The Brahma (Brm) complex of Drosophila melanogaster is a SWI/SNF-related chromatin remodeling complex required to correctly maintain proper states of gene expression through ATP-dependent effects on chromatin structure. The SWI/SNF complexes are comprised of 8-11 stable components, even though the SWI2/SNF2 (BRM, BRG1, hBRM) ATPase subunit alone is partially sufficient to carry out chromatin remodeling in vitro. The remaining subunits are required for stable complex assembly and/or proper promoter targeting in vivo. Our data reveals that SNR1 (SNF5-Related-1), a highly conserved subunit of the Brm complex, is required to restrict complex activity during the development of wing vein and intervein cells, illustrating a functional requirement for SNR1 in modifying whole complex activation functions. Specifically, we found that snr1 and brm exhibited opposite mutant phenotypes in the wing and differential misregulation of genes required for vein and intervein cell development, including rhomboid, decapentaplegic, thick veins, and blistered, suggesting possible regulatory targets for the Brm complex in vivo. Our genetic results suggest a novel mechanism for SWI/SNF-mediated gene repression that relies on the function of a ?core? subunit to block or shield BRM (SWI2/SNF2) activity in specific cells. The SNR1-mediated repression is dependent on cooperation with histone deacetylases (HDAC) and physical associations with NET, a localized vein repressor.

Wingless/Wnt signalling directs cell-fate choices during embryonic development. Inappropriate reactivation of the pathway causes cancer. In Drosophila, signal transduction from Wingless stabilizes cytosolic Armadillo, which then forms a bipartite transcription factor with the HMG-box protein Drosophila Tcf (dTcf) and activates expression of Wingless-responsive genes. Here we report that in the absence of Armadillo, dTcf acts as a transcriptional repressor of Wingless-responsive genes, and we show that Groucho acts as a corepressor in this process. Reduction of dTcf activity partially suppresses wingless and armadillo mutant phenotypes, leading to derepression of Wingless-responsive genes. Furthermore, overexpression of wild-type dTcf enhances the phenotype of a weak wingless allele. Finally, mutations in the Drosophila groucho gene also suppress wingless and armadillo mutant phenotypes as Groucho physically interacts with dTcf and is required for its full repressor activity.

Transcription repression mediated through histone deacetylase (HDAC) complexes is widespread, and mechanisms by which HDAC complexes act have been revealed by extensive studies in vitro and in cell culture. However, until recently, little has been known about the developmental roles of histone deacetylation. Mutants now exist for a number of members of the two major HDAC complexes (NuRD and SIN3) and some associated proteins. The emerging picture is that these complexes have specific functions in development, rather than being required for most cellular processes.

Acetylation of core histone N-terminal tails influences chromatin condensation and transcription. To examine how the SIN3-RPD3 deacetylase complex contributes to these events in vivo, we examined binding of SIN3 and RPD3 to DROSOPHILA: salivary gland polytene chromosomes. The binding patterns of SIN3 and RPD3 were highly coincident, suggesting that the SIN3-RPD3 complex is the most abundant chromatin-bound RPD3 complex in salivary gland cells. SIN3- RPD3 binding was restricted to less condensed, hypoacetylated euchromatic interbands and was absent from moderately condensed, hyperacetylated euchromatic bands and highly condensed, differentially acetylated centric heterochromatin. Consistent with its demonstrated role in transcriptional repression, SIN3-RPD3 did not co-localize with RNA polymer ase II. Chromatin binding of the complex, mediated by SMRTER, decreased upon ecdysone-induced transcriptional activation but was restored when transcription was reduced. These results implicate SIN3-RPD3 in maintaining histone acetylation levels or patterns within less condensed chromatin domains and suggest that SIN3-RPD3 activity is required, in the absence of an activation signal, to repress transcription of particular genes within transcriptionally active chromatin domains.

The Wnt signaling pathway controls cell proliferation and body patterning throughout development. A surprising number of cytoplasmic Wnt regulators (e.g., [beta]-catenin, Bcl-9/Lgs, APC, Axin) also appear, often transiently, in the nucleus. [beta]-Catenin is an integral component of E-cadherin complexes at intercellular adherens junctions, but also recruits chromatin remodeling complexes to activate transcription in the nucleus. The APC tumor suppressor is a part of the cytoplasmic [beta]-catenin destruction complex, yet also counteracts [beta]-catenin transactivation and histone H3K4 methylation at Wnt target genes. Furthermore, APC coordinates the cyclic exchange of Wnt coregulator complexes at the DNA. These opposing roles of APC and [beta]-catenin enable a rapid coordination of gene expression and cytoskeletal organization throughout the cell in response to signaling. 10.1101/gad.1424006

The Broad-Complex (BR-C) gene plays a key role in the ecdysone regulatory hierarchy. Together with other early ecdysone-inducible genes BR-C transmits the hormonal signal to a set of secondary response genes in a tissue-specific manner. Among its targets is the hsp23 gene. Previously we showed that expression of the hsp23 gene in late third instar is BR-C-dependent, and accompanied by the appearance of a BR-C-dependent DNase I hypersensitive site at position -1400 (DHS-1400). BR-C encodes a family of transcription factors, and we show here that at least three BR-C protein isoforms ? Z1, Z2, and Z3 ? bind to the sequences around DHS-1400 in vitro. A DNase I footprinting assay reveals five protected regions, designated site 1 to site 5, each of which specifically associates with one or several BR-C protein isoforms. We also show that a 100 bp region overlapping site 5, which binds all three isoforms in vitro, is required for hsp23 activity in vivo. The deletion of binding site 5 in a reporter gene construct reproduced the effect of the npr class mutations, that is, hsp23 is no longer expressed in any tissue tested except brain. Thus, BR-C regulates hsp23 expression via direct interaction of the predominant isoform with the distal regulatory element.

In Drosophila, all of the major metamorphic transitions are regulated by changes in the titer of the steroid hormone ecdysone. Here we examine how a key regulator of metamorphosis and primary ecdysone response gene, the Broad-Complex, transmits the hormonal signal to one of its targets, the Sgs-4 glue gene. We show that Broad-Complex RNAs accumulate in mid third instar larval salivary glands prior to Sgs-4 induction, as expected for the products of a gene that regulates the timing of Sgs-4 activation. The Broad-Complex codes for a family of zinc finger transcriptional regulators. We have identified a number of binding sites for these proteins in sequences known to regulate the timing of Sgs-4 induction, and have used these sites to derive a binding consensus for each protein. Some of these binding sites are required in vivo for Sgs-4 activity. In addition, rbp+, a genetically defined Broad-Complex function that is required for Sgs-4 induction, acts through these Broad-Complex binding sites. Thus, the Broad-Complex directly mediates a temporal and tissue-specific response to ecdysone as larvae become committed to metamorphosis.

The ensemble of tissue-specific changes that drivesDrosophilametamorphosis is initiated by the steroid hormone ecdysone and proceeds through a transcriptional cascade comprised of primary response transcriptional regulators and secondary response structural genes. TheBroad-Complex(BR-C) primary response early gene is composed of several distinct genetic functions and encodes a family of related transcription factor isoforms. Our objective in this study was to determine whether individual BR-C isoforms directly regulate secondary response target genes. A cluster of 10 salivary gland-specific secondary responseL71late genes are dependent on theBR-C rbp+genetic function. Transgenic animals expressing individual BR-C isoforms were tested for their ability to provide theBR-C rbp+genetic function by monitoring the transcriptional activation of theL71genes. We found that the BR-C Z1 isoforms could complement the transcriptional defects seen inrbpmutants but the Z2, Z3, and Z4 isoforms could not. We conclude that theBR-C rbp+function is provided by the BR-C Z1 isoform in prepupal salivary glands.L71gene rescue was restricted to the prepupal salivary gland, suggesting the involvement of additional factors inL71gene regulation. Interestingly, we found that the overexpression of Z3 or Z4 isoforms inBR-C+salivary glands repressedL71expression, indicating that BR-C proteins might also function as transcriptional repressors. Molecular mapping and characterization of the regulatory sequences that controlL71-6expression revealed several Z1 isoform binding sites. Mutagenesis of these Z1 binding sites resulted in the failure to activate late gene expressionin vivowhen measured by transgenic reporter genes. We conclude that theBR-Cearly gene directly activates late gene transcription by interacting with late genecis-acting regulatory elements and that this interaction is responsible for the temporal linkage of early and late ecdysone-induced gene expression.