Functional Analysis Of Sin3 Isoforms In Drosophila

he multisubunit SIN3 complex is a global transcriptional regulator. In Drosophila, a single Sin3A gene encodes different isoforms of SIN3, of which SIN3 187 and SIN3 220 are the major isoforms. Previous studies have demonstrated functional non-redundancy of SIN3 isoforms. The role of SIN3 isoforms in regulating distinct biological processes, however, is not well characterized. In addition, how the components of the SIN3 complex modulate the gene regulatory activity of the complex is not well understood. In this study, I identified the biological processes regulated by the SIN3 isoforms. Additionally, I explored how Caf1-55 impacts the gene regulatory activity of the SIN3 220 complex. For the purpose of the study, I developed a highly reproducible ChIP protocol using micrococcal nuclease (MNase)-mediated chromatin preparation from Drosophila cultured cells. This protocol can be used to perform ChIP to map both histones and non-histone chromatin binding proteins locally and globally across the genome. Next, we identified the biological processes regulated by the SIN3 isoforms. We established a Drosophila S2 cell culture model system in which cells predominantly express either SIN3 187 or SIN3 220. To identify genomic targets of SIN3 isoforms, we performed chromatin immunoprecipitation followed by deep sequencing. Our data demonstrate that upon overexpression of SIN3 187, the level of SIN3 220 decreased and the large majority of genomic sites bound by SIN3 220 were instead bound by SIN3 187. We used RNA-seq to identify genes regulated by the expression of one isoform or the other. In S2 cells, which predominantly express SIN3 220, we found that SIN3 220 directly regulates genes involved in metabolism and cell proliferation. We also determined that SIN3 187 regulates a unique set of genes and likely modulates expression of many genes also regulated by SIN3 220. Interestingly, biological pathways enriched for genes specifically regulated by SIN3 187 strongly suggest that this isoform plays an important role during the transition from the embryonic to the larval stage of development. Finally, I investigated the function of Caf1-55 in the SIN3 220 complex. Our data demonstrate that Caf1-55 localizes to SIN3 220 gene targets and is partly required for recruiting SIN3 220 to chromatin. In addition, we show that the C-terminal domain of SIN3 220 physically interacts with Caf1-55. We found that the interaction between SIN3 and Caf1-55 is significantly reduced upon mutating the histone H4 binding pocket of Caf1-55. Surprisingly, the reduced interaction between the histone H4 binding mutant of Caf1-55 and SIN3 220 is not sufficient to cause a change in the expression of SIN3 220 regulated genes. Together, these data provide evidence of a novel role of Caf1-55 in impacting recruitment of a component of a chromatin modifying complex to genomic loci. In summary, our research reveals important insights of how the SIN3 isoform specific complexes might function during the course of fly development

Genome-wide studies reveal novel and distinct biological pathways regulated by SIN3 isoforms

Detailed annotation of ChIP-seq peaks for SIN3 187HA (SIN3 187HA ceas) or SIN3 220HA (SIN3 220HA ceas) as determined by the cis-regulatory enrichment annotation (CEAS) system. This table is related to Fig. 2 (XLSX 4014 kb

SETDB1 mediated histone H3 lysine 9 methylation suppresses MLL-fusion target expression and leukemic transformation

Epigenetic regulators play a critical role in normal and malignant hematopoiesis. Deregulation, including epigenetic deregulation, of the HOXA gene cluster drives transformation of about 50% of acute myeloid leukemia. We recently showed that the Histone 3 Lysine 9 methyltransferase SETDB1 negatively regulates the expression of the pro-leukemic genes Hoxa9 and its cofactor Meis1 through deposition of promoter H3K9 trimethylation in MLL-AF9 leukemia cells. Here, we investigated the biological impact of altered SETDB1 expression and changes in H3K9 methylation on acute myeloid leukemia. We demonstrate that SETDB1 expression is correlated to disease status and overall survival in acute myeloid leukemia patients. We recapitulated these findings in mice, where high expression of SETDB1 delayed MLL-AF9 mediated disease progression by promoting differentiation of leukemia cells. We also explored the biological impact of treating normal and malignant hematopoietic cells with an H3K9 methyltransferase inhibitor, UNC0638. While myeloid leukemia cells demonstrate cytotoxicity to UNC0638 treatment, normal bone marrow cells exhibit an expansion of cKit+ hematopoietic stem and progenitor cells. Consistent with these data, we show that bone marrow treated with UNC0638 is more amenable to transformation by MLL-AF9. Next generation sequencing of leukemia cells shows that high expression of SETDB1 induces repressive changes to the promoter epigenome and downregulation of genes linked with acute myeloid leukemia, including Dock1 and the MLL-AF9 target genes Hoxa9, Six1, and others. These data reveal novel targets of SETDB1 in leukemia that point to a role for SETDB1 in negatively regulating pro-leukemic target genes and suppressing acute myeloid leukemia