Biol. two mutually exclusive HIC1 binding sites (HiRE) around the promoter. transcriptional activation induced by short-term serum starvation coincides with loss of occupancy of the distal sites by HIC1/MTA1 and HIC1/CtBP. Upon longer starvation, both complexes are found but on a newly identified proximal HiRE that is evolutionarily conserved and specifically enriched with repressive histone marks. Our results decipher a mechanistic link between two competitive posttranslational modifications of HIC1 and corepressor recruitment to specific genes, leading to growth control. (is usually a direct target gene of P53 (5, 24, 65). HIC1 directly represses the transcription of transcription in response to DNA damage, but SIRT1 binds and deacetylates E2F1 that inhibits E2F1-mediated gene activation (30, 66). In addition, E2F1 directly activates (27) and HIC1 directly represses the promoter in quiescent but not in G1 human fibroblasts, which contributes to the growth suppression induced by serum deprivation (71). Tmem14a Thus, HIC1 is placed at the intersection of complex regulatory loops modulating p53-dependent and E2F1-dependent cell survival, growth control, and stress responses (15). encodes a sequence-specific transcriptional repressor with five Krppel-like C2H2 zinc fingers mediating DNA binding to a HIC1 responsive element (HiRE), (C/G)NG(C/G)GGGCA(C/A)CC Talarozole (48). To date, (a proneuronal transcription factor) (4), (a receptor for the chemokine CXCL12) (62), and ephrin-A1 (a cell surface ligand for Eph receptors) (72) are the only characterized direct target genes of HIC1. Our earlier work exhibited that HIC1 contains two autonomous repression domains, an N-terminal BTB-POZ (16) and a central region that recruits CtBP corepressor complexes through a conserved GLDLSKK motif (17, 58). Notably, hypoxia promotes association of HIC1 with CtBP (58), whereas glycolysis inhibition by 2-deoxyglucose decreases it (70). Thus, through its conversation with the redox sensor CtBP, HIC1 can specifically link expression to free nuclear Talarozole NADH levels. SIRT1 positively regulates HIC1 repressive activity by orchestrating with HDAC4 a switch between acetylation and SUMOylation, two mutually exclusive posttranslational modifications on the same lysine K314 in the conserved K314xEP motif (59). Despite its functional importance, the mechanistic details including corepressor recruitment and gene-specific regulation through this acetylation/SUMOylation switch are not known. In the present study, by yeast two-hybrid (Y2H) screening, we identified MTA1 as a novel corepressor for HIC1. The MTA family of proteins consists of evolutionarily conserved integral subunits of the NuRD (nucleosome remodeling and histone deacetylase) complex (29). We show that endogenous HIC1 interacts with MTA1 and MBD3, two specific subunits of the NuRD complex, and recruits it to the promoter in WI38 fibroblasts. We further demonstrate that this acetylation/SUMOylation switch on K314 regulates the conversation with MTA1 and RBBP4 and thus the recruitment of the NuRD complex. Indeed, SUMOylation-deficient or constitutively acetylated mutants exhibiting Talarozole significantly reduced binding to MTA1 Talarozole displayed a decreased repression in transient reporter assays. However, this cross talk between two posttranslational modifications (PTMs) on HIC1 K314 has no effect on the recruitment of CtBP by the central region. Consistent with the role of HIC1 in growth control, we demonstrate that CtBP and NuRD complexes are differentially recruited on known HIC1 target genes in quiescent versus growing human fibroblasts WI38. In particular, HIC1/MTA1 complexes cooccupy the promoters of two new direct target genes, and promoter in quiescent versus proliferating WI38 cells. Notably, the evolutionarily conserved HiRE identified here is specifically enriched with repressive histone marks in quiescent cells. Thus, Talarozole our results provide a unique example of promoter-specific recruitment of repression complexes regulated through competitive posttranslational modifications of HIC1. MATERIALS AND METHODS Y2H screen. Yeast two-hybrid (Y2H) screening was performed by Hybrigenics, Paris, France. For bait cloning, the BTB-central region of HIC1 (positions 1 to 422) encompassing the two autonomous repression domains was PCR amplified and cloned in frame with a C-terminal LexA DNA-binding domain name in a Y2H vector. The bait construct was checked by sequencing the insert and was transformed in the L40GAL4 yeast strain. A human breast tissue random-primed cDNA library, transformed into the Y187 yeast strain and.