InteractiveFly: GeneBrief

Sin3A: Biological Overview | References


Gene name - Sin3A

Synonyms -

Cytological map position - 49B5-49B7

Function - Transcription factor; Chromatin protein

Keywords - transcriptional corepressor, cell cycle, histone deacetylase complex

Symbol - Sin3A

FlyBase ID: FBgn0022764

Genetic map position - 2R:8,462,743..8,480,397 [-]

Classification - SIN3

Cellular location - nucleus



NCBI links: Precomputed BLAST | EntrezGene

Recent literature
Gajan, A., Barnes, V. L., Liu, M., Saha, N. and Pile, L. A. (2016). The histone demethylase dKDM5/LID interacts with the SIN3 histone deacetylase complex and shares functional similarities with SIN3. Epigenetics Chromatin 9: 4. PubMed ID: 26848313
Summary:
Two histone-modifying enzymes, RPD3, a deacetylase, and dKDM5/LID, a demethylase, are present in a single complex, coordinated through the SIN3 scaffold protein. This study analyzed the developmental and transcriptional activities of dKDM5/LID in relation to SIN3. Knockdown of either Sin3A or lid resulted in decreased cell proliferation in S2 cells and wing imaginal discs. Conditional knockdown of either Sin3A or lid resulted in flies that displayed wing developmental defects. Interestingly, overexpression of dKDM5/LID rescued the wing developmental defect due to reduced levels of SIN3 in female flies, indicating a major role for dKDM5/LID in cooperation with SIN3 during development. Together, these observed phenotypes strongly suggest that dKDM5/LID as part of the SIN3 complex can impact previously uncharacterized transcriptional networks. Transcriptome analysis revealed that a significant affect was observed on genes required to mount an effective stress response. Together, the data provide a solid framework for analyzing the gene regulatory pathways through which SIN3 and dKDM5/LID control diverse biological processes in the organism.

Saha, N., Liu, M., Gajan, A. and Pile, L. A. (2016). Genome-wide studies reveal novel and distinct biological pathways regulated by SIN3 isoforms. BMC Genomics 17: 111. PubMed ID: 26872827
Summary:
The multisubunit SIN3 complex is a global transcriptional regulator. In Drosophila, a single Sin3A gene encodes different isoforms of SIN3, of which SIN3 187 and SIN3 220 are the major isoforms. Previous studies have demonstrated functional non-redundancy of SIN3 isoforms. The role of SIN3 isoforms in regulating distinct biological processes, however, is not well characterized. This study established a Drosophila S2 cell culture model system in which cells predominantly express either SIN3 187 or SIN3 220. To identify genomic targets of SIN3 isoforms, chromatin immunoprecipitation was performed followed by deep sequencing. The data demonstrate that upon overexpression of SIN3 187, the level of SIN3 220 decreased and the large majority of genomic sites bound by SIN3 220 were instead bound by SIN3 187. RNA-seq was used to identify genes regulated by the expression of one isoform or the other. In S2 cells, which predominantly express SIN3 220, it was found that SIN3 220 directly regulates genes involved in metabolism and cell proliferation. It was also determined that SIN3 187 regulates a unique set of genes and likely modulates expression of many genes also regulated by SIN3 220. Interestingly, biological pathways enriched for genes specifically regulated by SIN3 187 strongly suggest that this isoform plays an important role during the transition from the embryonic to the larval stage of development. These data establish the role of SIN3 isoforms in regulating distinct biological processes. This study substantially contributes to understanding of the complexity of gene regulation by SIN3.

Chaubal, A., Todi, S.V. and Pile, L.A. (2016). Inter-isoform-dependent regulation of the Drosophila master transcriptional regulator SIN3. J Biol Chem [Epub ahead of print]. PubMed ID: 27129248
Summary:
SIN3 is a transcriptional corepressor that acts as a scaffold for a histone deacetylase (HDAC) complex. The SIN3 complex regulates various biological processes, including organ development, cell proliferation and energy metabolism. Little is known, however, about the regulation of SIN3 itself. There are two major isoforms of Drosophila SIN3, 187 and 220, which are differentially expressed. Intrigued by the developmentally timed exchange of SIN3 isoforms, this study examined whether SIN3 187 controls the fate of the 220 counterpart. It was shown that in developing tissue there is interplay between SIN3 isoforms: when SIN3 187 protein levels increase, SIN3 220 protein decreases concomitantly. SIN3 187 has a dual effect on SIN3 220. Expression of 187 leads to reduced 220 transcript, while also increasing the turnover of SIN3 220 protein by the proteasome. These data support the presence of a novel, inter-isoform-dependent mechanism that regulates the amount of SIN3 protein, and potentially the level of specific SIN3 complexes, during distinct developmental stages.

Liu, M. and Pile, L. A. (2016). The transcriptional corepressor SIN3 directly regulates genes involved in methionine catabolism and affects histone methylation, linking epigenetics and metabolism. J Biol Chem [Epub ahead of print]. PubMed ID: 28028175
Summary:
Chromatin modification and cellular metabolism are tightly connected. Chromatin modifiers regulate the expression of genes involved in metabolism and, in turn, the levels of metabolites. The generated metabolites are utilized by chromatin modifiers to affect epigenetic modification. The mechanism for this cross-talk, however, remains incompletely understood. The corepressor SIN3 controls histone acetylation through association with the histone deacetylase RPD3. The SIN3 complex is known to regulate genes involved in a number of metabolic processes. This study finds that Drosophila SIN3 binds to the promoter region of genes involved in methionine catabolism, and that this binding affects histone modification, which in turn influences gene expression. Specifically, it was observed that reduced expression of SIN3 leads to an increase in S-adenosylmethionine (SAM), which is the major cellular donor of methyl groups for protein modification. Additionally, Sin3A knockdown results in an increase in global histone H3K4me3 levels. Furthermore, decreased H3K4me3 caused by knockdown of either SAM synthetase (Sam-S) or the histone methyltransferase Set1 is restored to near normal levels when SIN3 is also reduced. Taken together, these results indicate that knockdown of Sin3A directly alters the expression of methionine metabolic genes to increase SAM, which in turn leads to an increase in global H3K4me3. This study reveals that SIN3 is an important epigenetic regulator directly connecting methionine metabolism and histone modification.

BIOLOGICAL OVERVIEW

The transcriptional corepressor SIN3 is an essential gene in metazoans. In cell culture experiments, loss of SIN3 leads to defects in cell proliferation. Whether and how SIN3 may regulate the cell cycle during development has not been explored. To gain insight into this relationship, conditional knock down of Drosophila SIN3 was generated and effects on growth and development were analyzed in the wing imaginal disc. It was found that loss of SIN3 affects normal cell growth and leads to down regulation of expression of the cell cycle regulator gene String (Stg). A SIN3 knock down phenotype can be suppressed by overexpression either of Stg or of Cdk1, the target of Stg phosphatase. These data link SIN3 and Stg in a genetic pathway that affects cell cycle progression in a developing tissue (Swaminathan, 2010).

Histone acetylation levels are regulated by the opposing activities of histone lysine (K) acetyltransferases (KATs) and histone deacetylases (HDACs). The SIN3 complex is one of two major class I containing HDAC complexes present in cells. The corepressor SIN3 and the HDAC RPD3 (HDAC1 and 2 in mammals) are two important components of the multi-subunit complex (Silverstein, 2005). Mutations in either SIN3 or RPD3 result in lethality in both Drosophila and mouse model systems (Cowley, 2005; Dannenberg, 2005; David, 2008; Neufeld, 1998b; Pennetta, 1998). Accordingly, establishment and/or maintenance of histone acetylation levels are critical for metazoan development and viability (Swaminathan, 2010).

SIN3 has been shown to be important for cell proliferation. In Drosophila tissue culture cells, reduction of SIN3 protein expression by RNA interference (RNAi) resulted in a G2 phase delay in cell cycle progression (Pile, 2002). A comparison of gene expression profiles from wild type and RNAi-induced SIN3 knock down cells revealed differences in expression of genes encoding proteins that control multiple cellular processes, including cell cycle progression, transcription, mitochondrial activity and signal transduction (Pile, 2003). Expression of two genes critical for the G2/M transition of the cell cycle, String (Stg) and cyclin B (CycB), was reduced in the SIN3 knock down tissue culture cells. Stg is the Drosophila homolog of Schizosaccharomyces pombe Cdc25, a conserved protein phosphatase that dephosphorylates and activates the cyclin dependent kinase, Cdk1 (also known as DmCdc2), which is critical for entry into M phase. CycB interacts with Cdk1 and promotes the G2/M transition (Swaminathan, 2010).

In mouse, knock out of either SIN3 gene, mSin3a or mSin3b, by gene disruption revealed links to cell cycle regulation. Analysis of SIN3-deficient mouse embryonic fibroblasts (MEFs) indicated that mSin3A is important for cell proliferation (Cowley, 2005; Dannenberg, 2005). The mSin3A-deficient MEFs exhibited reduced proliferative capacity relative to their wild type counterparts. Analysis of the DNA content of the MEFs indicated a reduction in the number of cells in S phase with an increase in the number of cells in the G2/M phase of the cell cycle. Although mSin3b is highly similar to mSin3a, the proteins are non-redundant since loss of either gene by targeted gene disruption resulted in embryonic lethality (David, 2008). Furthermore, mSin3B-deficient, but not mSin3a-deficient, MEFs proliferated similarly to the wild type cells under standard culture conditions (David, 2008). Upon serum starvation, however, wild type cells ceased to proliferate while the mSin3B-deficient cells continued to cycle, indicating that mSin3B is necessary for cell cycle exit at the start of differentiation (Swaminathan, 2010).

Null mutations in Drosophila Sin3A result in embryonic lethality with only a few animals surviving to the first larval instar stage (Neufeld, 1998b; Pennetta, 1998). Using an RNAi conditional mutant, it has been determined that SIN3 is also necessary for post-embryonic development (Sharma, 2008). To study the role of SIN3 during the process of cellular proliferation and differentiation, an RNAi conditional mutant was used to eliminate SIN3 in wing imaginal disc cells. SIN3 knock down cells were analyzed during larval and adult stages of development. Loss of SIN3 resulted in fewer cells in the wing blade and a curled wing phenotype in the adult. The curly wing phenotype was partially suppressed by overexpression of the cell cycle regulator Stg and its target Cdk1. These data suggest that SIN3 and G2 to M regulators work in a similar pathway to affect cell cycle progression (Swaminathan, 2010).

Loss of SIN3 from wing imaginal disc cells resulted in a number of observable phenotypes, including smaller imaginal discs and smaller, curly adult wings. The SIN3 knock down curly wing phenotype could be modified by reduction in the level of PCAF, a KAT enzyme that carries out the opposing reaction to histone deacetylation. The curly wing phenotype was also partially suppressed by overexpression of the cell cycle regulatory factors Stg and Cdk1 (Swaminathan, 2010).

SIN3 and proteins associated with the SIN3 complex have been linked to cell cycle regulation in multiple model systems. Loss of Drosophila SIN3 or RPD3 in tissue culture cells resulted in loss of cell proliferation (Pile, 2003). SIN3 has also been implicated in cell survival or proliferation during eye development; generation of homozygous null SIN3 clones resulted in scars across the eye (Neufeld, 1998b). In mouse model systems, genetic knock out of mSin3a from embryonic fibroblasts resulted in loss of cell proliferation (Cowley, 2005; Dannenberg, 2005). Knock out of mSin3b from mouse embryonic fibroblasts resulted in loss of ability of the cells to exit the cell cycle at the start of differentiation (David, 2008). Recent work has indicated that mSin3 is recruited to cell cycle regulated E2F4 target genes in terminally differentiated myoblasts to keep these genes in a repressed state (van Oevelen, 2008). In this study it was observed that reduction of SIN3 in wing imaginal disc cells results in fewer mitotic cells in the wing disc and fewer cells in the adult wing. These results suggest that SIN3 is required for cell proliferation and/or cell survival in the context of a developing organism, as well as in tissue culture cells (Swaminathan, 2010).

Loss of SIN3 in both tissue culture cells and wing imaginal disc tissue results in a decrease of stg mRNA expression. Overexpression of Stg in the background of SIN3 knock down is able to partially suppress the small wing and curly wing phenotypes. Stg is a key regulator of the cell cycle, specifically of the G2 to M transition. Loss of Stg in clones in wing imaginal discs resulted in loss of cell proliferation while overproduction of dE2F resulted in increased Stg expression and accelerated cell proliferation, thus implicating dE2F as a transcriptional activator of stg (Neufeld, 1998a). stg has also been shown to be regulated at the level of transcription by the action of the activator Pointed and the repressor Tramtrack 69 (ttk69). Additional activators including eyes absent and Sine oculis were found to bind to the stg regulatory region in eye imaginal disc cells. Taken together, these results suggest that stg expression is likely regulated by the combinatorial action of multiple activators and repressors, the binding of which may vary with cell cycle stage and tissue (Swaminathan, 2010).

Because SIN3 is a transcriptional corepressor and loss of SIN3 leads to reduced stg expression rather than activation of stg, it is hypothesized that the effect of SIN3 on stg gene expression is indirect. One possible model to explain this effect is that loss of SIN3 leads to an increase in expression of a repressor of stg. If this model is accurate, then loss of this repressor may be able to suppress the SIN3 knock down curly wing phenotype. A second possible model is that loss of SIN3 leads to increased acetylation of a transcription factor necessary for appropriate stg expression. Numerous transcription factors, including p53, have been found to be acetylated. Acetylation of these factors can affect protein stability, localization, interactions with other proteins and DNA binding activity. Experiments to test the possible models linking SIN3 and Stg are currently underway (Swaminathan, 2010).

Genetic interactions were also observed between SIN3 and Cdk1, the substrate of Stg and another important G2/M regulatory factor. Overexpression of Cdk1 suppressed the SIN3 knock down curly wing phenotype. A reduction of Cdk1 levels using the cdc2c03495 allele resulted in enhanced abnormal adult wing morphology as compared to the SIN3 mutants alone. Cdk1 must be dephosphorylated by Stg in order for cells to pass from the G2 to M phase of the cell cycle. Increasing the amount of the substrate for Stg may permit formation of enough active CycB-Cdk1 complexes to drive cell proliferation in the SIN3 knock down cells. A similar suppression of a cell proliferation defect has been previously reported. In Aspergillus nidulans, introduction of an extra copy of cyclin B into a cdc25 (Stg homolog) mutant partially rescued the cell cycle defect of the cdc25 mutant cells (Swaminathan, 2010).

Overexpression of Stg does not fully suppress the SIN3 knock down phenotype, possibly because not all cells in larval wing imaginal discs are sensitive to ectopic Stg expression. Consistent with a cell type specific response to Stg, it was found that Stg overexpression in tissue culture cells is unable to suppress the strong RNAi-induced SIN3-deficient cell proliferation defect. It is also possible that other factors interact with SIN3 to affect wing morphology. Experiments are being conducted to identify other novel factors in the SIN3 regulatory network that may contribute to the role of SIN3 in development (Swaminathan, 2010).

The SIN3 complex is one of the two major class I HDAC complexes conserved from Drosophila to human. The current results have uncovered a genetic link between transcription repression by SIN3 and G2/M cell cycle progression by Stg and Cdk1. Further investigation of this interaction is expected to shed light on the role that histone acetylation plays in the regulation of cell proliferation and differentiation (Swaminathan, 2010).

The Drosophila Smad cofactor Schnurri engages in redundant and synergistic interactions with multiple corepressors

In Drosophila a large zinc finger protein, Schnurri, functions as a Smad cofactor required for repression of brinker and other negative targets in response to signaling by the transforming growth factor beta ligand, Decapentaplegic. Schnurri binds to the silencer-bound Smads through a cluster of zinc fingers located near its carboxy-terminus and silences via a separate repression domain adjacent to this zinc-finger cluster. This study shows that this repression domain functions through interaction with two corepressors, CtBP and Sin3A, and that either interaction is sufficient for repression. Schnurri contains additional repression domains that function through interaction with CtBP, Groucho, Sin3A and SMRTER. By testing for the ability to rescue a shn RNAi phenotype evidence is provided that these diverse repression domains are both cooperative and partially redundant. In addition Shn harbors a region capable of transcriptional activation, consistent with evidence that Schnurri can function as an activator as well as a repressor (Cai, 2009).

Whole-genome analysis of muscle founder cells implicates the chromatin regulator Sin3A in muscle identity

Skeletal muscles are formed in numerous shapes and sizes, and this diversity impacts function and disease susceptibility. To understand how muscle diversity is generated, gene expression profiling was performed of two muscle subsets from Drosophila embryos. By comparing the transcriptional profiles of these subsets, a core group of founder cell-enriched genes was identified. Mutants were screened for muscle defects and functions were identified for Sin3A and 10 other transcription and chromatin regulators in the Drosophila embryonic somatic musculature. Sin3A is required for the morphogenesis of a muscle subset, and Sin3A mutants display muscle loss and misattachment. Additionally, misexpression of identity gene transcription factors in Sin3A heterozygous embryos leads to direct transformations of one muscle into another, whereas overexpression of Sin3A results in the reverse transformation. These data implicate Sin3A as a key buffer controlling muscle responsiveness to transcription factors in the formation of muscle identity, thereby generating tissue diversity (Dobi, 2014).

Prior FACS and transcriptional profiling of Drosophila muscle cells has focused on identifying differences between founder cells (FCs) and fusion- competent myoblasts (FCMs) and examining global effects of identity gene misexpression. Two of these studies used genetic manipulation to enrich for FC and FCM populations. This study used a sorting strategy that purified very small populations of FC subsets from wild-type embryos for analysis. By this approach, significant overlap was observed in the transcriptional profiles of these two muscle subsets, defining a group of factors common to FCs and suggesting that a relatively small number of genes encode factors responsible for muscle diversity in each population, while the rest comprise a 'molecular signature' for all FCs. Homozygous mutant embryos were then screened for muscle defects. The strong combination of FACS, microarray, and mutant analysis has so far identified 11 factors with roles in Drosophila embryonic muscle development, 10 of which have already been shown to have mammalian homologs. While this work has focused on a number of factors that were found to be enriched in both FC subsets, examination of FC subset-specific factors is ongoing and promises to yield additional genes regulating morphogenesis of muscle subsets (Dobi, 2014).

The screen identified a number of ubiquitously expressed essential genes, which can be difficult to identify using forward genetic screening due to maternal loading and early functions during embryogenesis. Factors like Med13, Elo-B, and Sin3A are required for the regulation of transcription in most cells and tissues. This approach enabled the discovery of muscle-specific roles for these general transcriptional regulators, broadening understanding of the genetic network that controls muscle development and setting the stage for future studies into the molecular mechanisms that regulate the establishment of muscle diversity (Dobi, 2014).

The array and screen identified roles for 11 transcription factors and chromatin modifiers, including chn, Lid, crp, Gug, lola, Alh and Kdm2, effectively doubling the list of transcriptional regulators known to control muscle development in the Drosophila embryo. The conservation of these factors opens up the possibility that roles for these factors will be found in vertebrate myogenesis as well. The various mutant phenotypes observed suggest that the identified genes may have defined functions in specific aspects of muscle development: FC specification or cell viability for muscle loss, muscle guidance and myotendinous junction formation for muscle misattachment and FC specification, and cytoskeleton regulation for shape changes. Future work will focus on identification of specific regulatory targets to determine the distinct roles played by these factors at particular steps in development (Dobi, 2014).

Interestingly, although expression of Sin3A was detected in all FCs in the muscle pattern, phenotypic changes were detected in Sin3A08269 mutants primarily within the lateral transverse muscles (LTs). It is clear from analysis of the stronger Sin3Ae64 and Sin3AEP2387 alleles that Sin3A is required throughout the musculature for earlier myogenesis steps like myoblast fusion. The lack of defects in all 30 muscles in Sin3A08269 mutants is attributed to two causes: (1) the weaker nature of the Sin3A08269 allele and (2) the integration of Sin3A into the complicated genetic network of previously characterized identity genes. Subsequent experiments in which misexpression of Kr in Sin3A08269 heterozygotes leads to ventral acute (VA) muscle transformations supports the theory that Sin3A still plays a role in muscles even if no perturbations to those muscles are seen in Sin3A08269 homozygotes. Additionally, it was found that overexpression of the Sin3A-187 isoform in the mesoderm led to muscle transformations, while overexpression of the Sin3A-220 isoform did not. This result is consistent with previous studies showing that Sin3A-187 is the dominant isoform in differentiated tissues and has greater histone deacetylase activity than Sin3A-220. The data suggest that Sin3A-187 isoform is more active in the muscle tissue at genes required for the specification of muscle fate (Dobi, 2014).

How is muscle identity regulated by identity genes? The idea of a combinatorial code specifying muscle fate predicts that the DNA-binding transcription factors work cooperatively and/or antagonistically to generate a muscle-specific transcriptional profile. The incomplete overlap of identity gene expression generates some specificity, as does the timing of identity gene expression. But how do identity genes work once they have bound to the DNA? What factors ensure that the correct FCs respond to signals from identity genes? The results suggest Sin3A is a factor that titrates the responsiveness of muscles to identity genes. Genetic reduction of Sin3A levels leads VA1 to be susceptible to overexpression of the identity gene Kr, ectopically activating Slou expression in this muscle and resulting in VA1 to VA2 transformation. In contrast, overexpression of Sin3A prevents activation of Slou in VA2 and drives VA2 to VA1 transformation (Dobi, 2014).

One hypothesis to explain the results is that the ability of Kr to activate Slou expression in a given muscle depends on the histone modification landscape over the slou promoter, which is in turn regulated by Sin3A histone deacetylase activity. This model suggests that one critical factor contributing to muscle competence is chromatin structure. A model is built upon observations of specific single muscle transformations in the embryonic somatic musculature. The challenge going forward will be to design methods to monitor transcription factor binding, chromatin structure, and gene expression changes at the molecular level in a relatively small percentage of cells in the embryo. The muscle transformation results that were observed are similar to studies from the C. elegans nervous system, in which chromatin regulators have been shown to play important roles in the terminal differentiation of neurons. This work extends these findings to the Drosophila musculature, suggesting a more global role for chromatin regulation in tissue diversity and differentiation. It is possible that the other chromatin modifiers found in this screen, Lid and Kdm2, will be found to play roles in this process as well (Dobi, 2014).

The results draw a distinction between Sin3A and the well-characterized identity genes. While Sin3A is required for the correct formation of muscles, its absence does not change FC specification. This lack of phenotype likely indicates that other positive regulators of transcription are required to drive fate changes in these muscles; alternatively, it could also be that a stronger depletion of Sin3A would cause muscle transformations but that this phenotype is obscured by the earlier patterning defects observed with stronger alleles. It is important to note that Sin3A08269 homozygous mutant embryos do not display VA1 to VA2 transformations, despite overall derepression of slou transcript. While loss of Sin3A disrupts the muscle pattern, it is only in concert with identity gene misexpression that its role in muscle diversity was detected, underscoring the importance for the identity genes in prescribing final muscle characteristics (Dobi, 2014).

Additionally, though overexpression of Sin3A in the mesoderm can lead to VA2 to VA1 transformations, this change does not happen in every hemisegment. Sin3A is just one factor contributing to muscle identity in the context of each muscle’s specific network of gene expression. The experiments reveal the strong commitment to a specific cell fate each muscle makes during development. The somatic musculature is particularly resistant to perturbations; overexpression of Kr can only cause muscle transformations when a strong Gal4 driver is used. In this way, formation of muscle identity is analogous to a buffered solution. In this system, reduction of Sin3A is like removing the buffer, allowing more frequent muscle identity changes. Sin3A, then, positively contributes to the establishment of muscle diversity by fortifying the muscles against shifts in gene expression (Dobi, 2014).

Regulators of chromatin structure have been increasingly shown to play important roles in muscle development and disease. Sin3A, in particular, has been shown to regulate the expression of several genes that have been implicated in muscular dystrophies. While chromatin regulators have been implicated in muscle cell differentiation, Sin3A is the first to be shown to have a role in the formation of muscle identity. A key gap in knowledge about the specification of muscle identity has been that misexpression of identity genes results in relatively few direct muscle transformations. The current results add a layer of complexity to the combinatorial code model for the generation of muscle diversity, suggesting that the epigenetic landscape of a particular muscle sets the stage for identity gene response, either by modulating transcription factor binding or fine tuning the activity of factors once bound to mesodermal enhancers. This knowledge will be critical to synthesize muscles of specific sizes and shapes for use in stem cell transfer therapies to treat muscle disease (Dobi, 2014).

A role for mammalian Sin3 in permanent gene silencing

The multisubunit Sin3 corepressor complex regulates gene transcription through deacetylation of nucleosomes. However, the full range of Sin3 activities and targets is not well understood. This study has investigated genome-wide binding of mouse Sin3 and retinoblastoma (RB)-binding protein 2 (RBP2) as well as histone modifications and nucleosome positioning as a function of myogenic differentiation. Remarkably, this study found that Sin3 complexes spread immediately downstream of the transcription start site on repressed and transcribed genes during differentiation. RBP2 is part of a Sin3 complex, and on a subset of E2F4 target genes, the coordinated activity of Sin3 and RBP2 leads to deacetylation, demethylation, and repositioning of nucleosomes. This work provides evidence for coordinated binding of Sin3, chromatin modifications, and chromatin remodeling within discrete regulatory regions, suggesting a model in which spreading of Sin3 binding is ultimately linked to permanent gene silencing on a subset of E2F4 target genes (van Oevelen, 2008).

A mammalian Sin3B complex is reversibly recruited to cell-cycle genes in quiescent and early G1 cells (Balciunaite, 2005; Rayman, 2002), and the activity of Sin3B appears essential for transient cell-cycle withdrawal (David, 2008). This detailed analysis suggests a model in which Sin3 is also a critical component for the permanent repression of cell-cycle genes during differentiation. Several observations lend credence to this model. (1) In differentiated cells, distinct Sin3/E2F4 complexes (and on a subset of Sin3/E2F4 target genes, RBP2) bind to downstream regions and locally alter both histone modifications and chromatin structure. Moreover, genome-wide location, factor ablation, and bioinformatics analyses show a clear functional relationship between Sin3 and E2F4 at target sites. In addition, although the possibility that Sin3 associates with other repressors within the same region cannot be ruled out, extensive computational analysis with search algorithms designed to identify enriched motifs failed to identify enrichment of known Sin3-associated repressor proteins. (2) The activities of Sin3 and RBP2 result in the coordinated removal of acetylation and methylation marks, and this coincides with the timely acquisition of the repressive H3K27 methylation mark and altered nucleosome architecture. (3) Ablation of Sin3 activity leads to loss of nucleosome density, and this coincides with reacetylation, reflecting hyperacetylation and subsequent re-expression of cell-cycle-regulated genes in differentiated cells. (4) Consistent with a role in permanent gene silencing, recent conditional knockout studies in mice suggest both overlapping and redundant roles for Sin3 isoforms in maintenance of the differentiated state (van Oevelen, 2008).

How Sin3 stably associates with this downstream region is not known. Recruitment of the Sin3 complex creates localized regions of histone H3 hypoacetylation and methylation in a coordinated manner. Interestingly, in vitro experiments suggest that the Sin3 complex shows a preference for the hypoacetylated amino-terminal tail of histone H3, resulting in strong, repressor-independent anchoring of the Sin3 complex (Vermeulen, 2006). A mutual dependency on E2F4 and Sin3 was observed for the stable association of Sin3/E2F4 complexes. Thus, it is proposed that in differentiated myotubes, Sin3 is stably associated with downstream promoter regions by E2F4 and interaction with hypoacetylated histone tails. Other chromatin modifications, in particular H3K36 methylation, could possibly also play a role in directing mammalian Sin3 recruitment to target genes (van Oevelen, 2008).

These nucleosome density experiments and gene expression studies suggest that binding of Sin3 to cell-cycle-regulated genes has a profound effect on chromatin structure and gene expression. How do Sin3/E2F4 complexes alter chromatin structure? It is known that Sin3 and E2F/pocket protein complexes interact with a variety of factors involved in chromatin function. For example, the Sin3 complex was shown to interact with Brg1, the catalytic subunit of the Swi/Snf chromatin-remodeling enzyme (Nagl, 2007; Sif, 2001). Given this connection, it is reasonable to speculate that, in the proper setting, the recruitment and activity of Sin3-HDAC and Swi/Snf (or functionally related remodeling enzymes) are tightly coordinated to promote nucleosome repositioning (van Oevelen, 2008).

Nucleosome positioning within promoter regions plays a critical role in regulating gene expression by limiting the access of transcription factors. The presence of Sin3/E2F4 complexes could therefore physically block access of the preinitiation complex and recruitment of factors to promoters at least in part by increasing the density of nucleosomes locally. Such regions could represent localized, facultative heterochromatin in differentiated skeletal muscle cells, as has been observed the presence of the repressive H3K27 methylation mark on a subset of E2F4/pRb target genes, many of which have been shown to bind Sin3 in this study. This work also highlights an especially important role for nucleosomes immediately downstream of the TSS. Further work will be required to elucidate the importance of TSS-proximal nucleosomes implicated in this study, to define the chromatin marks associated with this region, to determine whether higher-order chromatin compaction can occur, and to examine whether PolII binding is blocked (van Oevelen, 2008).

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).

Drosophila SIN3 is required at multiple stages of development

SIN3 is a component of a histone deacetylase complex known to be important for transcription repression. While multiple isoforms of SIN3 have been reported, little is known about their relative expression or role in development. Using a combination of techniques, it was determined that SIN3 is expressed throughout the Drosophila life cycle. The pattern of expression for each individual isoform, however, is distinct. Knock down of all SIN3 expression reveals a requirement for this protein in embryonic and larval periods. Taken together, the data suggest that SIN3 is required for multiple developmental events during the Drosophila life cycle (Sharma, 2008).

Multiple histone deacetylases (HDACs) are present in Drosophila. SIN3 is a component of one multisubunit HDAC complex conserved from yeast to human (Silverstein, 2005). SIN3 is believed to serve as a scaffold protein for assembly of the complex and has been shown to be the major subunit that targets the complex to specific promoters (Silverstein, 2005). Null mutations in Drosophila Sin3A result in embryonic lethality with only a few animals surviving to the first larval instar stage (Neufeld, 1998b; Pennetta, 1998). For this reason, investigating the role of SIN3 in the regulation of specific developmental pathways has proven difficult. SIN3 is believed to be involved in various biological processes linked to development and cell cycle progression. For instance, SIN3 has been implicated in eye development as Drosophila Sin3A was isolated in a screen to identify factors involved in modulation of the rough eye phenotype caused by ectopic expression of seven in abstensia (Neufeld, 1998b). SIN3 has also been linked to developmental regulation through hormone signaling. SIN3 has been shown to associate with SMRTER, a 20-hydroxyecdysone (ecdysone) steroid hormone corepressor (Tsai, 1999). SIN3 colocalizes with SMRTER on polytene chromosomes isolated from Drosophila third instar larvae salivary glands (Pile, 2000). SIN3 binding to ecdysone steroid hormone regulated genes was shown to be dynamic and coincident with a developmental expression pattern of these genes in response to hormone signal. SIN3 has also been shown to be important for cell proliferation. Knock down of SIN3 in Drosophila tissue culture cells by RNA interference (RNAi) resulted in a G2 phase delay in cell cycle progression (Pile, 2002). Furthermore, comparison of gene expression profiles from wild-type and RNAi-induced SIN3-deficient cells revealed differences in expression of genes encoding proteins that control multiple cellular processes, including cell cycle progression, transcription, and signal transduction (Pile, 2003). Taken together, the phenotypes of the Drosophila mutants and the tissue culture knock down cells, along with the links to hormone signaling, suggest that SIN3 is a critical regulator of development and cell cycle progression (Sharma, 2008).

Drosophila Sin3A is represented by a single gene. Multiple cDNAs that correspond to alternatively spliced transcripts, however, have been isolated (Neufeld, 1998b; Pennetta, 1998). These transcripts are predicted to produce distinct protein isoforms that differ in amino acid sequence only at the carboxy (C)-terminus of each protein. An antibody raised against a region of the SIN3 protein common to all predicted isoforms recognized proteins of approximately 200 and 220 kDa in embryonic extracts (Pile, 2000). These protein sizes are consistent with the predicted molecular weights of the isoforms of 187, 190, and 220 kDa. Accordingly, the different SIN3 isoforms have been named SIN3 187, SIN3 190, and SIN3 220. Mammals have two SIN3 genes: SIN3A and SIN3B. Multiple alternatively spliced transcripts from mouse and human SIN3A and SIN3B have been reported (Yang, 2000; Alland, 2002). Interestingly, similar to the finding in Drosophila, the isoforms differ at the C-terminal region of the predicted proteins (Sharma, 2008).

To advance an understanding of the role of SIN3 in development, the expression pattern of SIN3 isoforms were examinted, and the consequences of elimination of SIN3 expression at various stages of the Drosophila life cycle was determined . It was found that while many tissues express multiple SIN3 isoforms, the relative expression of the isoforms varies in a developmental stage and tissue-specific manner. To assess the effect of loss of SIN3 at later stages of development, a conditional SIN3 knock down transgenic line was developed that circumvents the embryonic requirement for SIN3. Loss of SIN3 during postembryonic development leads to lethality, indicating that SIN3 is required at multiple stages of Drosophila development (Sharma, 2008).

To further the analysis of SIN3 isoform expression, protein expression was examined in multiple tissues during distinct time points of fly development. To determine the relative levels of the different isoforms during embryogenesis, protein extracts prepared from embryos collected at different stages were analyzed by Western blot using the SIN3 pan antibody. This antibody was raised against recombinant protein containing sequence common to all reported isoforms. Two protein bands were detected at all time points tested. One corresponded to SIN3 220, and the other corresponded to SIN3 187, SIN3 190, or a mixture of the two. The difference in molecular weight between SIN3 187 and SIN3 190 is too small to be resolved on an eight percent polyacrylamide gel (Sharma, 2008).

SIN3 220 expression was highest in extracts prepared from embryos at stages 12-16. Germ band retraction, dorsal closure, and ventral nerve shortening occur during these stages. SIN3 220 expression dropped at stage 17, the final stage of embryogenesis when the ventral nerve cord continues to retract and the majority of morphogenesis is complete. Expression of the lower molecular weight isoforms steadily increased as embryogenesis progressed, and remained high at the final stage 17. The relative levels of SIN3 220 and SIN3 187/190 thus change during embryogenesis. This finding that the SIN3 isoforms have a differential temporal expression pattern during embryonic development suggests that they may have different functions. It is possible that the different isoforms regulate distinct sets of genes that are required for specific developmental events (Sharma, 2008).

To analyze expression in different tissues and developmental stages of the fly, protein extracts prepared from various larval and adult tissues were analyzed by Western blot using the SIN3 pan antibody. Larval imaginal discs are comprised of committed cells that undergo differentiation resulting in the adult body structures. SIN3 220 is expressed at a higher level relative to the lower molecular weight isoforms in the wing and leg imaginal discs of third instar larvae. The isoforms are expressed at similar levels to each other in eye and antennal discs and larval brain tissue. SIN3 220 expression is much lower compared with the lower molecular weight isoforms in adult brain and total adult tissue. Of interest, in embryogenesis, larvagenesis, and adulthood, the ratio of SIN3 220 to the lower molecular weight isoforms is generally reflective of the ratio of undifferentiated to differentiated cells. SIN3 220 appears to be expressed equivalently to, or more than, the lower molecular weight isoforms in tissues that have numerous mitotic cells and that are less differentiated. In the most differentiated tissue, such as that of the final stage of embryogenesis, and in adults, SIN3 220 expression is very low. In support of this hypothesis, continuous Drosophila tissue culture cell lines primarily express SIN3 220 (Sharma, 2008).

Given that all isoforms were detected in the embryo, whether the expression of individual isoforms was restricted to specific cell types or localized to particular regions of the embryo was investigated. To determine the spatial expression patterns of individual SIN3 isoforms, either in situ hybridization or immunohistochemistry was used, depending on available reagents. To determine RNA expression patterns, in situ hybridization was performed in fly embryos of different stages. Using a probe that identifies all SIN3 isoform transcripts (SIN3 pan probe), it was found that SIN3 is ubiquitously expressed during embryogenesis. As embryonic development progressed, SIN3 RNA concentrated was observed in the germ band and high levels of staining were noted in the ventral nerve cord and the brain. To determine the expression pattern of SIN3 187 and 190, probes were designed specific for these isoforms. The SIN3 220 transcript is comprised of sequence that is also found in the 190 transcript, therefore, it was not possible to design a SIN3 220-specific riboprobe. The expression patterns of SIN3 187 and 190 observed using the isoform-specific probes are very similar to the pattern observed using the SIN3 pan probe. Expression of SIN3 187 and 190 is ubiquitous in embryos, and in more developed embryos, is concentrated in the germ band, with strong staining in the ventral nerve cord and the brain (Sharma, 2008).

To identify the expression pattern of SIN3 220, immunostaining in fly embryos of different stages was performed using the SIN3 220 antibody. Antibodies specific for the SIN3 220 isoform were generated using recombinant protein consisting of the unique C-terminal region of SIN3 220 (amino acids 1748-2062). The specificity of the antibody was tested by Western blot analysis. The antibody recognizes a protein of approximately 220 kDa in tissue culture and embryonic extracts. Reduced signal was detected in extracts prepared from SIN3-deficient tissue culture cells. In addition to the SIN3 220 antibody, immunohistochemistry was performed using the SIN3 pan antibody that recognizes SIN3 187, 190 and 220. A pool of 0-18 hr embryos was immunostained for SIN3 using either the SIN3 pan or the SIN3 220 antibody. The expression pattern observed using the SIN3 220 antibody is very similar to the pattern observed using the SIN3 pan antibody and to that observed for the SIN3 RNA transcripts. The SIN3 220 protein is ubiquitously expressed, and exhibits strong expression in the germ band, ventral nerve cord and brain during later development. Confocal microscopy images revealed that SIN3 is localized to the nucleus. The results generated from the in situ hybridization and immunohistochemistry experiments suggest that SIN3 187, SIN3 190, and SIN3 220 are expressed in similar spatial patterns during fly embryonic development. SIN3 is thus ubiquitously expressed during embryogenesis, consistent with a previous descriptive report (Penneta, 1998) of SIN3 embryonic expression (Sharma, 2008).

The expression pattern analysis indicates that the SIN3 187 and 220 isoforms are expressed throughout Drosophila development. Because SIN3 is required for embryogenesis, characterization of the role of SIN3 in larval and pupal development has not been possible (Neufeld, 1998b; Pennetta, 1998). Therefore, to determine whether SIN3 is also required for postembryonic development, a conditional knock down transgenic fly was designed. In Drosophila, conditional knock down can be achieved by developmental stage-specific induction of RNA interference (RNAi) using the GAL4-UAS system. A transgene (UAS-SIN3RNAi) was constructed designed to target the degradation of all SIN3 isoforms. Tubulin, actin, and heat shock GAL4 driver lines were used to knock down SIN3 expression in all tissues. Progeny resulting from the cross of UAS-SIN3RNAi lines to GAL4 driver lines are referred to as SIN3-deficient flies (Sharma, 2008).

To induce ubiquitous loss of SIN3, heterozygous tub-GAL4 or Act-GAL4 driver males were crossed to homozygous UAS-SIN3RNAi females. Half of the progeny are expected to be SIN3-deficient. Nine independent UAS-SIN3RNAi lines were tested. As a control, both UAS-SIN3RNAi females and GAL4 driver males were crossed to w1118 males and females respectively. Progeny of all crosses were allowed to develop to adulthood. Three independent test and control crosses were set up for each UAS-SIN3RNAi line. The minimum number of adults that were scored was 54. In eight of nine test crosses, no SIN3-deficient flies survived to adulthood. Flies from the single test cross that survived to adulthood showed no obvious phenotype. Western blot analysis revealed that these adult flies expressed SIN3 at levels comparable to control animals, suggesting that SIN3 is not being effectively knocked down in that single viable test cross. The finding that ubiquitous loss of SIN3 resulting from RNAi leads to lethality is consistent with previous reports (Neufeld, 1998b; Pennetta, 1998) demonstrating that SIN3 is essential during early stages of development (Sharma, 2008).

To determine the stage of development during which the SIN3-deficient flies die, the development was followed of embryos from the control and test crosses. In the control crosses, 100% of embryos survived to adulthood. In the test cross, 72% of embryos hatched into first instar larvae, but only 51% developed into wandering third instar larvae (consistent with the predicted ratio of progeny that will be SIN3-deficient). All surviving wandering third instar larvae developed into adults. All surviving adults in the test cross had stubble bristles, indicating that they were not SIN3-deficient flies. These results indicate that SIN3-deficient embryos die during embryonic and first, second, or early third instar larval development (Sharma, 2008).

To verify the knock down of SIN3, and to analyze the SIN3-deficient embryos, embryos from the test cross were immunostained for SIN3 using the SIN3 pan antibody, and DNA using DAPI. Initially a pool of 0-20 hr embryos was examined. Loss of SIN3 upon induction of RNAi is inferred by the decrease in SIN3 staining intensity in 37% of these embryos. This number is smaller than the predicted 50%. RNAi-induced loss of SIN3 expression is a consequence of degradation of transcribed RNA by the introduction of double stranded RNA (dsRNA) and of degradation of existing protein by normal cellular turnover. Depending on the stability of SIN3, the RNAi-induced effect may be delayed long after induction of the dsRNA from the UAS-SIN3RNAi transgene. To allow for protein turnover, embryos were collected for 2 hr, and allowed to age 18 hr. This pool of 18- to 20-hr embryos was immunostained with the SIN3 pan antibody. In this aged population, 48% of the embryos had little to no SIN3 staining. DNA staining of the 0- to 20-hr collection with DAPI revealed that the SIN3-deficient embryos fell into different phenotypic categories. Some of the SIN3-deficient embryos had wild-type morphology and the stage of development could thus be determined. 29% of the SIN3-deficient embryos were in stages 9-11, 26% in stages 12-14, and 11% in stage 15. The remaining 34% of the SIN3-deficient embryos had poor DNA staining by DAPI (4,6-diamidine-2-phenylidole-dihydrochloride), and in some, the cells of the embryo appeared to pull away from the periphery, suggesting embryo degeneration. Due to loss of recognizable cellular structure, a stage of development for these embryos could not be assigned. As no SIN3-deficient embryos at stage 16 or later were identified, ubiquitous loss of SIN3 by RNAi appears to allow development for approximately 13 hr, to stage 15. The finding that some embryos develop to stage 15 is likely due to the presence of maternally deposited SIN3 that is not targeted by the RNAi pathway (Pennetta, 1998). That some SIN3-deficient animals survive to larvae is possibly due to the presence of a low level of SIN3 that allows development to that stage. Eventually SIN3 is reduced to lethal levels in all animals having both the GAL4 driver and the UAS-SIN3RNAi transgenes. The SIN3-deficient larvae appeared phenotypically normal, but failed to continue to develop into wandering third instar larvae. Loss of SIN3 by RNAi in Drosophila tissue culture cells resulted in loss of cell proliferation, likely due to a G2 cell cycle block (Pile, 2002). Homozygous null SIN3 clones in the developing eye resulted in scars across the eye consistent with a role for SIN3 in cell survival or proliferation (Neufeld, 1998a). Given these previous findings, it was hypothesized that lethality following RNAi induced loss of SIN3 in developing Drosophila results either from loss of cell proliferation or cell viability (Sharma, 2008).

To determine the effect of loss of SIN3 on postembryonic development, SIN3 RNAi was induced at different stages of larval development by crossing hsp70-GAL4 males to UAS-SIN3RNAi females. Control crosses were set up as mentioned above. Embryos were collected and subjected to initial heat shock at different stages of development to induce SIN3 RNAi. The developing larvae were subjected to heat shock by incubating at 37°C for 1 hr. The larvae were subjected to heat shock each day, with a 24-hr recovery period at room temperature between heat shock treatments, until the larvae either died or developed into adults. The number of animals that survived to wandering third instar, pupae, and adulthood was determined. Induction of SIN3 RNAi in first or second instar larvae caused lethality before the wandering third instar, while inducing loss of SIN3 in wandering third instar larvae or pupae had no detectable effect on fly viability. Inspection of the SIN3-deficient dead larvae revealed no gross phenotypic abnormalities, and the surviving adults appeared phenotypically normal (data not shown) (Sharma, 2008).

The transitions from larva to prepupa, and from prepupa to pupa, are each driven by pulses of the steroid hormone ecdysone. Induction of loss of SIN3 in first, second, or early third instar, before the ecdysone pulse at the end of the third instar larval stage, results in lethality, while induction during or after the time frame of this pulse has no effect on fly viability. SIN3 has been found to bind to ecdysone-regulated genes on polytene chromosomes isolated from third instar larval salivary glands (Pile, 2000). It is possible that the larval lethality is due to aberrant expression of ecdysone responsive genes. Loss of SIN3 might lead to premature activation or lack of down-regulation of ecdysone target genes, resulting in altered expression of genes required for morphogenesis (Sharma, 2008).

Through the use of a conditional knock down transgenic fly system, it was established that SIN3 is required for both embryonic and early larval development. Results from these experiments have not detected a role for SIN3 in late larval, pupal, or adult development. The possibility was not, however, ruled out that SIN3 functions in these late stages of development, when, as in the early stages, expression is detected. Again, depending on the stability of SIN3, the RNAi-induced effect may occur after induction of the dsRNA from the UAS-SIN3RNAi transgene. Thus, even though RNAi was induced during the late larval and pupal stages, SIN3 protein levels may have remained at levels sufficient to allow development to the adult stage. It is also possible that in the latter stages of Drosophila development, other proteins are able to compensate for SIN3-deficiency (Sharma, 2008).

To confirm that the embryonic and larval lethality is a consequence of SIN3-deficiency and not due to an RNAi off target effect, a UAS-187 transgene (UAS driven expression of cDNA for the SIN3 187 isoform) was introduced into flies that are SIN3-deficient. Because the RNAi effect has been shown to be dose dependent, the expression of a SIN3 transgene should be able to rescue the RNAi-induced lethality. To test this hypothesis, flies were first generated carrying both the tub-GAL4 and UAS-187 trangenes on the third chromosome, over the TM3-Sb balancer chromosome. Half of the progeny from the cross of tub-GAL4, UAS-187/TM3-Sb X UAS-SIN3RNAi/UAS-SIN3RNAi would carry only the UAS-SIN3RNAi construct (identified by the presence of stubble bristles), while the other half would simultaneously express UAS-SIN3RNAi and over express SIN187 under the influence of tub-GAL4. Expression of SIN3 187 from UAS-187 increased survival of SIN3-deficient flies from 0 to 66% in males and 0 to 31% in females. Surviving adults appeared phenotypically normal. The rescue of lethality by the SIN3 187 transgene supports the idea that the dsRNA produced from the UAS-SIN3RNAi transgene specifically targets SIN3 RNA for degradation and that the RNAi-induced lethality is the result of SIN3-deficiency (Sharma, 2008).

Next a similar experiment was carried out to test rescue by SIN3 220. A UAS-220 transgene (UAS driven expression of cDNA for the SIN3 220 isoform) was introduced into flies that are SIN3-deficient. Interestingly, while SIN3 220 is also able to rescue SIN3 RNAi-induced lethality, the amount of rescue is not the same as that of SIN3 187. Expression of SIN3 220 from UAS-220 increased survival of SIN3-deficient flies from 0 to 53% in males and 0 to 55% in females. The difference in the ability of the individual isoforms to suppress the lethal phenotype is unlikely due to differences in the amount of expression from the two transgenes as the protein level of SIN3 187 and SIN3 220 in extracts prepared from adult females were similar. In contrast, the level of SIN3 220 in males compared with females was quite dissimilar. Like the SIN3 187 transgenic flies, surviving SIN3 220 transgenic adults appeared phenotypically normal. The finding that SIN3 187 or 220 can individually rescue the lethal phenotype due to knock down of expression of all SIN3 proteins suggests that SIN3 187 or 220 can partially substitute for the other isoforms. That the extent of rescue is different in males and females suggests the possibility that there may be sex-specific roles of the isoforms that cannot be compensated for by another isoform (Sharma, 2008).

It is well established that regulation and maintenance of histone acetylation levels are important for normal development. This study has investigated the expression and postembryonic requirement of SIN3, one component of a multisubunit HDAC complex. SIN3 isoforms were found to be expressed throughout development. The different isoforms have distinct patterns of expression. SIN3 187 has prominent expression in differentiated tissue such in the final stage 17 embryos and in adults. SIN3 220 expression is low in those differentiated tissues and high in proliferating cells such as larval imaginal discs and embryonic tissue culture cells. SIN3 190 has the most restricted pattern of the three; it was detected only in embryos and adult females. The distinct expression patterns and the finding that SIN3 187 and SIN3 220 rescue SIN3-deficiency to different levels lead to the possibility that these proteins regulate distinct genes that are required for specific developmental events. Consistent with their expression throughout development, elimination of SIN3 during embryonic and first, second, and early third instar larval development results in lethality. Further analysis of phenotypes resulting from SIN3-deficiency in specific tissues is anticipated to reveal the role of SIN3 in regulating specific developmental pathways (Sharma, 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. 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).

Sap18 is required for the maternal gene bicoid to direct anterior patterning in Drosophila melanogaster

Development of the insect head is a complex process that in Drosophila requires the anterior determinant, Bicoid. Bicoid is present in an anterior-to-posterior concentration gradient, and binds DNA and stimulates transcription of head-specific genes. Many of these genes, including the gap-gene hunchback, are initially activated in a broad domain across the head primordium, but later retract so that their expression is cleared from the anterior-most segmented regions. This study shows that retraction requires Bicoid-interacting protein, also known as Sap18, which is part of the Sin3A/Rpd3 histone deacetylase complex. In sensitized-mutant backgrounds (e.g., bcdE1/+, removal of maternal sap18 results in embryos that are missing labrally derived parts of the cephalopharyngeal skeleton. These sap18 mutant embryos fail to repress hb expression, and show reduced anterior cap expression of the labral determinant cap 'n' collar. These phenotypes are enhanced by lowering the dose of rpd3, which encodes the catalytic subunit of the deacetylase complex. The results suggest a model where, in labral regions of the head, Bicoid is converted from an activator into a repressor by recruitment of a co-repressor to Bicoid-dependent promoters. Bicoid's activity, therefore, depends not only on its concentration gradient, but also on its interactions with modifier proteins within spatially restricted domains (Singh, 2005).

Although SAP18 is conserved among humans, Caenorhabditis elegans, and flies, little is known about its function. No clear ortholog exists in yeast, and there are no compelling data on its exact role in any organism. No functional motifs are revealed by its sequence. It was discovered by biochemical fractionation of human cell extracts as an 18-kDa peptide that co-purifies with the Sin3A/Rpd3 HDAC complex, and enhances Sin3-mediated repression (Zhang, 1997). Drosophila Sap18 also interacts with GAGA and E(z) proteins, which are implicated in Trithorax- and Polycomb-mediated regulation of homeotic genes, respectively (Espinas, 2000 and Wang, 2002). Mammalian SAP18 interacts with Su(Fu), which is a repressor of the Gli transcription factor in the Hedgehog signaling pathway (Cheng, 2002). In each study, SAP18 was proposed to be an adaptor protein that bridges the interaction between a DNA-binding protein and a Sin3-HDAC co-repression complex. SAP18 was also found to be part of a complex known as ASAP, which contains both an RNA-binding protein (RNPS1) and a caspase (Acinus) (Schwerk, 2003), suggesting that it plays a role in RNA processing and apoptosis (Singh, 2005 and references therein).

This paper tested whether the interaction of Bicoid with Sap18 is important for embryonic head development, and whether Sap18 is required for retraction. To this end, P-element excision was used to generate a series of chromosomal deletions that removed the gene encoding Sap18 (sap18), and the consequences on both germline and embryonic development were examined. It was found that sap18 is required maternally and zygotically, and that embryos from bcd sap18 double mutant mothers fail to undergo normal retraction of the gap gene hb at the anterior tip of the embryo. Failure to down-regulate hb leads to loss of expression of the labral determinant cap 'n' collar, resulting in severe head defects. This phenotype is further enhanced by reducing the dosage of rpd3, indicating that repression by histone deacetylases is likely to be involved. Thus, Bicoid's activity is spatially regulated in the embryo, not only by its concentration gradient along the A-P axis, but also by its interaction with a modifier (co-repressor) protein that alters its activity. The results also reveal roles for Sap18 in oogenesis, and in larval and pupal development, that are independent of Bicoid (Singh, 2005).

Zygotic sap18 mutants arrested primarily as pupae that fail to eclose. Some larval lethality was also observed, but this occurred during the third instar larval stage. Therefore, it is likely that maternally derived Sap18 is sufficient for completion of embryogenesis, and that zygotic sap18 is not involved in bcd function. The pleiotropy observed in larval and pupal phenotypes is not surprising, given that Sap18 is a member of a general co-repressor complex that probably functions during multiple stages of development. The lethality of zygotic sap18 mutants is also conditional; whereas adults were readily recovered at 22°C, none were recovered at 25°C. Although the exact cause of this temperature sensitivity is not clear, it might be due to varying strengths of protein-protein interactions involving components of the Sin3-Rpd3 complex at 22°C and at 25°C (Singh, 2005).

Two major defects were observed in maternal sap18 mutants. (1) The vast majority of eggs laid by sap18 homozygous mothers did not initiate development, and appeared unfertilized. Using sap18R7−18 germline clones, this unfertilized egg phenotype was rescued so that most embryos initiated development normally. This indicates that sap18 is required in somatic cells during oogenesis, perhaps for proper formation of the vitelline membrane or chorion, defects that would prevent fertilization. (2) Maternal sap18 mutants displayed a variety of segmentation defects, including large holes in the anterior and thoracic cuticular pattern, head abnormalities, and deleted abdominal segments. The holes in the cuticles were similar to those seen in terminal system mutants and cell death mutants, while the head defects were reminiscent of phenotypes caused by some weak bcd alleles. The inability of zygotic sap18 to rescue these defects indicates that maternal sap18 is important for embryonic development. The variety of phenotypes observed suggests multiple roles for sap18 during embryogenesis (Singh, 2005).

Despite its importance, sap18 does not seem to be absolutely essential, as evidenced by the incomplete penetrance and variable expressivity of the sap18 mutant phenotypes. In addition, it was surprising that the expression patterns of most of the segmentation genes that were examined (>15) were not detectably altered in sap18 mutant embryos. Perhaps, this is because recruitment of the Sin3-Rpd3 to promoters also occurs via other proteins in the complex, or because other unrelated co-repressors such as Groucho or dCtBP play redundant roles (Singh, 2005).

In evolutionary terms, Bicoid is a relative newcomer to the network of regulatory proteins that pattern the insect head. In Drosophila, Bicoid apparently usurped some functions carried out by the Hunchback protein in more primitive insects. Bicoid has also evolved the ability to carry out multiple distinct functions. For example, in addition to its well-studied role in regulating RNA pol II transcription, it also represses caudal mRNA translation. To carry out these diverse functions and to limit its activity in different parts of the embryo, Bicoid is likely to interact with a discrete set of modifier proteins. Included in this set are the Bin3 protein methyltransferase, whose function is still unknown, eIF4E, and Sap18 (Singh, 2005).

Although Sap18 has been found in other complexes, several findings suggest that to modulate Bicoid activity, it recruits the Sin3-Rpd3 HDAC complex: (1) Sap18 downregulates Bicoid-dependent transcription in Drosophila S2 cells, and this inhibition is reduced by the addition of trichostatin A, a known HDAC inhibitor; (2) in the present study, hb retraction was impaired in bcd sap18 double heterozygotes, resulting in de-repression of hb mRNA levels at the anterior tip of the embryo; (3) mutation of rpd3 enhanced the bcd sap18 mutant phenotypes, resulting in greater loss of hb retraction and more severe head defects (Singh, 2005).

These results lead to the following model. At the anterior of the blastoderm embryo, just prior to cellularization, Bicoid interacts with maternal Sap18 thereby recruiting the Sin3-Rpd3 HDAC complex to repress hb transcription. Repression of hb allows the anterior cap expression of the labral determinant cnc, which is required for pharyngeal development. In this model, Bicoid-Sap18 interaction must be restricted to labral and perhaps acronal regions, despite the fact that maternal sap18 mRNA is present uniformly throughout the embryo (Zhu, 2001). This spatial restriction would ensure that zygotic hb can still be activated further down the Bicoid gradient. One possibility is that the Bicoid-Sap18 interaction is inherently weak, so that it only occurs at the anterior tip of the embryo where Bicoid concentrations are the highest. Alternatively, Bicoid-Sap18 interaction might be stimulated by the terminal system tor RTK pathway, whose activity is restricted to the poles of the embryo. Thus, the role of the terminal system might be to enhance co-repressor activity of a Bicoid-Sap18 HDAC complex, but this remains unproven (Singh, 2005).

Consistent with this model, bcd sap18 and bcd sap18 rpd3 mutant embryos phenocopied tor loss-of-function mutants. In tor mutants, the labrum is missing, the dorsal bridge is not formed, and there is a collapse of the head skeleton. This may be because hb retraction by Bicoid-Sap18 repression is tor-dependent. Or, the terminal system may act in a parallel and partially redundant manner with Bicoid (see below) (Singh, 2005).

The bcd sap18 and bcd sap18 rpd3 mutants also phenocopy an unusual bcd allele, bcdE5, which has a non-sense mutation at residue 264. bcdE5 mutant embryos have a normal thorax and posterior head, but they have defects in the anterior head. The phenotype of this bcdE5 allele has been dubbed 'dispersed deletion profile' and is contrary to the phenotypes of the normal bcd allelic series where weak alleles affect the thorax, intermediate alleles affect the thorax and posterior head, and strong alleles affect the thorax and both the posterior and anterior head. In bcdE5 embryos, Bicoid activity is only affected within a sub-region of the overall fate map that is under Bicoid control, a region similar to that controlled by Sap18 in the model presented in this study. It is possible that bcdE5 encodes a protein unable to interact with Sap18, and that bcdE5 would be indifferent to the removal of maternal sap18 (Singh, 2005).

Several observations suggest that the retraction of Bicoid-dependent gene expression is likely to be more complex than indicated by this model. (1) Retraction of otd and ems expression was not reduced in bcd, sap18, rpd3 mutant combinations, and thus does not seem to involve Bicoid-Sap18 repression. Therefore, conversion of Bicoid from an activator to a repressor by Sap18 appears to be promoter-dependent. For example, the spacing of Bicoid binding sites, which is known to affect Bicoid activity, or the presence of other co-regulators on the otd and ems promoters, might prevent repression by Bicoid-Sap18. This type of differential repression has been noted for another maternal determinant, Dorsal, in its interactions with the Groucho co-repressor. Also, otd retraction depends on hkb, a target of the terminal polarity system, whereas hb retraction does not. No changes were detected in hkb expression in sap18 mutants, consistent with the failure to see effects on otd retraction. Thus, retraction of hb and otd (and ems) may occur through independent pathways (Singh, 2005).

(2) Not all maternal sap18 mutant embryos showed head defects. Even in sensitized backgrounds, the sap18 mutant head phenotypes were incompletely penetrant and temperature-sensitive, showing observable phenotypes only at 29°C. One possibility is that Bicoid also interacts with other components of the HDAC complex such as Sin3, Sap30, or the catalytic subunit Rpd3, so that elimination of one bridge protein such as Sap18 would only reduce, but not abolish repression. Or, as suggested previously, there may be an inherent redundancy of co-repressor action such that under certain conditions, that is, reduced dosages of sap18 and rpd3, other co-repressors such as Groucho or dCtBP would substitute to repress hb transcription. Finally, it is possible that part of the repressive effects of Bicoid on hb during retraction are due to a self-inhibitory domain located outside the region that interacts with Sap18 (Singh, 2005).

In summary, these data show that Sap18 is required for Bicoid-dependent retraction of hb expression in the anterior head primordium, and that retraction is likely to be the result of recruitment of a histone deacetylase complex to the hb promoter. Retraction also requires the action of the terminal polarity system, but the mechanism by which this occurs remains obscure. The simplest explanation, that Bicoid activity is downregulated as a result of phosphorylation by the tor terminal system kinases, has been ruled out. An attractive alternative is suggested by results of the present study; that the tor kinase pathway regulates either the interaction of the Sap18-Sin3/Rpd3 HDAC complex with Bicoid, or its repression activity. Future experiments will examine these possibilities, and may help explain how the terminal and anterior polarity systems converge to specify head development (Singh, 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).

The SIN3 deacetylase complex represses genes encoding mitochondrial proteins: implications for the regulation of energy metabolism

Deacetylation of histones by the SIN3 complex is a major mechanism utilized in eukaryotic organisms to repress transcription. Presumably, developmental and cellular phenotypes resulting from mutations in SIN3 are a consequence of altered transcription of SIN3 target genes. Therefore, to understand the molecular mechanisms underlying SIN3 mutant phenotypes in Drosophila, full-genome oligonucleotide microarrays were used to compare gene expression levels in wild type Drosophila tissue culture cells versus SIN3-deficient cells generated by RNA interference. Of the 13,137 genes tested, 364 were induced and 35 were repressed by loss of SIN3. The approximately 10-fold difference between the number of induced and repressed genes suggests that SIN3 plays a direct role in regulating these genes. The identified genes are distributed throughout euchromatic regions but are preferentially excluded from heterochromatic regions of Drosophila chromosomes suggesting that the SIN3 complex can only access particular chromatin structures. A number of cell cycle regulators were repressed by loss of SIN3, and functional studies indicate that repression of string, encoding the Drosophila homologue of the yeast CDC25 phosphatase, contributes to the G2 cell cycle delay of SIN3-deficient cells. Unexpectedly, a substantial fraction of genes induced by loss of SIN3 is involved in cytosolic and mitochondrial energy-generating pathways and other genes encode components of the mitochondrial translation machinery. Increased expression of mitochondrial proteins in SIN3-deficient cells is manifested in an increase in mitochondrial mass. Thus, SIN3 may play an important role in regulating mitochondrial respiratory activity (Pile, 2003).

The SIN3/RPD3 deacetylase complex is essential for G(2) phase cell cycle progression and regulation of SMRTER corepressor levels

The SIN3 corepressor and RPD3 histone deacetylase are components of the evolutionarily conserved SIN3/RPD3 transcriptional repression complex. The SIN3/RPD3 complex and the corepressor SMRTER are required for Drosophila G2 phase cell cycle progression. Loss of the SIN3, but not the p55, SAP18, or SAP30, component of the SIN3/RPD3 complex by RNA interference (RNAi) causes a cell cycle delay prior to initiation of mitosis. Loss of RPD3 reduces the growth rate of cells but does not cause a distinct cell cycle defect, suggesting that cells are delayed in multiple phases of the cell cycle, including G2. Thus, the role of the SIN3/RPD3 complex in G2 phase progression appears to be independent of p55, SAP18, and SAP30. SMRTER protein levels are reduced in SIN3 and RPD3 RNAi cells, and loss of SMRTER by RNAi is sufficient to cause a G(2) phase delay, demonstrating that regulation of SMRTER protein levels by the SIN3/RPD3 complex is a vital component of the transcriptional repression mechanism. Loss of SIN3 does not affect global acetylation of histones H3 and H4, suggesting that the G2 phase delay is due not to global changes in genome integrity but rather to derepression of SIN3 target genes (Pile, 2002).

The consequences of the deacetylase inhibitor trichostatin A (TSA) on the development of Drosophila melanogaster was examined. When fed to flies, TSA causes lethality and delays development at concentrations as low as 5 microM, has stronger effects on males than females, and acts synergistically with mutations in the gene encoding the RPD3 deacetylase to cause notched wings, but does not appear to affect a SINA signaling pathway that is normally repressed by the SIN3 corepressor. These findings suggest that deacetylated histones play an important role in normal developmental progression and establish parameters for genetic screens to dissect the role of deacetylases in this process (Pile, 2001).

Drosophila Enhancer of zeste protein interacts with dSAP18

The Drosophila Enhancer of zeste [E(z)] gene encodes a member of the Polycomb group of transcriptional repressors. This study provides evidence for direct physical interaction between E(Z) and dSAP18, which previously has been shown to interact with Drosophila GAGA factor and Bicoid proteins. dSAP18 shares extensive sequence similarity with a human polypeptide originally identified as a subunit of the SIN3A-HDAC (switch-independent 3-histone deacetylase) co-repressor complex. Yeast two-hybrid and in vitro binding assays demonstrate direct E(Z)-dSAP18 interaction and show that dSAP18 is capable of interacting with itself. Co-immunoprecipitation experiments provide evidence for in vivo association of E(Z) and dSAP18. Gel filtration analysis of embryo nuclear extracts shows that dSAP18 is present in native protein complexes ranging from approximately 1100 to approximately 450 kDa in molecular mass. These studies provide support for a model in which dSAP18 contributes to the activities of multiple protein complexes, and potentially may mediate interactions between distinct proteins and/or protein complexes (Wang, 2002).

Drosophila SAP18, a member of the Sin3/Rpd3 histone deacetylase complex, interacts with Bicoid and inhibits its activity

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), 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).

Trithorax-like interaction with SAP18, a Sin3-associated polypeptide

Drosophila SAP18 (accepted FlyBase name: Bicoid interacting protein 1), a polypeptide associated with the Sin3-HDAC co-repressor complex, has been identified in a yeast two-hybrid screen as capable of interacting with the Drosophila GAGA factor. The interaction was confirmed in vitro by glutathione S-transferase pull-down assays using recombinant proteins and crude SL2 nuclear extracts. The first 245 residues of GAGA, including the POZ domain, are necessary and sufficient to bind dSAP18. In polytene chromosomes, Drosophila SAP18 and GAGA co-localize at a few discrete sites and, in particular, at the bithorax complex where GAGA binds some silenced polycomb response elements. When the Drosophila SAP18 dose is reduced, flies heterozygous for the GAGA mutation Trl67 show the homeotic transformation of segment A6 into A5, indicating that GAGA-dSAP18 interaction contributes to the functional regulation of the iab-6 element of the bithorax complex. These results suggest that, through recruitment of the Sin3-HDAC complex, GAGA might contribute to the regulation of homeotic gene expression (Espinas, 2000).

The identity of dSAP18 with either human (hSAP18) or C. elegans (cSAP18) SAP18 is high, ~60% and 47%, respectively. The three polypeptides show high homology throughout their sequences, except for the most N- (1-15) and C-terminal (138-150) residues, and a central region (residues 32-45). Two specific regions, RI (16-31) and RII (65-89), show a very high degree of conservation with a similarity >80%. A third region, RIII (123-137), also shows significant similarity (80%), but in this case the identity is lower (47%) than for regions RI (81%) and RII (67%) (Espinas, 2000).

GAGA is organized into several functionally distinct domains. A single zinc finger is involved in nucleic acid recognition. In addition to this central DNA binding domain (DBD), GAGA carries a C-terminal glutamine-rich domain (Q-domain), which is involved in transcription activation, and a highly conserved N-terminal POZ domain, which mediates protein-protein interactions. A relatively long (140 amino acids) region of unknown function(s) links the POZ and DBD domains. Little is known about the interaction of GAGA with other nuclear proteins. The POZ domain of GAGA has been shown to support homomeric as well as heteromeric interactions with other POZ-containing proteins, such as tramtrack (ttk). The first 245 residues of GAGA are necessary and sufficient for binding dSAP18, and efficient GAGA-dSAP18 interaction requires the contribution of both the POZ and linking domains of GAGA. Not all regions of dSAP18 contribute equally to its interaction with GAGA, and residues 73-113, which include most of the highly conserved RII region, are mainly responsible for binding to GAGA (Espinas, 2000).

SAP18 was identified as a polypeptide associated with the mammalian transcriptional repressor Sin3. The core mSin3 complex contains a total of seven polypeptides, which include the histone deacetylases HDAC1 and HDAC2, RbAp48 and RbAp46, and SAP30 and SAP18. Recruitment of the Sin3-HDAC complex to specific target genes appears to rely on its interaction with sequence-specific DNA binding proteins, since none of the known components of the complex are capable of binding DNA. SAP30 and SAP18 could mediate some of these interactions. mSAP30 binds both mSin3 and N-CoR, and is required for N-CoR-mediated repression by a set of sequence-specific DNA-binding transcription factors. SAP18 could also be involved in interactions with sequence-specific DNA binding proteins. It is known that SAP18 interacts directly with mSin3. Since POZ is a highly conserved structural domain, it is likely that similar interactions would be observed with other POZ domains. Actually, several sequence-specific transcriptional repressors carry POZ domains, some of which are also found to interact with N-CoR and SMRT. Most likely, formation of a stable complex requires multiple interactions between its various components (Espinas, 2000 and references therein).

Contrary to most POZ-containing proteins, the Drosophila GAGA factor acts as a transcriptional activator. Its interaction with a component of the Sin3 co-repressor complex indicates that GAGA might also act as a repressor in some cases. In this respect, the presence of GAGA at some silenced PREs of the bithorax and antennapedia complexes might be especially revealing. Interestingly, though the immunostaining patterns of GAGA and dSAP18 show only a limited general overlapping in polytene chromosomes, the two proteins co-localize at the region of the bithorax complex (BX-C), suggesting a possible contribution of GAGA-dSAP18 interaction to BX-C regulation. Consistent with this possibility, a genetic interaction between Trl and a deficiency that uncovers dSAP18 has been observed. Flies heterozygous for the Trl67 mutation and hemizygous for Df(3R)sbd26 show a homeotic transformation of the sixth abdominal segment into the fifth as indicated by the presence in the sixth sternite of several bristles in the vast majority of the individuals. Results presented indicate that GAGA-dSAP18 interaction has a significant contribution to the functional regulation of the iab-6 element of BX-C (Espinas, 2000).

The concurrent presence of GAGA and Polycomb at some silenced PREs is surprising since, as derived from genetic analysis, these two proteins are expected to have opposing functions on the regulation of the expression of the homeotic genes. Acting at the level of the core promoter elements, GAGA is likely to activate transcription of the homeotic genes. However, functional trithorax response elements (TREs) are frequently found in the vicinity of PREs and a contribution of GAGA to the functional regulation of several segment-specific cis-regulatory regions of the bithorax complex has been reported (Espinas, 2000 and references therein).

It is still uncertain whether GAGA helps to establish the repressed or the active state of these elements. GAGA has been shown to contribute to the relief of repression at the Fab-7 element, and the homeotic transformations described here and elsewhere are also consistent with a role in activation. However, in the case of the iab-7 and bxd PREs, GAGA has been shown to contribute to silencing and the genetic interactions observed between some Pc and Trl alleles also suggest a contribution to repression. The results presented here indicate that GAGA might participate in the recruitment of the Sin3-HDAC co-repressor complex to some PREs, but that contrary to what would be anticipated for such an interaction, it contributes to the relief of repression at the iab-6 element. The same phenotype is observed in flies homozygous for the hypomorph Trl13C allele. Interestingly, some rpd3 alleles behave as enhancers of PEV, also leading to an increase in repression. It is possible that by modifying chromatin structure, GAGA-SAP18 interaction could contribute to the establishment of the domain boundaries that insulate different cis-regulatory elements, rather than to the formation of the repressed or active states themselves (Espinas, 2000).

SMRTER, a Drosophila nuclear receptor coregulator, reveals that EcR-mediated repression is critical for development

The Ultraspiracle heterodimer is a Drosophila corepressor mediates transcriptional silencing of the Ecdysone receptor. SMRT-related ecdysone receptor-interacting factor (Smrter), also 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 (see Drosophila Rpd3), 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).

Genetics experments have provided the first evidence for the existence of a corepressor for EcR:Usp heterodimers. Previous genetic studies have shown that mutations in the ecdysone receptor give rise to lethal and developmental defects, indicating that this signaling pathway is essential for development. However, the means by which these mutations cause the observed defects remain unclear. The fact that the EcR:USP heterodimer can carry out its regulatory functions as both an activator and a potent repressor in vertebrate cultured cells (CV-1 cells) enabled the use mammalian cells to carry out initial studies on the molecular consequences of these EcR mutations. One of these EcR mutations, EcR with a mutation in alanine 483 (A483T), is of particular interest as it displays increased reporter activity both in the absence and the presence of hormone. In this experiment, EcR or its mutant derivative, EcRA483T, was cotransfected along with vp16-USP and a luciferase reporter that contains multimerized Ecdysone receptor response elements (hsp27EcREs). A potential explanation for its elevated activity is that EcR A483T fails to recruit an endogenous (vertebrate) repressor complex in CV-1 cells. To investigate whether repression by EcR in CV-1 cells is mediated by its association with a vertebrate corepressor and whether such an interaction, if it does occur, is impaired by the A483T mutation, a mammalian two-hybrid assay with Gal4-c-SMRT, the vertebrate corepressor, was conducted. No significant induction of the Gal4 reporter has been observed for Gal4-c-SMRT alone or in cells cotransfected with EcR or with vp16-USP. However, in the presence of both EcR and vp16-USP, the resulting heterodimerization shows strong association with Gal4-c-SMRT. The addition of hormone appears to completely dissociate SMRT from the heterodimer complex, therefore eliminating reporter activity. In contrast, EcR A483T alone or in the presence of vp16-USP is unable to associate with Gal4-c-SMRT, resulting in minimal activity in the presence or absence of ligand. Not only does this result suggest that A483 of EcR is critical for corepressor binding, but it also reveals that EcR:USP is a preferred binding complex for SMRT (Tsai, 1999).

Sequence alignment of EcR with the vertebrate TR, RAR, and v-erbA, an oncogenic TR variant, reveals that this alanine 483 is located within a highly conserved 23-amino acid (aa) loop region connecting helices 3 and 4, termed the LBD signature motif. Based on structural studies of vertebrate nuclear receptors, this alanine residue appears to be on the exposed surface, consistent with it being a potential corepressor binding site for nuclear receptors. In vivo studies indicate that EcRA483T is a semilethal allele. When EcRA483T is placed in trans with EcRE261st (an allele that removes both the DBD and LBD domains of EcR), lethality (greater than 95%) ensues. However, the few surviving EcRA483T/EcRE261st flies display significant delays in development, blistered wings, and defective tergites, indicating that EcR is involved in the development of these tissues. The ability of EcR to bind a vertebrate corepressor and the loss of this property in EcR A483T suggests that the defects observed in EcRA483T flies may result from the disruption of its interaction with a Drosophila corepressor (Tsai, 1999).

Although EcR readily interacts with vertebrate SMRT in both mammalian and yeast cells, repeated low-stringency hybridization screens failed to identify a Drosophila SMRT homolog. Given that no SMRT/N-CoR homolog is found in C. elegans, it was speculated that either a SMRT/N-CoR-like corepressor is not conserved in invertebrates or, alternatively, invertebrate corepressors may have diverged significantly from their vertebrate counterparts. To pursue the isolation of an EcR corepressor, a yeast interaction screen of a Drosophila embryonic cDNA library using EcR as bait was conducted. This screen resulted in the isolation of a clone, E52, whose protein product interacts with EcR as well as with the vertebrate RAR and TR, but notably not with USP. Intriguingly, unlike the interaction between E52 and RAR, which can be dissociated by all-trans retinoic acid, the interaction between E52 and EcR or the interaction between SMRT and EcR is not dissociated by Muristerone A (MurA). This result suggests that other factors essential for the dissociation of E52 from EcR, such as USP, are missing in yeast (Tsai, 1999).

Isolation of overlapping cDNA and genomic clones led to the identification of a full-length sequence encoding a large protein of 3446 amino acids. This protein contains several unusually long stretches of Gln, Ala, Gly, and Ser repeats. Comparative analysis reveals it to be a novel protein with limited regions of clear homology with the vertebrate nuclear receptor corepressors SMRT and N-CoR. This protein was named Smrter (SMRT-related ecdysone receptor-interacting factor). Northern blot analysis indicates that Smrter encodes large transcripts (greater than 12 kb) expressed broadly throughout the embryonic stage and three larvae stages, as well as in adult flies (Tsai, 1999)

To fulfill criteria necessary to establish Smrter as a corepressor of EcR, the Gal4-Smrter constructs were each examined for their ability to repress basal transcription. This resulted in the identification of three autonomous repressor domains termed SMRD1, SMRD2, and SMRD3, which respectively decrease basal activity of Gal4-DBD by approximately 17-fold, approximately 5-fold, and approximately 14-fold. Interestingly, SMRD1 contains the SNOR and SANT domains while SMRD2 overlaps with Ecdysone receptor interaction domain 1 (ERID1). Since repression by SMRT and N-CoR is mediated, at least in part, by the mSin3/HDAC complex and Smrter functions as a potent repressor in mammalian cells, it was asked whether the SMRD1, 2, or 3 could associate with vertebrate mSin3A. In the yeast two-hybrid assays, SMRD3 showed a strong association with mSin3A, while the interactions between SMRD1 or 2 and mSin3A are essentially undetectable. Using serial deletion constructs, a minimal Smrter-interacting domain has been localized to the first amphipathic helix of mSin3A. This region, PAH1, is also the primary site in mSin3 for the interaction with SMRT and N-CoR. These results indicate that at least one of the repression domains of Smrter can interact with vertebrate mSin3A and suggest that, despite their differences, insect and vertebrate corepressors employ a similar mechanism to recruit the repressor complex. However, the lack of significant interactions among SMRD1, SMRD2 and mSin3A raises the possibility that they may repress transcription using different strategies. The Drosophila mSin3A homolog (dSin3A) (Neufeld, 1998b and Pennetta, 1998) shows substantial sequence similarity to mSin3A in the four PAH domains as well as in the region between PAH3 and PAH4, which is required for interaction with histone deacetylase. The yeast two-hybrid screen was used in pairwise assays to identify Smrter interaction domains in dSin3A. The results reveal again that Smrter also interacts with dSin3A and that the region, including the PAH1 of dSin3A, is critical for associating with SMRD3 (Tsai, 1999).

Sequence comparison of SMRD3 with the defined Sin3-interacting domains of N-CoR and SMRT reveals a potential shared motif (the DALA motif) with N-CoR (aa 1835-1859), but not with SMRT. Deletion of a predicted helical secondary structure in N-CoR (aa 1833-1845), which includes the DALA motif, severs the interaction with mSin3A. The significance of this DALA motif is further strengthened by the finding that replacement of several conserved residues within the DALA motif of SMRD3 (mutations M2 and M3) destroys the interaction between SMRD3 and mSin3A/dSin3A in yeast two-hybrid experiments. In functional assays, these two mutated SMRD3s consistently fail to repress basal activity of Gal4-DBD, supporting the idea that association with Sin3A is a necessary component of Smrter-mediated repression (Tsai, 1999).

In keeping with the evidence that dSin3A is a component in the EcR regulatory pathway, whether dSin3A interacts genetically with EcR was examined using several previously characterized EcR and dSin3A mutants. In the experiment, in which female dSin3AK07401 mutants were crossed with male EcRE261st mutants, only (approximately) 14% of the scored EcRE261st/dSin3AK07401 progenies survived; this is significantly lower than the expected 33.3%. This suggests that a large portion of the EcRE261st/dSin3AK07401 animals either die prior to eclosion or fail to eclose. Additionally, surviving EcRE261st/dSin3AK07401 escapers show delayed development and wing defects, which are held horizontally at 45°-90° angle from the body axis. These results suggest that dSin3A shares an overlapping regulatory pathway with EcR. Strikingly, in a reverse genetic cross, in which female EcRE261st flies were crossed with male dSin3AK07401 flies, none of the EcRE261st/dSin3AK07401 flies survived to adulthood. Apparently, EcRE261st/dSin3AK07401 results in a genetically sensitized background. By halving the maternally deposited EcR in embryos descended from female EcRE261st/SM6b, the lethality for EcRE261st/dSin3AK07401 is further increased. These results reveal that, in addition to its previously known zygotic function, EcR also contributes maternally to Drosophila development (Tsai, 1999).

The selectivity of the point mutation A483T in disrupting Smrter association is noteworthy because it still allows heterodimer formation, DNA binding, ligand binding, and activation. Based on the crystal structure of vertebrate nuclear receptors, residue 483 of EcR is located at the surface exposed loop between helices 3 and 4 of the LBD. Given that this region is well conserved among nuclear receptors and that the EcRA483T mutation disrupts the binding of both Smrter and SMRT, this L3-4 represents a likely component of the binding site. If this is indeed the case, it is expected that the ability of TR and RAR to interact with SMRT or N-CoR may also be impaired by mutations at the corresponding residue of these two vertebrate nuclear receptors. In this study, the phenotype of the lethal EcRA483T mutant could be linked to a selective deficiency in corepressor binding, thus providing strong evidence as to the importance of nuclear receptor:corepressor complex in animal development. This result reveals the outcome of a constitutive dissociation of corepressor from nuclear receptor; this complements previous studies that have linked several oncogenic diseases to the constitutive association of corepressor to nuclear receptors. For example, v-erbA, an oncogenic TR variant, acts as a constitutive repressor due to its constant unregulated association with SMRT, and two translocation mutant forms of the RAR in humans, RAR-PLZF and RAR-PML, which are involved in acute promyelocytic leukemia, also repress transcription because of unregulated association with corepressor. Together, these results indicate that a coordinated interaction between corepressors and nuclear receptors is an essential feature of metazoan development and hormone action (Tsai, 1999 and references therein).

The Drosophila Sin3 gene encodes a widely distributed transcription factor essential for embryonic viability

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).


REFERENCES

Search PubMed for articles about Drosophila Sin3a

Alland, L., et al. (2002). Identification of mammalian Sds3 as an integral component of the Sin3/histone deacetylase corepressor complex. Mol. Cell. Biol. 22: 2743-2750. PubMed ID: 11909966

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Cai, Y. and Laughon, A. (2009). The Drosophila Smad cofactor Schnurri engages in redundant and synergistic interactions with multiple corepressors. Biochim. Biophys. Acta 1789(3): 232-45. PubMed ID: 19437622

Canudas, S., et al. (2005). dSAP18 and dHDAC1 contribute to the functional regulation of the Drosophila Fab-7 element. Nuc. Acids Res. 33(15): 4857-64. PubMed ID: 16135462

Cheng, S. Y. and Bishop, J. M. (2002). Suppressor of Fused represses Gli-mediated transcription by recruiting the SAP18-mSin3 corepressor complex. Proc. Natl. Acad. Sci. 99: 5442-5447. PubMed ID: 11960000

Cowley, S. M., et al. (2005). The mSin3A chromatin-modifying complex is essential for embryogenesis and T-cell development. Mol. Cell. Biol. 25: 6990-7004. PubMed ID: 16055712

Dannenberg, J. H., et al. (2005). MSin3A corepressor regulates diverse transcriptional networks governing normal and neoplastic growth and survival. Genes Dev. 19: 1581-1595. PubMed ID: 15998811

David, G., et al. (2008). Specific requirement of the chromatin modifier mSin3B in cell cycle exit and cellular differentiation. Proc. Natl. Acad. Sci. 105: 4168-4172. PubMed ID: 18332431

De Rubertis, F., et al. (1996). The histone deacetylase RPD3 counteracts genomic silencing in Drosophila and yeast. Nature 384(6609): 589-91. PubMed ID: 8955276

Dobi, K. C., Halfon, M. S., Baylies, M. K. (2014) Whole-genome analysis of muscle founder cells implicates the chromatin regulator Sin3A in muscle identity. Cell Rep 8(3):858-70. PubMed ID: 25088419

Espinas, M. L., et al. (2000). The GAGA factor of Drosophila interacts with SAP18, a Sin3-associated polypeptide. EMBO Rep. (3): 253-9. PubMed ID: 11256608

Matyash, A., et al. (2009). SAP18 promotes Krüppel-dependent transcriptional repression by enhancer-specific histone deacetylation. J. Biol. Chem. 284(5): 3012-20. PubMed ID: 19049982

Nagl, N. G., et al. (2007). Distinct mammalian SWI/SNF chromatin remodeling complexes with opposing roles in cell-cycle control. EMBO J. 26: 752-763. PubMed ID: 17255939

Neufeld, T. P. de la Cruz, A. F. Johnston, L. A. and Edgar, B. A. (1998a). Coordination of growth and cell division in the Drosophila wing. Cell 93: 1183-1193. PubMed ID: 9657151

Neufeld, T. P., Tang, A. H. and Rubin, G. M. (1998b). A genetic screen to identify components of the sina signaling pathway in Drosophila eye development. Genetics 148: 277-286. PubMed ID: 9475739

Pennetta, G. and Pauli, D. (1998). The Drosophila Sin3 gene encodes a widely distributed transcription factor essential for embryonic viability. Dev Genes Evol. 208(9): 531-6. PubMed ID: 9799435

Pile, L. A. and Wassarman, D. A. (2000). Chromosomal localization links the SIN3-RPD3 complex to the regulation of chromatin condensation, histone acetylation and gene expression. EMBO J. 19: 6131-6140. PubMed ID: 11080159

Pile, L. A., Lee, F. W. and Wassarman, D. A. (2001). The histone deacetylase inhibitor trichostatin A influences the development of Drosophila melanogaster. Cell Mol. Life Sci. 58(11): 1715-8. PubMed ID: 11706997

Pile, L. A., Schlag, E. M. and Wassarman, D. A. (2002). The SIN3/RPD3 deacetylase complex is essential for G(2) phase cell cycle progression and regulation of SMRTER corepressor levels. Mol. Cell. Biol. 22(14): 4965-76. PubMed ID: 12077326

Pile, L. A., Spellman, P. T., Katzenberger, R. J. and Wassarman, D. A. (2003). The SIN3 deacetylase complex represses genes encoding mitochondrial proteins: implications for the regulation of energy metabolism. J. Biol. Chem. 278(39): 37840-8. PubMed ID: 12865422

Rayman, J. B., et al. (2002). E2F mediates cell cycle-dependent transcriptional repression in vivo by recruitment of an HDAC1/mSin3B corepressor complex. Genes Dev. 16: 933-947. PubMed ID: 11959842

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Sif, S., et al. (2001). Purification and characterization of mSin3A-containing Brg1 and hBrm chromatin remodeling complexes. Genes Dev. 15: 603-618. PubMed ID: 11238380

Silverstein, R. A. and Ekwall, K. (2005). Sin3: a flexible regulator of global gene expression and genome stability. Curr. Genet. 47: 1-17. PubMed ID: 15565322

Singh, N., Zhu, W. and Hanes, S. D. (2005). Sap18 is required for the maternal gene bicoid to direct anterior patterning in Drosophila melanogaster. Dev. Biol. 278(1): 242-54. PubMed ID: 15649476

Swaminathan, A. and Pile, L. A. (2010). Regulation of cell proliferation and wing development by Drosophila SIN3 and String. Mech. Dev. 127: 96-106. PubMed ID: 19825413

Tsai, C.-C., et al. (1999). SMRTER, a Drosophila nuclear receptor coregulator, reveals that EcR-mediated repression is critical for development. Mol. Cell 4: 175-186. PubMed ID: 10488333

Vannier, D., Balderes, D. and Shore, D. (1996). Evidence that the transcriptional regulators SIN3 and RPD3, and a novel gene (SDS3) with similar functions, are involved in transcriptional silencing in S. cerevisiae. Genetics 144(4): 1343-53. PubMed ID: 8978024

van Oevelen, C., et al. (2008). A role for mammalian Sin3 in permanent gene silencing. Mol. Cell 32: 359-370. PubMed ID: 18995834

Vermeulen, M., et al. (2006). A feed-forward repression mechanism anchors the Sin3/histone deacetylase and N-CoR/SMRT corepressors on chromatin. Mol. Cell. Biol. 26: 5226-5236. PubMed ID: 16809761

Wang, L. Ding, L., Jones, C. A. and Jones, R. S. (2002). Drosophila Enhancer of zeste protein interacts with dSAP18. Gene 285: 119-125. PubMed ID: 12039038

Yang, Q., et al. (2000). The winged-helix/forkhead protein myocyte nuclear factor beta (MNF-beta) forms a co-repressor complex with mammalian sin3B. Biochem. J. 345(pt 2): 335-343. PubMed ID: 10620510

Zhang, Y., et al. (1997). Histone deacetylases and SAP18, a novel polypeptide, are components of a human Sin3 complex. Cell 89: 357-364. PubMed ID: 9150135

Zhu, W., Foehr, M., Jaynes, J. B. and Hanes, S. D. (2001). Drosophila SAP18, a member of the Sin3/Rpd3 histone deacetylase complex, interacts with Bicoid and inhibits its activity. Dev. Genes Evol. 211: 109-117. PubMed ID: 11455422


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date revised: 20 July 2010

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