Structural basis for the assembly of the SMRT/NCoR core transcriptional repression machinery

Abstract

Eukaryotic transcriptional repressors function by recruiting large co-regulatory complexes that target histone deacetylase enzymes to gene promoters/enhancers. Transcriptional repression complexes, assembled by the co-repressor NCoR, and its homologue SMRT, play critical roles in many processes including development and metabolic physiology. The core repression complex involves the recruitment of three proteins: HDAC3, GPS2 and TBL1 to a highly conserved repression domain within SMRT and NCoR. We have used a variety of structural and functional approaches to gain insight into the assembly, stoichiometry and biological role of this complex. We report the crystal structure of the tetrameric oligomerization domain of TBL1, which interacts with both SMRT and GPS2, and the NMR structure of the interface complex between GPS2 and SMRT. These structures, together with computational docking, mutagenesis and functional assays, reveal the assembly mechanism and stoichiometry of the co-repressor complex.The regulated repression of transcription plays a key role in many biological processes. These include cell fate decisions during development and cellular differentiation, as well as the maintenance of homeostasis. SMRT and NCoR are large homologous co-repressor proteins that were identified through their role in transcriptional repression by many 5 Corresponding author: john.schwabe@le.ac.uk. 4 These authors should be considered co-first authors. Author ContributionsThe contributions of J.O., L.F. & P.W. were critical to the final manuscript and these authors should be considered co-first authors. J.O. (assisted by J.Gooch) performed most of the protein cloning, expression, purification and interaction mapping, although important preliminary experiments were performed by B.K. J.O. prepared the GPS2-SMRT complex for NMR structure determination which was carried out by J-C.Y. and D.N. Crystallizations were performed primarily by J.O., with some later trials by J.Greenwood and L.F. Crystal structure determinations were performed by L.F., J.O. and J.W.R.S. The interaction motifs in GPS2 and SMRT were identified by J.W.R.S. and tested by pull-down experiments by J.O. Fluorescence polarization, co-immunoprecipitation and co-transfection/ purification assays were performed by P.W. The two-hybrid assays, gel filtrations and NMR comparisons of WT and MT TBL1 were performed by P.W., Z.C. and B.G. In silico docking experiments were performed by T.K. The laboratories of L.N. and D.N. provided experimental expertise for transfection and NMR studies respectively. J.W.R.S. planned and supervised the project and prepared the manuscript with assistance from the other authors. Accession Codes1H, 13C and 15N NMR resonance assignments for the SMRT(167-207) -GPS2(53-90) complex have been deposited at the BioMagResBank under accession code 17271, and the coordinates have been deposited under the pdb accession code 215G. The TBL1 X-ray structures have been deposited with the PDB codes (2XTC, 2XTD & 2XTE). When purified from HeLa cell extr...

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