Lori A. Pile

Chromosomal localization links the SIN3–RPD3 complex to the regulation of chromatin condensation, histone acetylation and gene expression

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 SIN3/RPD3 Deacetylase Complex Is Essential for G2 Phase Cell Cycle Progression and Regulation of SMRTER Corepressor Levels

ABSTRACT The SIN3 corepressor and RPD3 histone deacetylase are components of the evolutionarily conserved SIN3/RPD3 transcriptional repression complex. Here we show that 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 G2 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.

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, we used full-genome oligonucleotide microarrays 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 ∼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.

Localizing transcription factors on chromatin by immunofluorescence.

This article contains a detailed protocol for localizing transcription factors on Drosophila melanogaster polytene chromosomes by immunofluorescence. The large polytene chromosomes from third-instar larval salivary gland cells allow mapping of chromosome-associated proteins at high resolution. Thus, this method has been used to investigate how broadly transcription factors function and to identify and characterize cis-acting protein domains, trans-acting proteins, and trans-acting DNA elements that are necessary for chromosomal association of transcription factors. The ability to directly visualize transcription factors bound to chromosomes during a transcriptionally active stage of the cell cycle has greatly enhanced our understanding of how transcription factors function in vivo.

Analysis of Drosophila chromatin structure in vivo.

Publisher Summary This chapter presents the analysis of Drosophila chromatin structure in vivo . Gene activation in vivo is a complex process. The chapter discusses in detail approaches for mapping chromatin structure, both at the level of nucleosome arrays and at the level of base-pair resolution, using cells or nuclei isolated from Drosophila at different stages of the life cycle. An in vivo analysis of chromatin structure and its functional role in regulating the expression of a given gene requires the exploitation of genetic tools whereby the effects of sequence alterations within the putative regulatory region and of mutations in the proposed trans -acting regulatory proteins can be examined. The extensive genetic information available and the ability to return an altered gene to the genome by P-element transformation make Drosophila an excellent system for these types of functional studies. The chapter describes the process of identifying major embryonic chromatin structural features at specific loci.

Drosophila SIN3 Isoforms Interact with Distinct Proteins and Have Unique Biological Functions

The SIN3 corepressor serves as a scaffold for the assembly of histone deacetylase (HDAC) complexes. SIN3 and its associated HDAC have been shown to have critical roles in both development and the regulation of cell cycle progression. Although multiple SIN3 isoforms have been reported in simple to complex eukaryotic organisms, the mechanisms by which such isoforms regulate specific biological processes are still largely uncharacterized. To gain insight into how SIN3 isoform-specific function contributes to the growth and development of a metazoan organism, we have affinity-purified two SIN3 isoform-specific complexes, SIN3 187 and 220, from Drosophila S2 cells and embryos. We have identified a number of proteins common to the complexes, including the HDAC RPD3, as well as orthologs of several proteins known to have roles in regulating cell proliferation in other organisms. We additionally identified factors, including the histone demethylase little imaginal discs and histone-interacting protein p55, that exhibited a preferential interaction with the largest SIN3 isoform. Our experiments indicate that the isoforms are associated with distinct HDAC activity and are recruited to unique and shared sites along polytene chromosome arms. Furthermore, although expression of SIN3 220 can substitute for genetic loss of other isoforms, expression of SIN3 187 does not support Drosophila viability. Together our findings suggest that SIN3 isoforms serve distinct roles in transcriptional regulation by partnering with different histone-modifying enzymes.

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, we have 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. Developmental Dynamics 237:3040–3050, 2008.

GAGA Factor-dependent Transcription and Establishment of DNase Hypersensitivity Are Independent and Unrelated Events in Vivo

Using a Drosophila transgenic system we investigated the ability of GAGA factor, a putative anti-repressor, to modulate transcription-related events in the absence or presence of a bona fide activator, the Adf-1 transcription factor. In contrast to previous in vitro and in vivo data linking the binding of GAGA factor to the acquisition of DNase hypersensitivity at heat shock promoters, we observed that inserting multiple GAGA binding motifs adjacent to a minimal alcohol dehydrogenase (Adh) promoter led to strongly elevated embryonic transcription without creation of a promoter-associated DNase-hypersensitive (DH) site. Establishment of DNase hypersensitivity required the presence of both GAGA and Adf-1 binding sites and was accompanied by a further, synergistic increase in transcription. Because Adf-1 is capable neither of establishing a DH site nor of promoting efficient transcription by itself in embryos, it is likely that DH site formation depends on a GAGA factor-mediated binding of Adf-1 to chromatin, perhaps facilitated by a locally remodeled downstream promoter region. More generally we suggest that GAGA factor-binding sequences may operate in a promoter-specific context, with transcriptional activation, polymerase pausing, and/or DH site formation critically dependent on the nature of the sequences (and their binding partners) linked in cis.

Regulation of cell proliferation and wing development by Drosophila SIN3 and String.

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, we have generated conditional knock down of Drosophila SIN3 and analyzed effects on growth and development in the wing imaginal disc. We find 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.

The histone deacetylase inhibitor trichostatin A influences the development of Drosophila melanogaster

Abstract. We examined the consequences of the deacetylase inhibitor trichostatin A (TSA) on the development of Drosophila melanogaster. When fed to flies, TSA caused lethality and delayed development at concentrations as low as 5 μM, had stronger effects on males than females, and acted synergistically with mutations in the gene encoding the RPD3 deacetylase to cause notched wings, but did 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.