Regulation of transcriptional repression by the mammalian Sin3 corepressor complex.

摘要

Control of transcription is important for cell growth, division, and differentiation. Proper levels of transcription are balanced by opposing activities of activators and repressors. Generally, activators function by recruiting coactivator complexes containing chromatin modifying enzymes whose net effect is to relax nucleosomal constraints to facilitate access of the transcriptional machinery. Repressors oppose this process by recruiting corepressor complexes whose enzymatic activities restore the repressive effects of chromatin rendering the binding sites for the transcriptional machinery inaccessible.; mSin3A is an abundant, ubiquitously expressed corepressor, conserved from yeast to humans. Roles for mSin3A in differentiation, cell cycle, apoptosis, silencing, nuclear hormone signaling, and oncogenesis have been established through mSin3A's association with multiple transcriptional repressors, suggesting that mSin3A is a central player in the regulation of diverse cellular processes. mSin3A functions as a large corepressor complex consisting of at least seven tightly associated stoichiometric subunits. This dissertation describes the identification and characterization of several of these mS&barbelow;in3A-a&barbelow;ssociated p&barbelow;roteins (SAPs).; A candidate approach identified the histone deacetylases HDAC1 and HDAC2. Both proteins were enzymatically active and coprecipitated with mSin3A. When HDAC activity was chemically inhibited, mSin3A repression was reduced. Further, HDAC1 repressed transcription when targeted to DNA, but when its catalytic activity was abolished by a point mutation, HDAC1-mediated repression was not observed. Together the experiments demonstrated that mSin3A repression was due in part to the deacetylase activity of the associated proteins HDAC1 and HDAC2.; To identify additional SAPS a mSin3A complex was purified. Mass spectrometry and protein sequencing identified three novel copurifying proteins, SAP180, SAP130, and SAP45. All three proteins interacted with mSin3A-HDAC and repressed transcription when targeted to DNA. Together these proteins are predicted to stabilize the mSin3A complex, target the complex to DNA, and to recruit additional transcriptional regulators.; Finally, the corepressor TLE1 was shown to interact with the mSin3A complex and contribute to mSin3A repression, but not in the presence of enzymatically active HIPK2. HIPK2, identified by yeast two hybrid with the chromodomain of SAP180, inhibited both SAP180 and mSin3A repression by disrupting mSin3A-TLE1 interaction. As such, the data described a novel mechanism of transcriptional regulation.