histone eviction from promoter areas and facilitates recruitment of chromatin-specific coactivator complexes. (6) ASF1 itself is one of the targets of Tousled-like kinase (TLK), which controls cell-cycle progression and chromatin dynamics. (7) Finally, ASF1 is involved in developmental gene expression control by mediating transcriptional repression of Notch target genes. ASF1 is recruited to E(spl) genes by the sequence-specific DNA-binding protein Su(H) and its associated corepressor complex, harboring Hairless (H) and SKIP (Moshkin, 2009).

Notch is the central component of a highly conserved developmental signaling pathway that is present in all metazoans. Notch is a single-pass transmembrane protein that is activated through ligand binding, resulting in the release of the Notch intracellular domain (Nicd), which is targeted to the nucleus to activate gene expression. The CSL (CBF1, Su(H), and Lag1) family of sequence-specific DNA-binding proteins is the key targeting factor of Nicd and coactivators and, in the absence of Nicd, corepressors. The repression of Notch target genes involves multiple chromatin-modifying activities including histone deacetylases, H3K9 methyltransferases, CtBP, NcoR/SMRT, and Goucho (GRO). In the absence of the Nicd, loss of ASF1 leads to derepression of the E(spl) genes, revealing its essential role in silencing (Moshkin, 2009).

The molecular mechanism by which ASF1 achieves gene-specific transcription repression and the potential roles of other histone chaperones in developmental gene regulation remains largely unknown. To address these issues, a proteomics survey was performed of the protein interaction networks of ASF1, CAF1, HIRA, and NAP1 in Drosophila embryos. This analysis revealed that ASF1 and NAP1 interact with two related but distinct corepressor complexes: LAF and RLAF. LAF, comprising LID/KDM5 SIN3A, PF1, EMSY, and MRG15, associates with ASF1 (forming LAF-A). RLAF, comprising LAF plus RPD3, interacts with NAP1 (forming RLAF-N). Through a combination of biochemistry and developmental genetics, it was established that LAF-A and RLAF-N are tethered to Notch target genes by the Su(H)/H complex and mediate gene-selective silencing. Both ASF1 and NAP1 are required for the targeted removal of the positive H3K4me3 mark by facilitating LID/KDM5 recruitment to chromatin. Furthermore, NAP1 mediates nucleosome assembly at regulatory elements of Notch target genes and histone deacetylation by RLAF. These results uncover extensive crosstalk between distinct histone chaperones and histone-modifying enzymes in developmental gene regulation (Moshkin, 2009).

These results emphasize that, rather than generic, redundant factors, histone chaperones play highly specialized roles in gene-specific regulation. This study has dissected the molecular mechanism underpinning coordinate silencing of Notch target genes by the histone H3/H4 chaperone ASF1 and the H2A/H2B chaperone NAP1. ASF1 interacts with LAF, comprising SIN3A, PF1, EMSY, MRG15, and the histone H3K4me2/3 demethylase LID/KDM5, forming LAF-A. A closely related complex, RLAF that includes the deacetylase RPD3, does not bind ASF1. Instead, RLAF associates with NAP1, forming RLAF-N. The chaperones ASF1 and NAP1 link, respectively, LAF and RLAF to the Su(H)/H DNA-binding complex, tethering them to the E(spl) genes. Both ASF1 and NAP1 bind the SKIP subunit of the Su(H)/H complex (Goodfellow, 2007). Thus, at least in part, ASF1 and NAP1 facilitate H3K4me3 demethylation activity at the E(spl) genes through LID recruitment. Other LAFs might provide additional links to the Su(H)/H complex by contacting GRO and CtBP, which themselves associate with the Su(H)/H complex. For example, mammalian PF1, MRG15, and SIN3A have been reported to bind GRO. This study identified CtBP in LID, PF1, and NAP1 immunopurifications, providing an additional contact between the Su(H)/H complex and (R)LAF (Moshkin, 2009).

ASF1 does not bind RLAF and has no effect on histone H3 deacetylation by RPD3. In contrast, NAP1 does associate with RLAF and stimulates both H3K4 demethylation by LID and H3 deacetylation by RPD3. SIN3A had a mild effect, but the other LAF subunits played no apparent role in deacetylation. Finally, NAP1 depletion caused a dramatic loss of histones at the E(spl) regulatory elements, whereas ASF1 depletion had no effect on local histone density (Moshkin, 2009).

ASF1 has been proposed to function in chromatin assembly by acting as a donor that hands off the H3/H4 tetramer to either CAF1 or HIRA (De Koning, 2007). Because LAF-A does not associate with either CAF1 or HIRA, this might explain that ASF1 does not modulate nucleosome density at the E(spl) genes. In conclusion, the H3/H4 chaperone ASF1 mediates silencing of Notch target genes by (1) providing a connection between LAF and the Su(H)/H tether and (2) facilitating H3K4 demethylation by LID. The H2A/H2B chaperone NAP1 participates in E(spl) silencing by (1) linking RLAF to Su(H)/H, (2) facilitating H3K4 demethylation by LID, (3) facilitating H3 deacetylation by RPD3, and (4) directing high nucleosome density at repressed loci. The functioning of the H2A/H2B chaperone NAP1 in demethylation and deacetylation of histone H3 provides an example of trans-histone regulation (Moshkin, 2009).

LID and its interacting factors appear to work in a context-dependent manner. For example, LID facilitates activation of dMYC target genes in a manner independent of its demethylase activity. Suggestively, this study observed a genetic interaction between ASF1 and dMYC, indicating a potential role for LAF-A. Recently, it has been suggested that selective RLAF subunits could interact with a homolog of GATA zinc-finger domain-containing protein 1 to facilitate expression of targets by inhibition of RPD3 activity. In mammalian cells, LID homolog RBP2 and MRG15 have been implicated in transcription elongation by restricting H3K4me3 levels within transcribed regions. Identification of SIN3A as a LAF and RLAF subunit provides a molecular explanation for the recent observation that SIN3A is involved in genome-wide removal of both H3K4 methyl and acetyl marks. Collectively, these findings suggest that LID and RPD3 enzymatic activities can be modulated through association with specific partners. The proteomics analysis of the LID, PF1, and EMSY interaction networks further emphasizes the diverse involvement of LAFs in regulation of chromatin dynamics (Moshkin, 2009).

In conclusion, these results emphasize the close interconnectivity between distinct chromatin transactions and reveal cooperation between histone chaperones and targeted histone modifications during developmental gene control. The proteomic survey of ASF1, CAF1, HIRA, and NAP1 provides a starting point for the functional analysis of the regulatory networks in which these chaperones participate. As illustrated by the analysis of LAF-A and RLAF-N, specific protein-protein associations and gene targeting provide an intricate network of combinatorial gene expression control (Moshkin, 2009).

The H3K4 demethylase lid associates with and inhibits histone deacetylase Rpd3

JmjC domain-containing proteins have been shown to possess histone demethylase activity. One of these proteins is the Drosophila histone H3 lysine 4 demethylase Little imaginal discs (Lid), which has been genetically classified as a Trithorax group protein. However, contrary to the supposed function of Lid in gene activation, the biochemical activity of this protein entails the removal of a histone mark that is correlated with active transcription. To understand the molecular mechanism behind the function of Lid, a Lid-containing protein complex was purified from Drosophila embryo nuclear extracts. In addition to Lid, the complex contains Rpd3, CG3815/Drosophila Pf1, CG13367, and Mrg15. Rpd3 is a histone deacetylase, and along with Polycomb group proteins, which antagonize the function of Trithorax group proteins, it negatively regulates transcription. By reconstituting the Lid complex, it was demonstrated that the demethylase activity of Lid is not affected by its association with other proteins. However, the deacetylase activity of Rpd3 is greatly diminished upon incorporation into the Lid complex. Thus, these finding that Lid antagonizes Rpd3 function provides an explanation for the genetic classification of Lid as a positive transcription regulator (Lee, 2009).

To shed light on the molecular mechanism of how the histone demethylase Lid regulates transcription, a Lid-containing protein complex, which includes dPf1, Rpd3, CG13367, and Mrg15, was purified from Drosophila embryonic NE. Previous studies have shown that the activities of chromatin-modifying enzymes can be modulated through association with other proteins in a complex. Although no alteration was observed in histone demethylase activity upon the formation of the Lid complex compared to the activity of recombinant Lid alone, the possibility cannot be ruled out that additional factors are required to mediate this stimulatory effect. Since nucleosomes could not be used as a substrate for reasons of sensitivity, it is possible that the Lid complex is irresponsive to enhanced demethylase activity on methylated histones. In contrast, a different Lid-containing complex that is primarily responsible for histone demethylation may exist. Previously, Lid has been reported to interact with dMyc and another TrxG protein, Ash2, in larval eye imaginal discs, implying that other, tissue- and developmental stage-specific Lid-containing complexes may exist (Lee, 2009).

Intriguingly, inhibition of the HDAC activity of Rpd3 was observed in the Lid complex. In this respect, the major function of Lid in this particular complex may be to counteract the transcriptional repression mediated by the deacetylase activity of Rpd3. Notably, Rpd3 has been shown previously to interact with the PRC2 (Polycomb repressive complex 2) complex and to enhance PcG-mediated gene silencing through histone deacetylation. As the H3K4 demethylase activity of Lid is not required for odd gene activation, it is tempting to speculate that the genetic characterization of lid as a TrxG gene is due in part to its inhibitory effect on Rpd3. By inhibiting the HDAC activity of Rpd3, Lid may counteract the full extent of PcG-mediated suppression of gene expression, providing an explanation for the contradictory genetic classification of lid as a TrxG gene and the enzymatic activity of Lid to remove an active histone mark. From this point of view, it appears possible that the histone demethylase activity of Lid is developmentally dispensable. However, it was observed that lid homozygous mutant flies can be rescued only by a transgene encoding wild-type Lid and not by a transgene encoding a catalytically inactive mutant form of Lid, indicating that H3K4 demethylation is developmentally important. Thus, Lid appears to fulfill two possibly distinct functions during development, and these functions may act independently of each other. One function is to demethylate H3K4, whereas the other is to antagonize HDAC activity to promote transcription (Lee, 2009).

The findings of a recent study substantiate the antagonistic behavior of Lid toward Rpd3. Lloret-Llinares (2008) reported that lid mutant alleles act as an enhancer of position effect variegation, whereas some mutations in Rpd3 have been found to confer suppressor-of-variegation phenotypes. Moreover, polytene chromosomes of lid mutants have been shown to have reduced levels of AcH3 (Lloret-Llinares, 2008), which is consistent with the finding that the overexpression of Lid is able to reduce the binding of Rpd3 to polytene chromosomes. Thus, in the absence of Lid, the balance between Lid and Rpd3 would be tilted toward Rpd3, resulting in reduced levels of AcH3 (Lee, 2009).

A similar HDAC complex containing Pf1 and Mrg15 in mammals has been described previously. It is envisaged that Lid is recruited to a core HDAC complex consisting of Rpd3, dPf1, and Mrg15 (and possibly including additional factors that are part of the HDAC complex) and thereby inhibits the HDAC activity. The recruitment of Lid to the sites of the HDAC complex may act as a switch to turn on the expression of target genes during development. It was shown on a gene-specific level for the odd gene in S2 cells by ChIP analysis and on a global level by the immunostaining of polytene chromosomes that the overexpression of Lid results in a marked decrease in Rpd3 binding, suggesting that excessive Lid is able to interact with and displace Rpd3 from its target sites. It has to be pointed out, however, that these findings are based on conditions of robust overexpression of Lid and that the observations need to be confirmed for target genes of the Lid complex in the context of development (Lee, 2009).

It is surprising that Mrg15 was found to negatively regulate the HDAC activity of Rpd3 in vitro, because Mrg15 has been shown previously to contribute to transcription repression. In this regard, it is possible that the interaction solely between Rpd3 and Mrg15 results in enzymatic inhibition and that interaction with additional factors, such as Sin3, may be required to restore the HDAC activity. Provided that the Lid complex identified in this study does play a role in regulating dynamic histone methylation, another role for Mrg15 is conceivable. The chromodomain of Mrg15 may potentially be involved in recruiting the Lid complex to target genes. The trimethylation of H3K4 peaks in the promoter region, whereas the trimethylation of H3K36 is enriched in the 3′ region of genes. During the process of transcription, the chromodomain of Mrg15 may target the Lid complex to the bodies of genes through its interaction with H3K36me3 and induce the removal of H3K4 trimethylation, resulting in the enrichment of the 5′ region of genes with this modification. In the absence of Lid, this distinct border of the different methyl marks would not be sustained and transcription efficiency would deteriorate, thus offering an explanation for the function of Lid in active transcription (Lee, 2009).

Future genome-wide location studies of Lid and the other components of the complex will reveal which target genes are controlled by this complex. Furthermore, it will be interesting to find out where within target genes the complex is located. Does the complex bind to the bodies of genes to demethylate H3K4, or does the binding take place at promoter regions to regulate dynamic histone deacetylation? The identification of Lid-associated proteins has set the stage for these detailed studies, which will reveal insight into the mechanism underlying transcription regulation by Lid (Lee, 2009).

Atrophin-Rpd3 complex represses Hedgehog signaling by acting as a corepressor of CiR

The evolutionarily conserved Hedgehog (Hh) signaling pathway is transduced by the Cubitus interruptus (Ci)/Gli family of transcription factors that exist in two distinct repressor (CiR/GliR) and activator (CiA/GliA) forms. Aberrant activation of Hh signaling is associated with various human cancers, but the mechanism through which CiRGliR properly represses target gene expression is poorly understood. This study used Drosophila and zebrafish models to define a repressor function of Atrophin (Atro) in Hh signaling. Atro directly binds to Ci through its C terminus. The N terminus of Atro interacts with a histone deacetylase, Rpd3, to recruit it to a Ci-binding site at the decapentaplegic (dpp) locus and reduce dpp transcription through histone acetylation regulation. The repressor function of Atro in Hh signaling is dependent on Ci. Furthermore, Rerea, a homologue of Atro in zebrafish, represses the expression of Hh-responsive genes. It is proposed that the Atro-Rpd3 complex plays a conserved role to function as a CiR corepressor (Zhang, 2013).

The RNA processing exosome is linked to elongating RNA polymerase II in Drosophila

RNA surveillance factors are involved in heterochromatin regulation in yeast and plants, but less is known about the possible roles of ribonucleases in the heterochromatin of animal cells. This study shows that RRP6, one of the catalytic subunits of the exosome, is necessary for silencing heterochromatic repeats in the genome of Drosophila melanogaster. It was shown that a fraction of RRP6 is associated with heterochromatin, and the analysis of the RRP6 interaction network reveals physical links between RRP6 and the heterochromatin factors HP1a, SU(VAR)3-9 and RPD3. Moreover, genome-wide studies of RRP6 occupancy in cells depleted of SU(VAR)3-9 demonstrates that SU(VAR)3-9 contributes to the tethering of RRP6 to a subset of heterochromatic loci. Depletion of the exosome ribonucleases RRP6 and DIS3 stabilizes heterochromatic transcripts derived from transposons and repetitive sequences, and renders the heterochromatin less compact, as shown by micrococcal nuclease and proximity-ligation assays. Such depletion also increases the amount of HP1a bound to heterochromatic transcripts. Taken together, these results suggest that SU(VAR)3-9 targets RRP6 to a subset of heterochromatic loci where RRP6 degrades chromatin-associated non-coding RNAs in a process that is necessary to maintain the packaging of the heterochromatin (Eberle, 2015).

Approximately 30% of the genome of Drosophila melanogaster is heterochromatic and is made up of transposons, transposon fragments and repetitive sequences with different degrees of complexity. The heterochromatin contains high levels of heterochromatin-specific proteins, such as Heterochromatin Protein 1a (HP1a), and is enriched in certain patterns of post-translational modifications of the histone tails. Heterochromatin formation involves a cascade of histone modifications that are targeted to specific regions of the genome by complex protein-protein and protein-nucleic acid interactions. In the switch from euchromatin to heterochromatin, acetylated H3K9 (H3K9ac) is deacetylated by histone deacetylases such as RPD3/HDAC1. H3K9 is subsequently methylated by histone methyltransferases, and the methylated H3K9 (H3K9me) acts as a binding site for HP1a. The properties of the heterochromatin can spread along the chromatin fiber, and HP1a plays a central role in this process. The ability of HP1a to dimerize, to interact with the methyltransferase SU(VAR)3-9, and to bind H3K9me provides the basis for the spreading of heterochromatin. An additional level of complexity in the establishment of heterochromatic states is provided by the fact that HP1a can also bind RNA in both D. melanogaster and Schizosaccharomyces pombe. Recent studies on Swi6, the HP1a ortholog of S. pombe, have shown that the interaction of Swi6 with RNA interferes with the binding of Swi6 with H3K9me (Eberle, 2015).

Small non-coding RNAs are essential components of the regulation of chromatin packaging in different organisms. Fission yeast uses siRNAs to silence heterochromatic sequences through the recruitment of the H3K9 methyltransferase Clr4. RNAi-dependent mechanisms of heterochromatin assembly exist also in plants, where siRNAs direct de novo DNA methyltransferases to specific genomic sequences. Animal cells use instead the piRNA pathway to trigger heterochromatin assembly and transposon silencing in the germ line. In D. melanogaster, non-coding RNAs transcribed from transposon-rich regions are processed into piRNAs, and a 'Piwi-piRNA guidance hypothesis' has been recently proposed for the recruitment of SU(VAR)3-9 and HP1a to heterochromatin. The Piwi-piRNA system is active during early development and it directs the initial establishment of heterochromatin states not only in the germ line but also in somatic cells. Recent studies suggest that after embryogenesis, the patterns of heterochromatin packaging are maintained through cell divisions via piRNA-independent mechanisms (Eberle, 2015).

An important player in the regulation of non-coding RNAs is the exosome, a multiprotein complex with ribonucleolytic activity. In D. melanogaster, the core of the exosome associates with two catalytic active subunits, RRP6 and DIS3. In the cell nucleus, the exosome is involved in the processing of many non-coding RNAs, including pre-rRNAs, and in the quality control of mRNA biogenesis. The exosome ribonucleases also degrade a large variety of unstable, non-coding RNAs in various organisms including S. cerevisiae, plants, and animals. Moreover, recent studies have revealed that RRP6 participates in the regulation of enhancer RNAs and in the degradation of unstable transcripts synthesized at DNA double-strand breaks (Eberle, 2015).

The exosome has been functionally linked to the methylation of H3K9 in heterochromatin. In S. pombe, RRP6 participates in the assembly of centromeric heterochromatin through an RNAi-independent mechanism, and collaborates with the RNAi machinery to silence developmentally regulated loci and retrotransposons. Much less is known about the possible links between RRP6 and heterochromatin in animals. This study found that a fraction of RRP6 is associated with heterochromatin in the genome of D. melanogaster, and physical interactions has been identifiedf between RRP6 and several heterochromatin factors, including HP1a, SU(VAR)3-9, and RPD3. These results show that SU(VAR)3-9 promotes the targeting of RRP6 to transposon-rich heterochromatic loci. In these loci, RRP6 contributes to maintaining the structure of the heterochromatin by degrading non-coding RNAs that would otherwise compromise the packaging of the chromatin (Eberle, 2015).

This study shows that RRP6 interacts physically with HP1a and SU(VAR)3-9, and that RRP6 is associated with a subset of heterochromatic regions of the genome. Less RRP6 is bound to the heterochromatin in cells with reduced levels of SU(VAR)3-9, which indicates that SU(VAR)3-9 contributes to the targeting of RRP6 to heterochromatin. Although the RNAi experiments do not reveal whether the effect of SU(VAR)3-9 knockdown on RRP6 occupancy is direct or indirect, the fact that RRP6 and SU(VAR)3-9 colocalize and can be co-immunoprecipitated suggests that SU(VAR)3-9 facilitates the recruitment of RRP6 to the heterochromatin, or stabilizes the interaction of RRP6 with other chromatin components, through a physical interaction (Eberle, 2015).

This study has focused on RRP6, and the existence of multiple exosome subcomplexes in cells of D. melanogaster makes it difficult to establish whether the entire exosome has a role in the heterochromatin. However, two observations suggest that this is the case. Firstly, the simultaneous depletion of both catalytic subunits of the exosome, RRP6 and DIS3, gave additive effects on the levels of chromatin-associated RNAs and on the association of HP1a to heterochromatic RNAs. Secondly, it was previously shown that a fraction of RRP4, a core exosome subunit, is also associated with chromatin. Altogether, these observations suggest that the entire exosome, not RRP6 alone, is targeted to heterochromatic loci through an interaction with SU(VAR)3-9 (Eberle, 2015).

Depletion of RRP6 or simultaneous depletion of RRP6 and DIS3 led to a local increase in heterochromatic transcripts associated with subtelomeric and pericentromeric regions, without a significant increase in the density of RNA Pol-II at those regions. This suggests that under normal conditions the RRP6 and DIS3 degrade pervasive RNAs that are transcribed from the heterochromatin. Direct MNase assays and PLA-based assays designed to measure the compaction of the chromatin revealed that the depletion of the exosome ribonucleases loosens the structure of the heterochromatin in the regions that accumulate heterochromatic non-coding RNAs, without affecting the levels of H3K9 methylation or the association of SU(VAR)3-9 with the chromatin. In S. pombe, deletion of the rrp6 gene leads to a derepression of heterochromatin, and this effect is partly due to the fact that in the absence of RRP6 activity, aberrant RNA species accumulate in S. pombe and recruit the siRNA machinery in competition with the RNAi-dependent pathways of H3K9 methylation. The situation is different in D. melanogaster, as no change in H3K9me2 or SU(VAR)3-9 recruitment occurred when RRP6 and DIS3 were depleted (Eberle, 2015).

What is then the mechanism by which the exosome ribonucleases influence the compaction of the heterochromatin in D. melanogaster? The HP1a ortholog in S. pombe, Swi6, is an RNA-binding protein, and non-coding RNAs can cause the eviction of Swi6 from the S. pombe heterochromatin by competing with H3K9me for Swi6. The HP1a protein of D. melanogaster interacts with several RNA-binding proteins and can bind directly to RNA. This study has shown that depletion of RRP6 and DIS3 results in increased levels of non-coding transcripts associated with heterochromatin in D. melanogaster cells. HP1a-RIP signals at selected heterochromatic loci are also increased in cells depleted of RRP6 and DIS3. Altogether, these observations are consistent with a model in which RRP6, and perhaps also DIS3, participate in the degradation of heterochromatic non-coding RNAs that, if stabilized, would outcompete the binding of HP1a to the methylated H3K9 and would thereby disrupt the packaging of the heterochromatin (Eberle, 2015).

Heterochromatin domains are characterized by high levels of H3K9me2 and by the presence of HP1a and SU(VAR)3-9. These results show that RRP6 interacts with SU(VAR)3-9 and that this interaction is important to tether RRP6 to the heterochromatin. Transcripts derived from sporadic transcription of heterochromatic repeat sequences are kept at low levels by RRP6 degradation. Failure to degrade such transcripts results in increased levels of chromatin-associated transcripts, increased binding of HP1 to the chromatin-associated transcripts, and chromatin decondensation (Eberle, 2015).

Specialized protein-protein interactions target RRP6 to different chromatin environments RRP6 and the exosome act on many different types of transcripts and participate in many essential biological processes. The existence of multiple mechanisms to target RRP6 to different types of transcripts, or even to different nuclear compartments, is thus not unexpected. The association of the exosome-or exosome subunits- with genes transcribed by RNA polymerase II (Pol-II) is mediated by interactions with different types of proteins. Co-immunoprecipitation experiments in D. melanogaster identified SPT5 and SPT6, two transcription elongation factors, as interaction partners for the exosome, which led to the proposal that the exosome is tethered to the transcription machinery during transcription elongation. In D. melanogaster, the exosome is also tethered to protein-coding loci through interactions with the hnRNP protein HRP59/RUMP. In human cells, a NEXT complex containing MTR4, the Zn-knuckle protein ZCCHC8, and the putative RNA binding protein RBM7 mediates an interaction between the exosome and Pol-II transcripts through the nuclear cap-binding complex. In many cases, these intermolecular interactions target the exosome to genomic loci that produce relatively stable transcripts, for instance protein-coding transcripts or stable non-coding RNAs. In these loci, the role of the exosome is primarily linked to RNA surveillance, not turnover (Eberle, 2015).

Much less is known about the mechanisms that target the exosome or its individual subunits to non-protein coding RNAs in the heterochromatin. This study of the RRP6 interactome in cells of D. melanogaster has revealed interactions between RRP6 and heterochromatin factors, and has established an important role for SU(VAR)3-9 in determining RRP6 occupancy. Depletion of SU(VAR)3-9 has a profound effect on the association of RRP6 with a subset of chromatin regions, including many transposon loci. The present findings suggest that these regions, that can be referred to as 'SUV-dependent', produce transcripts that are actively degraded by RRP6. SU(VAR)3-9 has less impact on the targeting of RRP6 to euchromatic protein-coding genes, where interactions with the Pol-II machinery and with mRNA-binding proteins play instead a decisive role. Altogether, the picture that emerges from many studies is that specialized protein-protein interactions target RRP6 to specific genomic environments where RRP6 participates in the processing, surveillance or degradation of specific RNA substrates (Eberle, 2015).

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