InteractiveFly: GeneBrief

CoRest: Biological Overview | References

BIOLOGICAL OVERVIEW

The Notch signaling pathway plays important roles in a variety of developmental events. The context-dependent activities of positive and negative modulators dramatically increase the diversity of cellular responses to Notch signaling. In a screen for mutations affecting the Drosophila follicular epithelium, a mutation was isolated in CoREST that disrupts the Notch-dependent mitotic-to-endocycle switch of follicle cells at stage 6 of oogenesis. Drosophila CoREST positively regulates Notch signaling, acting downstream of the proteolytic cleavage of Notch but upstream of Hindsight activity; the Hindsight gene is a Notch target that coordinates responses in the follicle cells. CoREST genetically interacts with components of the Notch repressor complex, Hairless, C-terminal Binding Protein and Groucho. In addition, it was demonstrated that levels of H3K27me3 and H4K16 acetylation are dramatically increased in CoREST mutant follicle cells. The data indicate that CoREST acts as a positive modulator of the Notch pathway in the follicular epithelium as well as in wing tissue, and suggests a previously unidentified role for CoREST in the regulation of Notch signaling. Given its high degree of conservation among species, CoREST probably also functions as a regulator of Notch-dependent cellular events in other organisms (Domanitskaya, 2012).

The highly conserved Notch signaling pathway plays a crucial role in a broad array of developmental events, including the maintenance of stem cells, cell fate specification, control of proliferation and apoptosis. Misregulation of the Notch pathway is associated with a number of diseases, including different types of cancer. The binding of the transmembrane ligands DSL (Delta, Serrate, LAG-2) to the extracellular domain of Notch, exposed on a neighboring cell, activates the signaling cascade by triggering a sequence of proteolytic cleavages of Notch protein. Extracellular cleavage (S2) leads to the formation of an intermediate membrane-bound C-terminal fragment of Notch, called NEXT. This event is followed by an intramembranous cleavage (S3) by the γ-secretase complex. The intracellular domain of Notch (NICD) then translocates to the nucleus and binds to a transcription factor of the CSL family [CBF-1, Su(H), LAG-1], converting it from a transcriptional repressor to an activator. In the canonical Notch pathway, Su(H) directly activates Notch target genes in response to signaling. Despite the relative simplicity of the Notch transduction pathway, the presence of a large number of proteins that positively or negatively influence Notch signaling dramatically increases the complexity of the Notch pathway and its cellular responses. For instance, extracellular modulators, such as Fringe, alter ligand-specific Notch activation, whereas cytoplasmic modulators, such as Numb, restrict signal transduction. Nuclear modulators, for instance Mastermind, influence the transcriptional activity of the NICD-containing complex. In addition, there is increasing evidence of the importance of the epigenetic regulation of Notch targets, which can cause differential cellular responses upon Notch activation (Domanitskaya, 2012).

Drosophila serves as an excellent model system to dissect the regulation of the Notch pathway. The Drosophila genome contains only a single Notch protein and two ligands [Delta (Dl) and Serrate (Ser)]. The Notch pathway is involved in several aspects of Drosophila development. The role of Notch in lateral inhibition during neurogenesis has been extensively studied; it restricts neural cell fates in the embryo, and leads to restriction of sensory-organ formation and induction of boundary formation in the wing discs. Notch activity is also required for many aspects of oogenesis, such as the establishment of egg chamber polarity, polar cell formation, control of follicle cell (FC) proliferation, differentiation, cell fate specification and morphogenesis. The Drosophila FCs are somatically derived epithelial cells that form a monolayer covering the germline cells during oogenesis. FCs divide mitotically from stage 2 to stage 6 of oogenesis, followed by the switch from the mitotic cycle to the endocycle (the M/E transition). Endocycles take place from stage 7 to stage 10A of oogenesis and include three rounds of DNA duplication without subsequent cell division. The M/E switch is triggered upon Notch pathway activation. Dl produced in the germline binds to its receptor Notch, expressed in the FCs, and induces activation of the canonical Notch signaling pathway. Removal of Dl from germline cells, or of Notch from FCs, maintains follicle cells in the mitotic cycle throughout oogenesis. NICD complexed with Su(H) activates transcription of downstream target genes required for the M/E switch, such as Hindsight (Hnt). Hnt then mediates the Notch-dependent downregulation of Cut, String (Stg) and Hedgehog (Hh) signaling in the FCs, thus promoting the M/E switch (Domanitskaya, 2012).

This study describes the identification of the transcriptional cofactor Corepressor for element-1-silencing transcription factor (CoREST) as a positive modulator of Notch signaling in the FCs and during wing development. CoREST is required for the promotion of the M/E switch during oogenesis. CoREST acts downstream of NICD release but upstream of Hnt activity, and it is a previously unidentified modulator of the Notch pathway. The genetic interactions between CoREST and Hairless (H), CtBP and Groucho (Gro), members of the Notch repressor complex, suggest that CoREST might influence the activity of either Notch transcriptional repressor or activator complexes. In addition, CoREST specifically affects tri-methylation of lysine 27 of histone 3 (H3K27) and acetylation of H4K16 in FCs, because these chromatin modifications show elevated levels in the CoREST mutant cells. These findings point to a possible role of CoREST in regulation of the activity of the Notch repressor-activator complexes and/or epigenetic regulation of the components of the repressor-activator complexes or of factors involved in the transduction of the signaling or directly of target genes of the Notch signaling pathway (Domanitskaya, 2012).

Initially, CoREST was identified in humans as a corepressor with REST (RE1 silencing transcription factor) in mediating repression of the proneuronal genes, and thus as an important factor in the establishment of non-neural cell specificity (Andres, 1999; Lunyak, 2002). Subsequently, CoREST was identified in a variety of vertebrate and invertebrate species, and was shown to play a functionally conserved role in neurogenesis (Tontsch, 2001; de la Calle-Mustienes, 2002; Jarriault, 2002; Dallman, 2004). Recent studies show that CoREST regulates a very broad range of genes by both REST-dependent and REST-independent means, including genes encoding members of key neural developmental signaling pathways, such as BMP, SHH, Notch, RA, FGF, EGF and WNT (Abrajano, 2009a; Abrajano, 2010; Qureshi, 2010). Analysis of CoREST downstream target genes and their developmental expression profiles suggested that the liberation of CoREST from gene promoters is associated with both gene repression and activation depending on the cell context (Abrajano, 2009a; Abrajano, 2009b; Abrajano, 2010). In the work reported in this study, a lethal allele of Drosophila CoREST was isolated, and the contribution of CoREST to the development of FCs, a process that involves cell proliferation and differentiation, was analyzed. This study has implicated CoRESTin the regulation of Notch signaling, and acts as a positive modulator of the Notch pathway in Drosophila FCs (Domanitskaya, 2012).

This study has identified a role for CoREST in the Notch-mediated regulation of the M/E switch during stage 6 of oogenesis. Loss of CoREST activity in FCs primarily disrupts the Notch signaling pathway. We further demonstrated that CoREST regulates the Notch pathway downstream of NICD release and upstream of Hnt. The misexpression of Hnt in the CoREST mutant clones rescues the failure in the M/E switch. Furthermore, the role of CoREST in Notch pathway regulation is not restricted to FCs: CoREST also interacts with Notch during wing development. Interestingly, CoREST was identified as a negative modulator of Notch signaling in Caenorhabditis elegans in a genetic screen for suppressors of the developmental defects in sel-12 presenilin mutants (Eimer, 2002; Jarriault, 2002; Lakowski, 2006). Presenilin is a component of the γ-secretase complex that performs the S3 cleavage of Notch. Mutations in spr-1, the C. elegans homolog of CoREST, suppress the developmental defects observed in sel-12 animals by derepressing the transcription of the other functionally redundant presenilin gene, hop-1 (Jarriault, 2002; Lakowski, 2006). Therefore, CoREST acts as a negative regulator of the γ-secretase complex in C. elegans, and hence proteolytic cleavage of Notch and release of NICD. By contrast, Drosophila CoREST does not affect the processing of the Notch receptor in the follicle cells, and instead acts as a positive modulator of the Notch pathway functioning downstream of NICD release (Domanitskaya, 2012).

CoREST plays transcriptional and epigenetic regulatory roles: it can promote gene activation in addition to repression, as well as being able to modify the epigenetic status of target gene loci distinct from its effects on transcription (Qureshi, 2010). Several possible scenarios of how CoREST could be involved in the regulation of Notch signaling are discussed, based on the previous knowledge about CoREST and considering the current data (Domanitskaya, 2012).

hnt, the downstream target gene of Notch signaling in FCs, fails to be properly upregulated upon Notch activation in the CoREST mutant cells. CoREST might therefore act as a transcriptional repressor for an unknown factor, which is in turn involved in the transcriptional repression of hnt. Alternatively, CoREST could be directly involved in the transcriptional regulation of hnt and act as an activator. hnt was shown to be a putative direct target of Notch signaling in DmD8 cells (Krejci, 2009) from the analysis of genes for which mRNA levels increase within 30 minutes of Notch activation, and which contain regions occupied by Su(H). If hnt is a direct target of Notch in FCs, its transcription would be regulated by the balance between Notch repressor and activator complexes, and CoREST might be involved in the regulation of stability or activity of either of these. Interestingly, CoREST was shown to interact with CtBP1 in mammals (Kuppuswamy, 2008), and to bind to the SIRT1-LSD1-CtBP1 complex, which is required for the repression of certain Notch target genes (Mulligan, 2011). Thus, Drosophila CoREST might similarly directly bind to the repressor complex containing CtBP and modify its activity or destabilize it. However, CoREST could be involved in the transcriptional regulation of the components of Notch repressor or activator complexes. In this scenario, in CoREST mutant FCs, upregulation of negative regulator(s) would lead to greater activity of negative than positive regulators, resulting in disruption of Notch signaling. Both suggested models of the direct and indirect transcriptional role of CoREST are consistent with the current results, given that the CoREST mutant phenotype could be suppressed by removal of one copy of H, CtBP or Gro, components of the Notch repressor complex (Domanitskaya, 2012).

More recently, epigenetic mechanisms have emerged as an important interface regulating context-dependent and stage-specific gene regulation. Mammalian CoREST acts as a scaffold for recruitment of transcriptional regulators such as REST, and epigenetic factors such as the enzymes HDAC1, HDAC2 and LSD1 (Lakowski, 2006; Qureshi, 2010). In Drosophila, using two-hybrid interaction, CoREST was also shown to interact with Su(VAR)3-3 (Drosophila homolog of LSD1) and Rpd3 (HDAC1) (Dallman, 2004). This study has shown that the levels of H3K27me3 and H4K16 acetylation are significantly and specifically increased in the CoREST mutant FCs. Recently, the H3K27me3 demethylase UTX was shown to act as a suppressor of Notch- and Rb-dependent tumors in Drosophila eyes (Herz, 2010), and in addition to increased level of H3K27me3 staining, an excessive activation of Notch was detected in Utx mutant eye discs. The observation of increased levels of H3K27me3 coupled to cell overproliferation and modified Notch signaling in both of these cases [(Herz, 2010) and this study] suggests that the increased H3K27me3 results in epigenetic regulation of genes involved in Notch signaling and/or of Notch target genes. However, in the eye tumor system, this increase in H3K27me3 promotes Notch signaling, whereas in the follicle cells, it reduces Notch signaling. This indicates a strong context-dependent effect on Notch signaling by certain chromatin modifications. Thus, these chromatin modifications might be involved in cell-context-dependent Notch target gene silencing and/or activation (Schwanbeck, 2011). Interestingly, many Notch-regulated genes are highly enriched in a characteristic chromatin modification pattern, termed a bivalent domain, consisting of regions of H3K4me3, a marker for actively expressed genes, and H3K27me3, a marker for stably repressed genes; and Notch signaling could be involved in resolving these domains, leading to gene expression (Schwanbeck, 2011). Therefore, the increased level of H3K27me3 in CoREST mutant FCs might lead to a repression of certain Notch target genes, for instance hnt (Domanitskaya, 2012).

To further understand the function of the Drosophila CoREST in Notch pathway regulation, identification of other CoREST essential and specific binding partners would be useful. One previously identified partner for CoREST is Chn (Tsuda, 2006). Given that wild-type expression of Hnt and Cut was observed in chn mutant cells, this factor does not appear to partner CoREST in regulation of Notch signaling in FCs. Using yeast two-hybrid analyses and an embryonic cDNA fusion protein library, it was shown that all three splice variants of Drosophila CoREST interact with the unique C-terminus of Tramtrack88 (Ttk88), a known repressor without homology to REST (Dallman, 2004). In addition, a Ttk69 splice variant can form a complex with CoREST and Ttk88 (Dallman, 2004). However, Ttk88 was not detected in the ovary by immunofluorescence or western blot analysis, and disruption of Ttk88 does not have any impact on oogenesis. Conversely, Ttk69 is steadily expressed in FCs before stage 10 and it is required for the M/E transition. However, in contrast to CoREST, which acts upstream of Hnt, Hnt expression is not affected in ttk^1e11 mutant FCs, indicating a role of Ttk69 downstream of Hnt in the control of the M/E switch. Additionally, Ttk69 is not required for cell differentiation, as expression of FasIII, a cell fate marker for immature follicle cells, is normal in ttk^1e11 mutant FCs. From these important phenotypic differences between Ttk69, Ttk88 and CoREST, it appears that CoREST plays a Ttk-independent role in Notch pathway regulation in the FCs. Future work to identify transcription regulators that act as binding partners of CoREST will help in determining the precise biochemical role of CoREST in modulating Notch signaling (Domanitskaya, 2012).

These results demonstrate an unexpected role for CoREST in positively regulating Notch signaling. The effect of the loss of CoREST is particularly strong in the PFCs and relatively mild in the lateral and anterior follicle cells. This implies that CoREST is crucially required in cells that are more sensitive to loss of Notch signaling. The difference between the PFCs and the other follicle cells is established at approximately stages 6-7 of oogenesis by EGF receptor activation in response to Gurken produced by the oocyte. EGF signaling, therefore, is active around the same time as the Notch pathway and hence it is probable that downstream effector(s) of EGFR signaling result in the increased sensitivity of PFCs to the loss of CoREST. In the model of CoREST negatively affecting a repressor of Notch signaling, EGFR signaling would be expected to act positively to enhance expression and/or activity of a Notch repressor. Thus, loss of CoREST from the PFCs would occur in a cell type where repressor activity is already augmented, which would explain the observation of differential loss of Notch signaling in the PFCs (Domanitskaya, 2012).

In summary this study has shown that CoREST, a component of transcriptional repressor complexes, acts positively in Notch signaling in the ovarian follicle cells of Drosophila. The results also show that different cell types are differentially sensitive to loss of this repressor. Future identification of partners and targets of CoREST in the follicle cells should further elucidate how activity of EGFR and other signaling pathways are integrated in this process (Domanitskaya, 2012).

LINT, a Novel dL(3)mbt-Containing Complex, Represses Malignant Brain Tumour Signature Genes

Mutations in the l(3)mbt tumour suppressor result in overproliferation of Drosophila larval brains. Recently, the derepression of different gene classes in l(3)mbt mutants was shown to be causal for transformation (Richter, 2011). However, the molecular mechanisms of dL(3)mbt-mediated gene repression are not understood. This study identified LINT, the major dL(3)mbt complex of Drosophila. LINT has three core subunits -- dL(3)mbt, dCoREST, and l(3)mbt interacting protein 1 (dLint-1) -- and is expressed in cell lines, embryos, and larval brain. Using genome-wide ChIP-Seq analysis, it was shown that dLint-1 binds close to the TSS of tumour-relevant target genes. Depletion of the LINT core subunits results in derepression of these genes. By contrast, histone deacetylase, histone methylase, and histone demethylase activities are not required to maintain repression. These results support a direct role of LINT in the repression of brain tumour-relevant target genes by restricting promoter access (Meier, 2012).

LINT subunit composition differs from the human L3MBTL1 complex which contains pRb, HP1γ, H1b and core histones. dLint-1 has no apparent homolog in mammals. The mammalian homologs of dCoREST exist in complexes containing LSD1 and HDAC1/2. dLsd1 and dRpd3 are not stably associated with LINT. Nevertheless, the LINT subunit dLint-1 associates with dCoREST, dLsd1 and dRpd3 arguing for the existence of complexes in Drosophila that are related to mammalian CoREST/LSD1 complexes. Two observations are consistent with the view that these complexes might associate with chromatin and occupy sites that are not bound by LINT. First, dLint-1 is associated with approximately 50 bands on polytene chromosomes that show no dL(3)mbt binding. Second, ChIP-Seq analysis has revealed 2,902 dL(3)mbt binding sites but more than 8,000 dLint-1 binding sites. The functional relationship between these different dLint-1-containing complexes is unclear (Meier, 2012).

Comparison of genomewide binding profiles of dL(3)mbt in larval brain and dLint-1 in S2 and Kc cells strongly argues that LINT subunits bind to a large set of common binding sites. In particular, MBTS germline-related genes are bound and often repressed by the three LINT subunits. The finding that LINT exists in larval brain strongly implies that it is the LINT complex that is inactivated in l(3)mbt^ts mutants. In addition to malignant brain tumour signature (MBTS) genes, genes targeted by the Salvador-Warts-Hippo (SWH) pathway have recently been shown to be deregulated in l(3)mbt^ts brains (Richter 2011). Although binding of dLint-1 to about half of the SWH targets was detected, changes in SWH target gene expression following depletion of dL(3)mbt or dLint-1 has not been detected in Kc cells. It is possible that protein depletion was not sufficient to derepress these genes under the conditions used. Also, SWH target genes might be regulated differently in larval brain compared to cell lines (Meier, 2012).

The results suggest that maintenance of MBTS germline gene repression by LINT is largely independent of repressive histone modifying activities. Depletion of the dLint-1-associated histone demethylase dLsd1 and dRpd3 enzymes does not lead to derepression of LINT targets. An increase of the active H3K4me2 mark was detected at derepressed LINT target genes but this is most likely a result of active transcription rather than a direct consequence of the loss of LINT associated chromatin modifying activities. In agreement with this view, depletion of dLsd1 does not result in changes of H3K4me2 levels at LINT target genes. Microarray analysis also did not detect significant changes in the expression of genes recently shown to be repressed by dLsd1 in S2 cells and developing flies. This suggests that LINT and dLsd1 target different sets of genes (Meier, 2012).

Chromatin association and the repressive potential of human L3MBTL1 is enhanced by PR-SET7 and H4K20 monomethylation (Trojer, 2007; Kalakonda, 2008). Depletion of dPR-Set7, the sole Drosophila enzyme responsible for H4K20 monomethylation, did not result in derepression of LINT targets. Also no significant levels of H4K20me1 was detected at promoters of LINT target genes. This strongly suggests that even though dL(3)mbt can bind H4K20me1 in vitro this interaction does not play an important role in LINT complex targeting and repression (Meier, 2012).

dL(3)mbt does also bind to H4K20me2 in vitro. Indeed, H4K20me2 is present at LINT-regulated genes. However, H4K20me2 levels are are not elevated at LINT target gene promoters compared to control regions. This finding was not surprising given that 85%-90% of all histone H4 molecules are dimethylated at K20 and, therefore, H4K20me2 levels might be expected to be uniformely high along the chromosome. This makes it unlikely that an interaction between the MBT domains and H4K20me2 specifically directs the LINT complex to its target genes. However, it remains possible that after recruitment of LINT by other means, an interaction between dL(3)mbt and H4K20me2 contributes to transcriptional repression (Meier, 2012).

Depletion of other enzymes setting repressive histone marks such as H3K9me3 and H3K27me3 has likewise no effect on LINT-mediated repression. Although it was not possible to test all histone modifying enzymes for their roles in LINT target gene repression, the results argue for a largely histone modification independent mode of repression. LINT subunits bind predominantly near TSSs suggesting that LINT might inhibit transcription by restricting the access of RNA polymerase II or transcription factors to promoters. In support of this model, recruitment of LINT subunits to the promoter of a reporter gene is sufficient for repression even under conditions where the levels of repressive histone modification enzymes are reduced. Two modes of promoter access restriction by LINT can be envisioned that are not mutually exclusive. First, LINT might bind to the promoter segments required for RNA polymerase II recruitment. Second, as has been suggested for human L3MBTL1, LINT might locally compact nucleosomes. Two of the findings are inconsistent with the latter hypothesis. Nucleosome compaction by L3MBTL1 is dependent on the presence of the H4K20me1 modification. However, as discussed above, ablation of this modification does not result in derepression of LINT target genes. In addition, as a consequence of nucleosome compaction at LINT bound promoters one might expect a local increase in nucleosome density. However, histone H3 ChIP experiments have shown that the promoters of LINT target genes are generally depleted of nucleosomes. While these findings do not rule out a local nucleosome compaction that is - once established - independent of H4K20 monomethylation and undetectable by H3 ChIP, we favour the simpler hypothesis that LINT association with promoter sequences prevents transcription factors and RNA polymerase II from promoter binding (Meier, 2012).

The dL(3)mbt and dCoREST subunits of LINT are well conserved. Similar to the derepression of germline-related genes in l(3)mbt^ts tumours, misexpression of testis-specific genes (so-called cancer testis antigens) have been described in many human tumours. Based on this study, it is conceivable that L3MBTL1 or CoREST play a role in the repression of cancer testis antigens (Meier, 2012).

Drosophila LSD1-CoREST demethylase complex regulates DPP/TGFβ signaling during wing development

The choice and timing of specific developmental pathways in organogenesis are determined by tissue-specific temporal and spatial cues that are acted upon to impart unique cellular and compartmental identities. A consequence of cellular signaling is the rapid transcriptional reprogramming of a wide variety of target genes. To overcome intrinsic epigenetic chromatin barriers to transcription modulation, histone modifying and remodeling complexes are employed. The deposition or erasure of specific covalent histone modifications, including acetylation, methylation, and ubiquitination are essential features of gene activation and repression. This study has found that the activity of a specific class of histone demethylation enzymes is required for the specification of vein cell fates during Drosophila wing development. Genetic tests revealed that the Drosophila LSD1-CoREST complex is required for proper cell specification through regulation of the DPP/TGFβ pathway. An important finding from this analysis is that LSD1-CoREST functions through control of rhomboid expression in an EGFR-independent pathway (Curtis, 2013).

The Su(var)3-3 gene (CG17149) encodes the Drosophila LSD1 homolog. Mutations in Su(var)3-3 result in aberrant histone methylation and heterochromatin formation, with increased global levels of H3K4me2 and impaired heterochromatic gene silencing. A physical association between LSD1 and CoREST has been described in Drosophila (Dallman, 12 2004), revealing that the critical relationship between these proteins is conserved. LSD1 has an important role in organogenesis and germ line maintenance, such as during mouse anterior pituitary development (Wang, 2007) and Drosophila ovary and wing development. LSD1 also regulates neural stem cell proliferation by modulating signaling via the orphan nuclear receptor TLX (Sun, 2010)¸ and LSD1 appears to have distinct functions in mammalian neuronal morphogenesis (Fuentes, 2012; Zibetti, 2010) as well as stem cell self-renewal and differentiation (Adamo,2011). In humans, loss of LSD1 has been strongly correlated with several types of cancer and high-risk tumors, including prostate cancer, breast cancer and neuroblastomas). In contrast, overexpression of LSD1 has also been linked to some cancers. As a consequence of the emerging links between histone demethylase functions and disease, an understanding how LSD1 contributes to specific cell-cycle regulation and developmental processes is crucial (Curtis, 2013).

The Drosophila wing provides an outstanding in vivo model system to identify factors that regulate cell-fate determination as alterations in cell-fate can often be observed at the single cell level. Multiple conserved signaling pathways contribute to wing patterning and development and are regulated, in part, by the coordinated activities of chromatin remodeling complexes and epigenetic modifying enzymes. Previously work has identified histone lysine demethylase enzymes as coregulators of Brm complex remodeling activities in a genetic screen for factors that influenced a wing patterning phenotype associated with a conditional loss-of-function mutation in the snr1 gene that encodes a core regulatory subunit of the Brm complex. Genetic interaction tests indicated that lsd1 (Su[var]3-3) most likely interacted with the PBAP subtype of the Brm complex (Curtis, 2011). This report further addresses how LSD1 contributes to the cell-type and developmental time-point specific regulation of conserved signaling pathways by understanding its contribution to wing patterning and development (Curtis, 2013).

Recently, it was suggested that LSD1 regulates notch signaling during Drosophila wing development (Mulligan, 2011). This study presents evidence from genetic interaction analyses and tissue or cell-type specific targeted depletion experiments that suggest LSD1 and CoREST/CG42687 (synonymous with CG33525) may also regulate the DPP/TGFβ signaling pathway in a noncanonical manner, by regulating expression of rhomboid, a key player in canonical EGFR signal transduction. This is the first demonstration of LSD1-CoREST regulated DPP/TGFβ signaling and the results further define important roles of the LSD1-CoREST complex in tissue patterning (Curtis, 2013).

The appropriate elaboration of wing vein and intervein cell fates depends on the interplay of factors that promote and those that repress or block vein cell differentiation. In this study, we provide genetic evidence suggesting an important role for lsd1 and CoRest in repressing vein-promoting genes in intervein cells. Ectopic vein development can result from either the loss of a factor required for repressing vein cell differentiation or the gain of a factor that promotes vein cell fate in intervein cells. The experimental results suggest that lsd1 and CoRest utilize the first mechanism, since the aos hypomorphic mutation (aos^w11), a factor known to repress vein fate, is enhanced by CoRest^EY14216 and lsd1^ΔN and targeted depletion by shRNAi of lsd1 and CoRest throughout the entire developing wing imaginal disc resulted in ectopic veins rather than loss of vein phenotypes. It was reasoned that if the LSD1-CoREST complex normally functions as a positive factor to promote vein development as proposed by the second mechanism, then mutations in lsd1 and CoRest or shRNAi depletion in the wing imaginal disc should produce a loss of vein phenotype. Based on the evidence presented in this manuscript, and on the recent finding that LSD1 is important for the regulation of NOTCH signaling in the wing (Di Stefano, 2011), it is proposed that the requirements of LSD1-CoREST are temporal and cell-type specific, and possibly dependent on the physical associations between LSD1 and several multiprotein complexes (Curtis, 2013).

An elaborate signaling network regulates wing patterning, where considerable cross-talk and functional redundancy connects five developmental pathways. For example, during pupal development, the main role of EGFR and DPP activation is to coordinately promote and maintain differentiation into vein cells while NOTCH activation establishes the provein-intervein boundary. However, DPP and NOTCH pathways are codependent, since expression of the NOTCH ligand, DELTA (DL) and its downstream target, ENHANCER OF SPLIT, (E(spl)mβ), require DPP signaling. LSD1 has been shown to interact directly with the histone deacetylase SIRT1 to repress NOTCH targets, suggesting important epigenetic functions for these co-repressors in metazoan development. However, recently it was shown that CoREST could function as a positive regulator of NOTCH in Drosophila follicle cells and wings (Domanitskaya, 2012). Therefore, there is growing precedent for the LSD1-CoREST complex to have both positive and negative roles in regulating gene expression depending on developmental context (Curtis, 2013).

LSD1 and CoREST depletion in the developing wing causes bifurcated or duplicated crossveins, a phenotype previously observed with Hairless (H) loss of function mutations. Because H both antagonizes NOTCH and promotes EGFR signaling, it is difficult to decipher the individual pathway regulated by LSD1-CoREST. Furthermore, the broadened vein delta phenotype observed at the wing margin in wing-specific LSD1-CoREST depleted and lsd1^ΔN null flies (Di Stefano, 2011) is similar to Notch and DPP receptor (tkv) loss of function phenotypes (Curtis, 2013).

It is proposed that during the initial stages of wing vein development and differentiation, LSD1 negatively regulates NOTCH signaling. This is based on the observation that loss of lsd1 function suppresses the notched wing phenotype associated with mutations in suppressor of hairless (Su[H^T4]) (Mulligan, 2011). However, later in development during vein refinement and maintenance, LSD1 appears to undergo a regulatory switch to positively regulate NOTCH signaling, since lsd1^ΔN suppresses the short vein phenotype associated with the gain-of-function N^Ax-16 mutation. Additionally, the increased expression of downstream E(spl) targets in N^Ax-16 mutants is reversed by lsd1^ΔN (Di Stefano, 2011). It was also recently shown that a transheterozygous mutant allele of CoRest (CoRest^GF60) could enhance the wing phenotypes of flies carrying alleles of Dl and N (Domanitskaya, 2012), suggesting positive functions in regulating NOTCH signaling. Concurrently, LSD1 and CoREST repress vein cell differentiation by regulating components of the DPP signaling pathway at multiple points. For example, lsd1^ΔN and CoRest^EY14216 genetically interact with both dpp and genes encoding its receptors (e.g., dpp, tkv, sax), consistent with upstream functions. Strong genetic interactions were observed with downstream DPP signaling components (e.g., mad, med, ara, caup, shn), which suggests that the LSD1-CoREST complex has important regulatory functions in controlling the expression of DPP pathway targets. This conclusion is further supported by ectopic expression of the DPP-specific downstream signaling component, p-MAD, was observed in LSD1-CoREST-depleted animals. Activated DPP signaling is confined to proveins largely by the overexpression of TKV, a member of the TGFβ receptor family, in intervein boundary cells. TKV binds and sequesters the DPP morphogen. When TKV is downregulated, DPP spreads into regions of the wing destined to become intervein cells, resulting in ectopic veins. It is predicted that TKV is the most likely target of LSD1-CoREST complex regulation, since genetic interactions were observed between lsd1^ΔN and CoRest^EY14216 and almost all loss of function mutations in DPP signaling components, and tissue-specific LSD1-CoREST depletion lead to the development of ectopic veins, similar to phenotypes observed with loss of function alleles of tkv. Because activation of NOTCH and repression of DPP signaling are both required to repress vein promoting genes in differentiating intervein cells, LSD1 appears to have cell-type and context-specific activities to differentially regulate these pathways (Curtis, 2013).

Coimmunoprecipitation experiments suggested a complex forms between the HDAC1/2 class protein RPD3, LSD1, CoREST, and two TTK splice variants TTK88 or TTK69 (Dallman, 2004). Complexes containing CoREST/TTK69 or CoREST/TTK88 independently localize on polytene salivary glands, suggesting differential gene targeting (Dallman, 2004). TTK and REST are likely functional homologs. Orthologs of tramtrack only exist in invertebrates, whereas REST orthologs are vertebrate-specific (Dallman, 2004). TTK69 is a transcription factor that can recognize and bind to a specific DNA RE-1 consensus sequence (CCAGGACG), resulting in gene transcription (Dallman, 2004). Unpublished observations suggest that TTK69, but not TTK88, function to negatively regulate vein cell development, since an incomplete vein phenotype is observed when TTK69 is overexpressed, whereas overexpression of TTK88 results in the development of ectopic veins. Therefore, it is predicted that LSD1-CoREST-TTK69 form a complex in developing wing tissue to negatively regulate DPP signaling in intervein cells. Furthermore, in mammals, the Brg1 complex chromatin remodeling capacity and recruitment specificity depends on formation of a LSD1-CoREST-REST-BRG1 complex (Ooi 2006). Because LSD1 can physically associate with the Brm chromatin remodeling complex in Drosophila (Curtis, 2011), it is predicted that the Brm complex-LSD1-CoREST-TTK69 super-complex regulates genes essential for wing patterning, possibly through co-localization or recruitment to RE-1 consensus binding sites. Intriguingly, RE-1 consensus sites are present in both the rho and tkv gene loci, making these exciting targets for future investigation (Curtis, 2013).

Widespread regulatory specificities between transcriptional co-repressors and enhancers in Drosophila

Gene expression is controlled by the precise activation and repression of transcription. Repression is mediated by specialized transcription factors (TFs) that recruit co-repressors (CoRs) to silence transcription, even in the presence of activating cues. However, whether CoRs can dominantly silence all enhancers or display distinct specificities is unclear. This work reports that most enhancers in Drosophila can be repressed by only a subset of CoRs, and enhancers classified by CoR sensitivity show distinct chromatin features, function, TF motifs, and binding. Distinct TF motifs render enhancers more resistant or sensitive to specific CoRs, as was demonstrated by motif mutagenesis and addition. These CoR-enhancer compatibilities constitute an additional layer of regulatory specificity that allows differential regulation at close genomic distances and is indicative of distinct mechanisms of transcriptional repression (Jacobs, 2023).

Animal development and homeostasis critically depend on differential gene expression, enabled by the precise regulation of transcriptional activation and repression. Although repression is often associated with heterochromatin, genes can also be silenced in transcriptionally permissive euchromatin by repressive transcription factors (TFs), also termed repressors, that bind to DNA and recruit corepressors (CoRs). As CoRs can suppress transcription even in the presence of activators, this mode of gene silencing is termed active transcriptional repression. Active repression is critical and its failure can cause developmental defects and diseases like cancer; and it is conceptually intriguing as it requires the fast and efficient overriding of activating cues. However, the modes and mechanisms of this process are unclear, and whether a regulatory code coordinates repression and activation, is unknown. Given that transcriptional activation can occur via distinct and mutually incompatible modes, it is intriguing to speculate whether distinct modes of active transcriptional repression exist. Examples of specificities between repressors and activators have indeed been observed. The CoR Retinoblastoma protein (Rb) as part of the DREAM complex for example can repress the TFs E2F, Mip120 and PU.1 but not others like SP-1, and repressors can have different activities in distinct transcriptional contexts. Comprehensive studies allowing to define different modes of active repression and uncover their regulatory rules are however lacking. This study determined the mutual compatibilities between five known CoRs and a genome-wide library of active enhancers by measuring enhancer-activity changes upon CoR tethering in otherwise unperturbed cells, similar to activator-bypass experiments. It was reasoned that testing each enhancer with each CoR in all possible combinations should reveal CoR-enhancer combinations that lead to decreased enhancer activity (enhancers are sensitive) and those that do not (resistant), indicative of compatible and incompatible pairings (Jacobs, 2023).

First, this study tested whether distinct specificities between CoRs and enhancers exist, whereby certain enhancers are sensitive to repression by a given CoR, while other enhancers are resistant. For this, it was necessary to systematically measure the effects that selected CoRs have on the activity of a large number of enhancers. The comprehensive mapping of CoR-enhancer compatibilities, by examining all combinations of CoRs and enhancers, requires highly controllable quantitative high-throughput assays. Therefore the massively parallel enhancer-activity assay STARR-seq was modified to enable the function-based testing of genome-wide enhancer candidate libraries with different CoRs. Briefly, four upstream-activating-sequence (UAS) motifs were introduced immediately downstream of the enhancer library, which leave the enhancer sequence intact yet allows for the direct tethering of selected CoRs via the Gal4 DNA-binding domain. The tethering of CoRs next to active enhancers directly assesses whether CoRs can override existing activating cues, a process akin to active repression (Jacobs, 2023).

Drosophila S2 cells were chosen as a model system, and a panel of five CoRs; CoRest, CtBP, Rbf, Rbf2 and Sin3A was tested. These CoRs represent different protein complexes, repressive pathways, enzymatic functions, and distinct groups with context-specific functions. Testing diverse CoRs casts a wide net and should increase the ability to detect compatible and incompatible CoR-enhancer pairs. For each CoR, two independent UAS-STARR- seq screens were performed where the UAS-STARR-seq library was co-transfect with a vector that expresses the Gal4-CoR (or Gal4-GFP as neutral control), and spike-in-controls were used for normalization. As spike-in controls a distinct STARR-seq library was used containing 18 Drosophila pseudoobscura enhancers, cloned without the UAS motifs, and hence not targeted by the Gal4- -CoRs. In all cases, the two independent replicates correlated well. In order to reliably assess repression (which requires a high baseline activity), it was decided to evaluate enhancer-activity changes for 3094 enhancers that were highly active in Gal4-GFP controls (Jacobs, 2023).

First, which of the 3094 enhancers could be silenced by the highly conserved CoR CtBP was determined, by assessing the enhancer activity changes when tethering Gal4-CtBP versus Gal4-GFP. This revealed that some enhancers, like the enhancers near Orct, Orct2 and Pka-C3, were reproducibly repressed by Gal4-CtBP, whereas others, like the enhancer near CG10516, were unaffected. A differential analysis based on the two replicates per condition using edgeR showed that CtBP significantly repressed 759 enhancers but not the remaining 2335. Thus, CtBP could only repress ~25% of the enhancers, suggesting that CtBP displays preferences or specificities towards some enhancers but not others (Jacobs, 2023).

The enhancer-activity changes upon recruiting Rbf2, a CoR from the retinoblastoma protein family was determined. Interestingly, Rbf2 was also able to repress only a subset of the enhancers (1733, 56%), including the enhancers near Orct and Pka-C3, which were also repressed by CtBP, and CG10516, which was not repressed by CtBP. In contrast, Rbf2 was unable to repress the aforementioned Orct2 enhancer and others, indicating that the specificities of Rbf2 differ from those of CtBP. Indeed, while 502 enhancers were repressed by CtBP and Rbf2, 1231 enhancers were repressed by Rbf2 but not by CtBP and, vice versa, 257 enhancers were repressed by CtBP but not by Rbf2. These enhancer-CoR specificities were validated in luciferase reporter assays, in which the CoRs were recruited upstream of the enhancer and promoter. This assay confirmed that an enhancer near serpent was specifically repressed by Gal4-CtBP but not Gal4-Rbf2, an enhancer near CG2116 was specifically repressed by Gal4-Rbf2 but not Gal4-CtBP, and an intronic enhancer of kay was repressed by both CoRs, each in agreement with the STARR-seq results. Taken together, the CoRs CtBP and Rbf2 are each able to repress a specific subset of enhancers but not others, indicating the existence of distinct CoR-enhancer specificities (Jacobs, 2023).

Screening three additional prominent CoRs; CoRest, Rbf and Sin3A revealed that each of them was able to repress a specific subset of enhancers. CoRest was the strongest repressor, repressing 1452 enhancers and often reducing their activity to background levels. The overall repression profiles of CoRest and Sin3A were similar to that of CtBP and clearly different from Rbf2. Rbf's repression profile was not similar to any of the other tested CoRs. In general, the five tested CoRs were each able to repress a subset of enhancers and displayed distinct specificities (Jacobs, 2023).

Given that enhancers were differentially sensitive to some of the tested CoRs, and resistant to others, whether these differential sensitivities were related to other enhancer properties was examined. For this, the enhancers were clustered into groups of similar repression patterns. A self-organizing tree algorithm (SOTA) was used with the PCC as distance metric to cluster the enhancers into five groups based on their sensitivity to the CoRs. Cluster 1 contains CoRest and CtBP resistant enhancers while enhancers in cluster 2 are resistant to CtBP and Sin3A. Cluster 3 contains enhancers that are resistant to Rbf2, enhancers in cluster 4 are sensitive to all CoRs while enhancers in cluster 5 are overall very sensitive to repression but resistant to Rbf. Hence, the enhancers of S2 cells could be divided into five groups, defined by their differential response to the tested CoRs (Jacobs, 2023).

Enhancers clustered by corepressor sensitivity differ in chromatin marks, transcription factor motif content and binding Next, whether these enhancer clusters differ in additional properties that correlate with their behaviour towards the CoRs was tested. Initial enhancer activity, as measured by UAS- STARR-seq with Gal4-GFP, was similar for all five clusters. Also H3K27ac, a histone modification that marks active enhancers, was similarly enriched at the endogenous enhancer loci of all clusters. It was inferred that the distinct specificities did not stem from differences in initial enhancer strengths. The histone modifications H3K4me1 and H3K4me3 did however show differential and complementary trends: H3K4me1 was the highest enriched at cluster 5, followed by 4 and 3, whereas H3K4me3 was more highly enriched at enhancer clusters 1 and 2. High H3K4me1 levels combined with high H3K27ac levels have been associated with distal regulatory regions or cell type-specific enhancers, suggesting that enhancers with the highest H3K4me1 levels (cluster 5) might be most specific to S2 cells. Analysing chromatin accessibility and H3K27ac levels in other cell types and tissues indeed revealed that enhancers from cluster 5 were highly cell type-specific. Interestingly, these enhancers were also the most strongly repressed by CoRest, CtBP and Rbf2, suggesting that developmental or cell type-specific enhancers might intrinsically be more sensitive to repression by certain CoRs, while more globally active or housekeeping enhancers might be more resistant. As active enhancers are known to function through a variety of different TFs, it was hypothesised that their differential response to the CoRs might be linked to the specific TFs they bind (Jacobs, 2023).

Using published TF ChIP-seq data, it was observed that prominent TFs were bound to enhancers from specific clusters and absent from others. The TFs DREF and M1BP for example bound almost exclusively to enhancers from clusters 1 and 2, with DREF preferring cluster 1 and M1BP cluster 2. Trithorax-like (Trl, also called GAGA factor or GAF) on the other hand was absent from these clusters and instead mainly bound to enhancers from cluster 3 and to a lower extent to cluster 4. The distribution of these three TFs suggests that differential CoR sensitivities might relate to distinct TFs. To identify associations between CoR sensitivities and TFs in a more comprehensive manner, TF-motif enrichment analyses was performed for the enhancers of the five clusters using the 6502 TF motifs from the iRegulon database. Consistent with the ChIP-seq results, motifs for DREF, M1BP and Trl were specifically enriched in clusters 1, 2 and 3, respectively. Additional motifs were also differentially enriched between the clusters: Rsc30 and E-box motifs were enriched in cluster 3, Sim motifs were enriched in cluster 4, while ETS and GATA motifs were enriched in cluster 5. Other prominent TF motifs were enriched over several clusters, including AP-1, Ato and CrebB. Taken together, the different enhancer clusters, defined by their sensitivity towards CoRs, associate with distinct chromatin features and TF motif content (Jacobs, 2023).

To directly test the association between TF motifs and sensitivity towards each of the CoRs,the enrichment of TF motifs within enhancers that were sensitive ) versus those that were resistant to each CoR were evaluated. For CtBP, for example, it was found that AP-1, Trl and GATA motifs were strongly enriched in sensitive enhancers, whereas DREF, Ohler1 and Mip120 motifs were enriched in the resistant enhancers. Motif enrichment profiles across all five CoRs revealed an intricate relationship between enhancer-CoR sensitivity and TF motifs with highly distinctive enrichments. Consistent with the conserved role of Rb proteins as part of the DREAM complex, E2F, DP, and Mip120 motifs were enriched in Rbf sensitive enhancers. Interestingly, these three motifs as well as DREF and M1BP motifs were enriched in enhancers that were resistant to CoRest, CtBP and Sin3A repression. On the other hand, motifs for the developmental TFs GATA, AP1 and TEAD were enriched in enhancers sensitive to CoRest, CtBP and Sin3A and resistant to Rbf and Rbf2. Furthermore, Trl and ETS motifs were specifically enriched in enhancers that were resistant to Rbf2 and Rbf respectively, while sensitive to all other CoRs. These results indicate that, for each CoR, certain TF motifs are specifically and strongly enriched in sensitive and resistant enhancers (Jacobs, 2023).

Given the differential motif enrichments, whether TF motif content is predictive of an enhancer's CoR sensitivity was tested. For this, a Generalized Linear Model (GLM) was trained using TF motif counts as features and enhancer sensitivity to a given CoR as response variable. For each CoR, using 10-fold cross-validation, the models based on motif counts performed well and were able to predict an enhancer's sensitivity and resistance to a given CoR. Overall, these results establish a strong association between CoR sensitivity and TF motif content that is predictive and might correspond to causal relationships (Jacobs, 2023). It was noticed that DREF TF motifs were enriched in enhancers that were resistant to CoRest and CtBP. Indeed, enhancers bound by DREF, such as enhancers near the RYBP and jupiter genes, are specifically resistant to repression by CoRest and CtBP but sensitive to repression by Rbf. Ranking all enhancers based on their sensitivity to repression by CoRest or CtBP confirmed that resistant enhancers were significantly enriched for DREF motifs and bound by DREF according to ChIP-seq. Given the correlation between CoRest and CtBP resistance and presence of DREF, it was considered that DREF might protect enhancers against CoRest and CtBP-mediated repression. To test whether DREF motifs are indeed required for the resistance, 65 DREF-motif containing enhancers, mutated the DREF motifs, were tested, and the enhancers' sensitivity to repression was assessed. The mutated enhancers were significantly more sensitive to repression by CoRest and CtBP, while their sensitivity towards Rbf did not change. It is inferred that DREF motifs are required for resistance to CoRest and CtBP. Several other TF motifs were predicted to confer resistance to repression by specific CoRs. ETS- family motifs, for example, were specifically enriched in enhancers that were resistant to Rbf repression was observed. The ETS motifs were mutated in 157 enhancers and indeed increased sensitivity towards Rbf-mediated repression. Remarkably, the opposite trend was observed for the other CoRs, which were all able to repress the wildtype enhancers containing ETS motifs better than their mutated counterparts, suggesting that ETS TFs are in general sensitive to repression. Similarly, Trl motifs were enriched in enhancers that were specifically resistant to Rbf2 repression and mutating these motifs in 127 enhancers led to a slight but specific increase in sensitivity towards Rbf2, but not towards the other CoRs (Jacobs, 2023).

In general, specific TF motifs are required to protect resistant enhancers against a given CoR. As these motifs and enhancers are however still sensitive to other CoRs, an intricate pattern emerges, which suggests that the interplay between repressors and activators can be highly specific (Jacobs, 2023).

Given that specific TF motifs are required for CoR resistance, it was asked whether these motifs are also sufficient to cause CoR resistance. To test this, two 'resistant' TF motifs (10 bp each) were introduced into CoR-sensitive enhancers at positions deemed unimportant for enhancer activity, and the enhancers' sensitivity to repression was evaluated. As a control for the addition of motifs, two neutral control TF motifs were introduced at the same positions (of the ACE2 and FOX TFs), which were predicted to not impact the sensitivity to repression. Since DREF motifs were required for resistance to CoRest and CtBP-mediated repression, whether they were sufficient to render CoRest and CtBP-sensitive enhancers resistant was tested. Overall, the introduction of two DREF motifs was sufficient to protect the enhancers from CoRest and CtBP-mediated silencing and this protection was specific and due to the DREF motifs, as control motifs had no effect on sensitivity. Similarly, ETS motifs were necessary and sufficient to desensitize enhancers from repression by Rbf, and Trl motifs were necessary and sufficient to specifically generate Rbf2-resistant enhancers (Jacobs, 2023).

In each case, the gain in resistance due to the addition of selected TF motifs was highly CoR specific, as these new motifs had little effect on the repression by other CoRs or could even sensitize the enhancers to other CoRs. The addition of ETS or Trl motifs for example made enhancers more sensitive to all other CoRs except for Rbf or Rbf2 respectively, while DREF motifs increased sensitivity towards Rbf. Hence, introducing specific TF motifs changed the enhancer's sensitivity to repression by a given CoR in a specific and predictable manner. Taken together, it is concluded that specific TFs are necessary and sufficient to confer resistance to repression by a given CoR, while other CoRs are able to repress these TFs, indicating that certain TFs can directly counteract some CoRs and their repressive function or that different modes of transcriptional activation require different modes of repression (Jacobs, 2023).

This work has shown that transcriptional enhancers are differentially susceptible to active repression by prominent CoRs. The regulatory specificities between active enhancers and five CoRs were mappedon a genome-wide scale, and it was discovered that enhancers across the genome are repressed by just a subset of specific CoRs. This additional layer of regulatory specificities enables the differential repression of closely spaced enhancers and genes as they frequently occur in the Drosophila genome. At the fs(1)h/mys locus for example, CoRest could repress the intronic mys enhancer without affecting the neighboring fs(1)h gene, whereas Rbf could do the opposite. Similarly, at the kay/fig locus some CoRs like CtBP could repress both closely spaced enhancers, whereas Rbf2 could specifically repress the enhancer closest to fos intronic gene (fig) but not the others (Jacobs, 2023).

The uncovered specificities between repressors and enhancers not only suggest that repression occurs via distinct mechanisms, but also reveal a previously unappreciated layer of 'resistance' against repression. Enhancers that are sensitive or resistant towards a given CoR display clear differences in their TF motif content, and motif mutagenesis experiments changed enhancer sensitivities in a predictable manner. Specific motif combinations of activating and repressive TFs, together with the regulatory proteins present in each cell type, will largely determine when and where a regulatory region is active or repressed. In addition, the ability of TFs to confer resistance also allows for enhancers and genes to be activated by de- repression via TF recruitment, as has been demonstrated by motif addition. This additional layer of regulation circumvents the requirement to remove the involved repressors and the interplay between TFs and CoRs provides more flexibility to the regulatory system. Importantly, the fact that one mode of activation can be sensitive to one repressor but resistant to another implies that distinct activation and repression mechanisms must converge. Active repression might directly interfere with specific factors or defined steps of transcriptional activation. Certain CoRs might for example inactivate specific TFs but not others (e.g. via posttranslational modifications), or counteract the TFs' downstream activating mechanisms. Alternatively, certain TFs might directly counteract a repressor's function or bypass the rate-limiting step of initiation- or elongation controlled by the CoR. Discerning the distinct mechanisms of activation and repression and how they intersect and coordinate will be of great future interest (Jacobs, 2023).

An NRSF/REST-like repressor downstream of Ebi/SMRTER/Su(H) regulates eye development in Drosophila

The corepressor complex that includes Ebi and SMRTER is a target of epidermal growth factor (EGF) and Notch signaling pathways and regulates Delta (Dl)-mediated induction of support cells adjacent to photoreceptor neurons of the Drosophila eye. A mechanism is described by which the Ebi/SMRTER corepressor complex maintains Dl expression. charlatan (chn) is repressed by Ebi/SMRTER corepressor complex by competing with the activation complex that includes the Notch intracellular domain (NICD). Chn represses Dl expression and is critical for the initiation of eye development. Thus, under EGF signaling, double negative regulation mediated by the Ebi/SMRTER corepressor complex and an NRSF/REST-like factor, Chn, maintains inductive activity in developing photoreceptor cells by promoting Dl expression (Tsuda, 2006).

The corepressor complex that includes Ebi, SMRTER and Su(H) is required for expression of Dl in Drosophila photoreceptor cells. To identify genetic loci that are transcriptionally repressed by the Ebi corepressor, a screen was set up using an ectopic gene expression system (Gene Search System). Insertion of a Gene Search (GS) vector, a modified P-element carrying the Gal4 upstream activating sequence (UAS_G) near its 3' end, causes overexpression of a nearby gene under the control of the Gal4-UAS_G system. GS insertions into the chn locus were identified, whose overexpression phenotype in the eye using an eye-specific Gal4 driver (GMR-Gal4) was modified by reducing ebi activity. Thus the regulation of chn by Ebi-dependent transcriptional repression was studied (Tsuda, 2006).

In third instar larval-stage eye discs, the chn transcript is highly expressed in the morphogenetic furrow (MF), where photoreceptor differentiation initiates, but is downregulated in cells in the later stage photoreceptor development. In ebi mutant eye discs, however, chn expression becomes detectable in differentiating photoreceptor cells, and its expression in the MF is increased, suggesting that Su(H) in association with Ebi and SMRTER represses chn transcription in the eye disc (Tsuda, 2006).

To reveal the role of Su(H) as an activator, chn expression was examined when the level of Su(H) expression was reduced. Removing one copy of Su(H) suppresses the loss-of-Dl expression phenotype in ebi mutants. It was found that reducing one copy of Su(H) suppresses ectopic chn expression in ebi mutants, suggesting that ectopic expression of chn in ebi mutants is Su(H)-dependent. RT-PCR analysis of chn expression in ebi^- eye discs differing in the dosage of Su(H) gene also supported these results. Strong reduction of Su(H) expression alone reduced expression of chn in the MF; this expression became weaker and was slightly broader. The phenotype of ebi, Su(H) double mutants is almost the same as Su(H) single mutants , suggesting that Su(H) acts as an activator in the absence of Ebi. This might be due to dual functions of Su(H) as an activator or repressor. Hence, reducing the amount of Ebi in the corepressor complex involving Su(H) might convert Su(H) to an activator by permitting the replacement of the corepressor complex with NICD (Tsuda, 2006).

To reveal the molecular nature of transcriptional regulation of chn by Su(H), Su(H) target sites were sought in the genomic region of chn. Since Su(H) binds slightly degenerate sequences, it was not easy to identify the functional Su(H) binding region from a simple genomic search. An alternative approach was taken to map the chn genomic region, which is regulated by Su(H) in the normal chromosomal context. Ebi-mediated repression involves SMRTER, a corepressor that recruits histone deacetylases and induces the formation of inactive chromatin, which spreads from the site where Su(H) recruits the corepressor complex. Promoters near the Su(H)-binding site are thus expected to be downregulated in an Ebi-dependent manner. Four insertion lines of the GS vector were identified in the chn promoter region. All these GS lines caused ectopic expression of chn with consequent abnormal eye morphology when they were crossed with GMR-Gal4. If the effect of the Ebi/SMRTER corepressor complex reaches the UAS_G in those insertions, reduction of Ebi activity will derepress UAS_G and further enhance activation by GMR-Gal4. One copy of a dominant-negative construct of ebi (GMR-ebi^DN) caused only a mild defect in eye morphology and weak, if any, ectopic expression of chn. GMR-ebi^DN strongly enhanced the overexpression phenotype of chn^GS17605 and chn^GS11450, which contained GS vector insertions (-474 and -734, respectively) upstream of the transcriptional start site. However, GMR-ebi^DN failed to enhance the overexpression phenotype of other GS lines (chn^GS2112 and chn^GS17892) that were inserted downstream (+773 and +1040, respectively) of the first exon. From these results, it is concluded that Ebi-dependent transcriptional repression is targeted to the proximity of the transcriptional initiation site of the chn promoter (Tsuda, 2006).

Chn is a 1108-amino-acid protein with multiple C2H2-type zinc-finger motifs. Although no highly homologous gene within the mammalian genome could not be detected using BLAST, a small sequence of similarity between the N-terminal zinc-finger motif of Chn and the fifth zinc-finger of human NRSF/REST was found. Chn has several structural and functional similarities to human NRSF/REST, as follows. First, Chn and NRSF/REST each contain an N-terminal region with multiple zinc-finger motifs (five motifs in 264 residues in Chn and eight motifs in 251 residues in NRSF/REST), followed by a cluster of S/T-P motifs (serine or threonine followed by a proline) and a single zinc-finger motif at the C terminus. Second, the C-terminal region of NRSF/REST binds a corepressor, CoREST, which serves as an adaptor molecule to recruit a complex that imposes silencing activities. The Drosophila homolog of CoREST (dCoREST) (Andres, 1999; Dallman, 2004) can associate with the C-terminal half of Chn in cultured S2 cells. Finally, NRSF/REST binds to NRSE/RE1, a 21-bp sequence located in the promoter region of many types of neuron-restricted genes, via the N-terminal zinc-finger motifs. It was found that a recombinant protein containing the N-terminal zinc-fingers of Chn bound specifically to the NRSE/RE1 sequence in vitro. Thus, the structural similarity to NRSF/REST, binding to dCoREST and the DNA-binding specificity of Chn suggest that it is a candidate for a functional Drosophila homolog of NRSF/REST (Tsuda, 2006).

If Chn acts as a regulator of neural-related functions, as suggested for NRSF/REST, then Chn would be expected to bind to a regulatory region common to many types of neural-related genes in Drosophila. Numerous sequences similar to NRSE/RE1 were identified in the Drosophila genome, and their binding to Chn was assessed by EMSA. Using these sequences, a consensus binding sequence for Chn (Chn-binding element (CBE), 5'-BBHASMVMMVCNGACVKNNCC-3') was derived. 26 CBEs were identified within 10 kb of annotated genes from the Drosophila genome. Binding to Chn was confirmed for 18 CBEs using EMSA competition assay. Genes containing the CBE include dopamine receptor 2 (DopR2) and the potassium channel, ether-a-go-go, for which the mammalian homologs are target genes of NRSF/REST. These results suggest that the CBE is a good indicator of Chn binding sites and that Chn regulates many types of neural-related genes, as is implicated for NRSF/REST. However, it was found that divergent forms of CBE adjacent to hairy and extramacrochaetae were bound specifically by Chn. Likewise, some of the CBE sites failed to bind to Chn. Thus, a further refinement will be necessary to predict a definitive set of Chn binding sites in the Drosophila genome (Tsuda, 2006).

Although it has been established that mammalian NRSF/REST is a key regulator of neuron-specific genes, attempts to isolate invertebrate homolog of NRSF/REST have so far failed to identify a true homologous factor in invertebrates. The properties of Chn, including the similarity in DNA-binding specificity, association with CoREST and transcriptional repressor activity, suggest that Chn is a strong candidate for a functional Drosophila homolog of NRSF/REST. chn was originally identified by its requirement in the development of the PNS. This study identified a number of candidate target genes of Chn, a large fraction of which is implicated in neural function and/or gene expression. It is expected that further analysis of these candidates will provide valuable information about chn function in vivo, which may be extended to the understanding of NRSF/REST (Tsuda, 2006).

The Chn mutation blocks eye development by preventing the initiation of MF, a process requiring Notch signaling. This phenotype is likely owing to a loss of Notch function, because elevated Dl expression is known to block Notch signaling. The function of Chn during the early stage of eye development might be to regulate Notch signaling at an appropriate level by downregulating Dl. It is possible that Chn-mediated modulation places a variety of Notch functions in eye under the influence of EGFR signaling and provides flexibility in its regulation (Tsuda, 2006).

Although chn is expressed in the MF, genetic analyses show that small clones of chn mutant cells permit progression of the MF and photoreceptor differentiation. It is speculated that the repressive effect of Chn is overcome by other signals in the MF, such as hedgehog signaling, which strongly induces Dl (Tsuda, 2006).

Developing photoreceptor cells are exposed to the EGFR ligand, Spitz, and the Notch ligand, Dl, and each cell must assess the level of the two signals and respond appropriately to perform each task of photoreceptor cell specification and induction of non-neural cone cells. This question was investigated by studying the expression of Dl in photoreceptor cells. chn was identified as a direct target of Ebi/SMRTER-dependent transcriptional repression and as a repressor of Dl expression. The abrogated expression of Dl in ebi mutants was recovered by reducing one copy of chn, suggesting that the negative regulation of chn by ebi is indeed prerequisite for photoreceptor cell development (Tsuda, 2006).

Genetic data suggest that Su(H) may activate or repress chn expression. This idea is supported by data showing that Ebi/SMRTER and NICD are recruited to the promoter region of chn. The Ebi/SMRTER complex formed in this region did not contain any detectable level of the intracellular domain of Notch (NICD), suggesting that the binding of Ebi/SMRTER and NICD to this region may be mutually exclusive, and therefore it is expected that a regulatory system controls the balance between the active and repressive states of Su(H). Taken together, these results suggest that chn is a key factor in the crosstalk between two major signal transduction pathways: the EGFR-dependent pathway and the Notch/Delta-dependent pathway (Tsuda, 2006).

In the mammalian system, competition between SMRT and NICD for interaction with RBPJkappa determines the state of RBPJkappa-dependent transcriptional activity. Extracellular signaling may modulate this competition; diverse signaling pathways modulate the functions of N-CoR/SMRT. The current findings would prompt investigations of potential interaction of two repression systems of NRSF/REST and N-CoR/SMRT, and their regulation by Notch and EGF signaling in mammalian neuronal differentiation (Tsuda, 2006).

A conserved role but different partners for the transcriptional corepressor CoREST in fly and mammalian nervous system formation

Identification of conserved proteins that act to establish the neuronal phenotype has relied predominantly on structural homologies of the underlying genes. In the case of the repressor element 1 silencing transcription factor (REST), a central player in blocking the neuronal phenotype in vertebrate non-neural tissue, the invertebrate homolog is absent, raising the possibility that distinct strategies are used to establish the CNS of invertebrates. Using a yeast two-hybrid screen designed specifically to identify functional analogs of REST, this study shows that Drosophila melanogaster uses a strategy that is functionally similar to, but appears to have evolved independently of, REST. The gene at the center of the strategy in flies encodes the repressor Tramtrack88 (Ttk88), a protein with no discernable homology to REST but that nonetheless is able to interact with the same transcriptional partners. Ttk88 uses the REST corepressor Drosophila CoREST to coordinately regulate a set of genes encoding the same neuronal hallmarks that are regulated by REST in vertebrates. These findings indicate that repression is an important mechanism for regulating neuronal phenotype across phyla and suggest that co-option of a similar corepressor complex occurred to restrict expression of genes critical for neuronal function to a compartmentalized nervous system (Dallman, 2004).

MicroRNA targeting of CoREST controls polarization of migrating cortical neurons

The migration of cortical projection neurons is a multistep process characterized by dynamic cell shape remodeling. The molecular basis of these changes remains elusive, and the present work describes how microRNAs (miRNAs) control neuronal polarization during radial migration. This study shows that miR-22 and miR-124 are expressed in the cortical wall where they target components of the CoREST/REST transcriptional repressor complex, thereby regulating doublecortin transcription in migrating neurons. This molecular pathway underlies radial migration by promoting dynamic multipolar-bipolar cell conversion at early phases of migration, and later stabilization of cell polarity to support locomotion on radial glia fibers. Thus, this work emphasizes key roles of some miRNAs that control radial migration during cerebral corticogenesis (Volvert, 2014)

Search PubMed for articles about Drosophila CoRest

Abrajano, J. J., Qureshi, I. A., Gokhan, S., Zheng, D., Bergman, A. and Mehler, M. F. (2009a). Differential deployment of REST and CoREST promotes glial subtype specification and oligodendrocyte lineage maturation. PLoS One 4: e7665. PubMed ID: 19888342

Abrajano, J. J., Qureshi, I. A., Gokhan, S., Zheng, D., Bergman, A. and Mehler, M. F. (2009b). REST and CoREST modulate neuronal subtype specification, maturation and maintenance. PLoS One 4: e7936. PubMed ID: 19997604

Abrajano, J. J., Qureshi, I. A., Gokhan, S., Molero, A. E., Zheng, D., Bergman, A. and Mehler, M. F. (2010). Corepressor for element-1-silencing transcription factor preferentially mediates gene networks underlying neural stem cell fate decisions. Proc Natl Acad Sci U S A 107: 16685-16690. PubMed ID: 20823235

Adamo, A., Sese, B., Boue, S., Castano, J., Paramonov, I., Barrero, M. J. and Izpisua Belmonte, J. C. (2011). LSD1 regulates the balance between self-renewal and differentiation in human embryonic stem cells. Nat Cell Biol 13: 652-659. PubMed ID: 21602794

Andres, M. E., Burger, C., Peral-Rubio, M. J., Battaglioli, E., Anderson, M. E., Grimes, J., Dallman, J., Ballas, N. and Mandel, G. (1999). CoREST: a functional corepressor required for regulation of neural-specific gene expression. Proc Natl Acad Sci U S A 96: 9873-9878. PubMed ID: 10449787

Curtis, B. J., Zraly, C. B., Marenda, D. R. and Dingwall, A. K. (2011). Histone lysine demethylases function as co-repressors of SWI/SNF remodeling activities during Drosophila wing development. Dev Biol 350: 534-547. PubMed ID: 21146519

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Dallman, J. E., Allopenna, J., Bassett, A., Travers, A. and Mandel, G. (2004). A conserved role but different partners for the transcriptional corepressor CoREST in fly and mammalian nervous system formation. J Neurosci 24: 7186-7193. PubMed ID: 15306652

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date revised: 12 June 2021

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