Lateral inhibition is critical for cell fate determination and involves the functions of Notch (N) and its effectors, the Enhancer of Split Complex, E(spl)C repressors. Although E(spl) proteins mediate the repressive effects of N in diverse contexts, the role of phosphorylation was unclear. The studies we describe implicate a common role for the highly conserved Ser/Thr protein kinase CK2 during eye and bristle development. Compromising the functions of the catalytic (alpha) subunit of CK2 elicits a rough eye and defects in the interommatidial bristles (IOBs). These phenotypes are exacerbated by mutations in CK2 and suppressed by an increase in the dosage of this protein kinase. The appearance of the rough eye correlates, in time and space, to the specification and refinement of the 'founding' R8 photoreceptor. Consistent with this observation, compromising CK2 elicits supernumerary R8's at the posterior margin of the morphogenetic furrow (MF), a phenotype characteristic of loss of E(spl)C and impaired lateral inhibition. We also show that compromising CK2 elicits ectopic and split bristles. The former reflects the specification of excess bristle SOPs, while the latter suggests roles during asymmetric divisions that drive morphogenesis of this sensory organ. In addition, these phenotypes are exacerbated by mutations in CK2 or E(spl), indicating genetic interactions between these two loci. Given the centrality of E(spl) to the repressive effects of N, our studies suggest conserved roles for this protein kinase during lateral inhibition. Candidates for this regulation are the E(spl) repressors, the terminal effectors of this pathway.

Brahma (BRM) is a novel anticancer gene, which is frequently inactivated in a variety of tumor types. Unlike many anticancer genes, BRM is not mutated, but rather epigenetically silenced. In addition, histone deacetylase complex (HDAC) inhibitors are known to reverse BRM silencing, but they also inactivate it via acetylation of its C-terminus. High-throughput screening has uncovered many compounds that are effective at pharmacologically restoring BRM and thereby inhibit cancer cell growth. As we do not know which specific proteins, if any, regulate BRM, we sought to identify the proteins, which underlie the epigenetic suppression of BRM. By selectively knocking down each HDAC, we found that HDAC3 and HDAC9 regulate BRM expression, whereas HDAC2 controls its acetylation. Similarly, we ectopically overexpressed 21 different histone acetyltransferases and found that KAT6A, KAT6B and KAT7 induce BRM expression, whereas KAT2B and KAT8 induce its acetylation. We also investigated the role of two transcription factors (TFs) linked to either BRM (GATA3) or HDAC9 (MEF2D) expression. Knockdown of either GATA3 and/or MEF2D downregulated HDAC9 and induced BRM. As targets for molecular biotherapy are typically uniquely, or simply differentially expressed in cancer cells, we also determined if any of these proteins are dysregulated. However, by sequencing, no mutations were found in any of these BRM-regulating HDACs, HATs or TFs. We selectively knocked down GATA3, MEF2D, HDAC3 and HDAC9, and found that each gene-specific knockdown induced growth inhibition. We observed that both GATA3 and HDAC9 were greatly overexpressed only in BRM-negative cell lines indicating that HDAC9 may be a good target for therapy. We also found that the mitogen-activated protein (MAP) kinase pathway regulates both BRM acetylation and BRM silencing as MAP kinase pathway inhibitors both induced BRM as well as caused BRM deacetylation. Together, these data identify a cadre of key proteins, which underlie the epigenetic regulation of BRM.

Rhabdoid sarcomas are highly malignant tumors that usually occur in young children. A key to the genesis of this tumor is the mutational loss of the BAF47 gene as well as the widespread epigenetic suppression of other key anticancer genes. The BRM gene is one such epigenetically silenced gene in Rhabdoid tumors. This gene codes for an ATPase catalytic subunit that shifts histones and opens the chromatin. We show that BRM is an epigenetically silenced gene in 10/11 Rhabdoid cell lines and in 70% of Rhabdoid tumors. Moreover, BRM can be induced by BAF47 re-expression and by Flavopiridol. By selective shRNAi knockdown of BRM, we show that BRM re-expression is necessary for growth inhibition by BAF47 re-expression or Flavopiridol application. Similar to lung cancer cell lines, we found that HDAC3, HDAC9, MEF2D and GATA3 controlled BRM silencing and that HDAC9 was overexpressed in Rhabdoid cancer cell lines. In primary BRM-deficient Rhabdoid tumors, HDAC9 was also found to be highly overexpressed. Two insertional BRM promoter polymorphisms contribute to BRM silencing, but only the -1321 polymorphism correlated with BRM silencing in Rhabdoid cell lines. To determine how these polymorphisms were tied to BRM silencing, we conducted ChIP assays and found that both HDAC9 and MEF2D bound to the BRM promoter at or near these polymorphic sites. Using BRM promoter swap experiments, we indirectly showed that both HDAC9 and MEF2D bound to these polymorphic sites. Together, these data show that the mechanism of BRM silencing contributes to the pathogenesis of Rhabdoid tumors and appears to be conserved among tumor types.

Chromatin remodeling complex SWI/SNF plays important roles in many cellular processes including transcription, proliferation, differentiation and DNA repair. In this report, we investigated the role of SWI/SNF catalytic subunits Brg1 and Brm in the cellular response to cisplatin in lung cancer and head/neck cancer cells. Stable knockdown of Brg1 and Brm enhanced cellular sensitivity to cisplatin. Repair kinetics of cisplatin DNA adducts revealed that downregulation of Brg1 and Brm impeded the repair of both intrastrand adducts and interstrand crosslinks (ICLs). Cisplatin ICL-induced DNA double strand break repair was also decreased in Brg1 and Brm depleted cells. Altered checkpoint activation with enhanced apoptosis as well as impaired chromatin relaxation was observed in Brg1 and Brm deficient cells. Downregulation of Brg1 and Brm did not affect the recruitment of DNA damage recognition factor XPC to cisplatin DNA lesions, but affected ERCC1 recruitment, which is involved in the later stages of DNA repair. Based on these results, we propose that SWI/SNF chromatin remodeling complex modulates cisplatin cytotoxicity by facilitating efficient repair of the cisplatin DNA lesions.

Our results, using endogenous mutants and Gal4-UAS driven transgenes, implicate multisite phosphorylation in repression by E(spl)M8. We propose that these phosphorylations occur in the morphogenetic furrow (MF) to reverse an auto-inhibited state of M8, enabling repression of Atonal during R8 specification. Our studies address the paradoxical behavior of M8*, the truncated protein encoded by E(spl)D. We suggest that differences in N signaling in the bristle versus the eye underlie the antimorphic activity of M8* in N + (ectopic bristles) and hypermorphic activity in N spl (reduced eye). Ectopic M8* impairs eye development (in N spl ) only during establishment of the atonal feedback loop (anterior to the MF), but is ineffective after this time point. In contrast, a CK2 phosphomimetic M8 lacking Groucho (Gro) binding, M8SDΔGro, acts antimorphic in N + and suppresses the eye/R8 and bristle defects of N spl , as does reduced dosage of E(spl) or CK2. Multisite phosphorylation could serve as a checkpoint to enable a precise onset of repression, and this is bypassed in M8*. Additional implications are discussed.