Structural Insights into the Recruitment of SMRT by the Corepressor SHARP under Phosphorylative Regulation

Abstract

The transcriptional corepressors SMRT/NCoR, components of histone deacetylase complexes, interact with nuclear receptors and many other transcription factors. SMRT is a target for the ubiquitously expressed protein kinase CK2, which is known to phosphorylate a wide variety of substrates. Increasing evidence suggests that CK2 plays a regulatory role in many cellular events, particularly, in transcription. However, little is known about the precise mode of action involved. Here, we report the three-dimensional structure of a SMRT/HDAC1-associated repressor protein (SHARP) in complex with phosphorylated SMRT, as determined by solution NMR. Phosphorylation of the CK2 site on SMRT significantly increased affinity for SHARP. We also confirmed the significance of CK2 phosphorylation by reporter assay and propose a mechanism involving the process of phosphorylation acting as a molecular switch. Finally, we propose that the SPOC domain functions as a phosphorylation binding module.

A phospho-dependent mechanism involving NCoR and KMT2D controls a permissive chromatin state at Notch target genes

“…By site-directed mutagenesis of the SPOC-domain of SHARP we would gain further mechanistic insights into how phospho-NCoR and KMT2D recruitment is regulated. For this purpose, cocrystalisation of the SPOC domain ( 42 ) together with phosphorylated NCoR or the most recent NMR-structure of SPOC-domain in complex with phosphorylated SMRT-peptide ( 59 ) could be the basis.…”

Integrative genomic analysis in K562 chronic myelogenous leukemia cells reveals that proximal NCOR1 binding positively regulates genes that govern erythroid differentiation and Imatinib sensitivity

“…Furthermore, increased corepressor binding also promotes direct association with the transcriptional repressor ZBTB33/KAISO ( 33 ) and targets increased DNA methylation ( 33 – 35 ). More recently, NCOR2/SMRT binding to SPEN/SHARP ( 36 ) has been shown to be important for gene silencing mediated by Xist ( 37 ).…”

PHF3 regulates neuronal gene expression through the new Pol II CTD reader domain SPOC

“…PHF3 SPOC did not bind the unphosphorylated CTD diheptapeptide ( Figure S2A), but phosphorylation of S2 within both repeats was sufficient to confer strong binding ( Figure 2B To uncover the basis of the CTD heptapeptide-SPOC interaction, we determined the structure of PHF3 SPOC in the apo form (2.6 Å), and bound to pS2 (2.0 Å), pS2pS7 (1.75 Å) and pS2pS5 (2.85 Å) diheptapeptides ( Figure 2D-I, Figure S2I-L). Superimposition of PHF3 SPOC with SPOC domains from SHARP (Mikami et al, 2014) and FPA (Zhang et al, 2016) revealed that PHF3 SPOC has a distorted β-barrel fold comparable to other structurally characterized SPOCs, with seven β-strands and four helices connected with various loop regions ( Figure 2D). The pS2-and pS2pS7-diheptapeptides were bound between strands of the β-barrel and along the α1 helix, in an extended conformation with trans isomer configuration of prolines ( Figure 2E The ε-amino groups K1267 and K1309 from the first patch on SPOC form a hydrogen bond with O1P from the N-terminal pS2a, whereas guanidinium nitrogens from R1248 and R1297 from the second patch form hydrogen bonds with O1P and O3P from the C-terminal pS2b ( Figure 2G,H).…”

“…These residues are required for the SPOC-containing protein SHARP to interact with the co-repressor complex SMRT/NCoR (Ariyoshi and Schwabe, 2003). Serine phosphorylation of the LSD motif within SMRT or NCoR increases their binding affinity for the conserved Arg within the SHARP SPOC domain, suggesting that the SPOC domain is a phospho-serine binding module (Mikami et al, 2014;Oswald et al, 2016).…”