A Tale of Two Epigenetic Protein Pairs: Characterizing the Sin3aTET1 Complex and the Decrotonylation Activity of HDAC1/2
thesisposted on 12.09.2018, 11:27 authored by Aditya Chandru
Characterizing the Sin3a-TET1 Complex: TET 1 permits active DNA demethylation, promoting a transcriptionally permissible environment by preventing aberrant methylation. Contrastingly, Sin3a is the scaffold-protein central to the eponymous co-repressor complex. Histone deacetylases 1 and 2 (HDACl/2) form the enzymatic core of this protein-multiplex where they function to increase DNA:histone cohesion, thereby repressing gene expression. Despite these divergent activities, TET1 was found to bind Sin3a and recruit it to target genes. Mapping this interaction was integral to characterizing the TET1:Sin3a complex. An evolutionarily conserved 892-VAIEALTQLS-901 region in TET1 was identified. This a-helix was confirmed to bind to Sin3a's PAH1 domain. Complex formation was found to be equimolar and NMR illustrated the association to the resolution of individual residues. The TET1 Sin3a interaction domain (SID)'s bulky hydrophobic residues, and smaller adjacent alanines, bound to 18 PAH1 amino acids in a manner that resembles a key fitting a lock. The SAP25-SID interacts with these same PAH1 residues and binds in an identical orientation to TETI. While it initially seems curious that Sin3a and TET1 should bind, TETI operates as a net transcriptional repressor in cells. This activity appears independent of TET1's enzymatic function and instead likely relies on HDAC recruitment through the Sin3a co-repressor complex. This was confirmed in a reporter assay where Gal4 TET1-SID fusions repressed gene expression in a manner that was proportional to the fusion's ability to bind Sin3a. Characterizing the Decrotonylation Activity of HDAC1/2: In addition to Lys-Acetylation, a constellation of different Lys-Acylations have recently been discovered, including crotonylation. The ubiquity and nuclear localization of HDAC1/2 suggested their involvement in decrotonylation. HDAC1/2 DKO ESCs confirmed HDAC1/2's absence led to hypercrotonylation of the core histones. A novel decrotonylase assay established HDAC1/2 as potent lysine decrotonylases and that this activity could be recruited to lysine residues, by LSD1, via the CoREST complex.