Structural and biochemical investigation of the Sin3a : HDAC1 : SDS3 transcriptional co-repressor complex
thesisposted on 10.07.2018, 09:36 authored by Irene Nigi
The Sin3a complex is a large multi-protein complex that is involved in embryonic development, cell cycle regulation, proliferation, oncogene regulation and senescence. The Sin3a protein acts as a scaffold protein and the complex contains several proteins including HDAC1 and HDAC2 as catalytic subunits, SDS3 and various SAPs. The Sin3a complex is one of the most evolutionarily conserved HDAC complexes regulates transcription by altering chromatin condensation. To date only few structures of Sin3a-Paired Amphipathic Helices domains interacting with various proteins have been published, and it therefore remains unknown how Sin3a interacts with HDAC1 and how the activity of the complex is regulated. In this study a stable Sin3a ternary complex containing Sin3a : HDAC1 : SDS3 was expressed using a mammalian expression system and purified using two alternative methods. A structural model was built by combining data from cross-linking, Small Angle X-Ray Scattering and negative stain Electron Microscopy providing the first structural information on the assembly of the Sin3a complex. SDS3 was found to be essential to allow the Sin3a complex to assemble as a dimer. The model also suggests that Sin3a interacts with HDACl through a structured central region, while the N- and C-termini of the HID domain wrap around the catalytic subunits to secure a stable interaction. Enzymatic assays showed that the Sin3a complex, unlike other HDAC complexes, is not regulated by inositol tetrakis phosphate and suggest a distinct regulatory mechanism that might involve the presence of different proteins within the Sin3a complex. In conclusion, both structural and functional results suggest that the Sin3a complex appears to be distinct from other Class I HDACs with both its structure and regulatory mechanism.