Eukaryotic gene transcription is a coordinated process involving the participation of a large number of proteins. Sequence-specific DNA-binding transcription factors function as activators or repressors of transcription and frequently recruit transcriptional coregulators that lack specific DNA-binding activity. These multi-protein complexes with intrinsic chromatin-modifying and chromatin-remodeling activities have emerged as key regulators of eukaryotic gene transcription. The Sin3 complex was one of the first corepressor complexes to be identified and is found in organisms ranging from yeast to human. In mammals, it is thought to be composed of at least ten polypeptides besides Sin3A/B. The Sin3 proteins appear to serve a dual role as molecular adapters bridging the enzymatic components of the complex with promoter-bound repressors and also as molecular scaffolds for the assembly of the complexes. To better understand how these multi-subunit complexes are recruited to specific loci on the genome and how the core components of the complexes are built into functional assemblies, I have conducted structure-function analysis toward the mammalian Sin3A corepressor complex using solution-state NMR spectroscopy as the primary tool. My results suggested that the PAH1 and PAH2 domains in the mammalian Sin3 proteins are structurally independent and ruled out any heterotypic association between the paralogous mSin3A and mSin3B proteins via interactions involving the mSin3A PAH2 domain. Preliminary results from structure determination of the mSin3A PAH3-SAP30 SID complex indicated that the PAH3 domain forms a four-helix bundle similar to other PAH domains, but with helix a3 much shorter than in the PAH1 and PAH2 domains. In addition, the SAP30 SID domain adopts a structure comprising three helices, which is a departure from the structural motif used by proteins targeting the mSin3A PAH1 and PAH2 domains. Furthermore, I discovered and determined the structure of a novel zinc finger motif in SAP30, which appears to function as a nucleic acid binding domain and implies a previously unknown function for the Sin3 corepressor complex. Finally, a bottom-up approach has been taken to incrementally reconstitute and characterize a sub-complex comprising subunits specific to the Sin3A/B corepressor complexes, and a portion of the co-expressed proteins remained in a soluble form. These studies have furthered our understanding of the mechanisms of recruitment and complex assembly for Sin3 while providing paradigmatic basis for characterization of other HDAC-associated corepressor complexes.

Last modified

• 09/07/2018

Creator

• Yuan He

DOI

• https://doi.org/10.21985/N2RF0T

Subject

• Interdepartmental Biological Sciences Program

Keyword

• Biophysics

Date created

• 2008-05-07

Resource type

• Dissertation

Rights statement

• In Copyright