Differential regulation of HIC1 target genes by CtBP and NuRD, via an acetylation/SUMOylation switch, in quiescent versus proliferating cells

Abstract

The tumor suppressor gene HIC1 encodes a transcriptional repressor involved in regulatory loops modulating P53-dependent and E2F1-dependent cell survival, growth control, and stress responses. Despite its importance, few HIC1 corepressors and target genes have been characterized thus far. Using a yeast two-hybrid approach, we identify MTA1, a subunit of the NuRD complex, as a new HIC1 corepressor. This interaction is regulated by two competitive posttranslational modifications of HIC1 at lysine 314, promotion by SUMOylation, and inhibition by acetylation. Consistent with the role of HIC1 in growth control, we demonstrate that HIC1/MTA1 complexes bind on two new target genes, Cyclin D1 and p57KIP2 in quiescent but not in growing WI38 cells. In addition, HIC1/MTA1 and HIC1/CtBP complexes differentially bind on two mutually exclusive HIC1 binding sites (HiRE) on the SIRT1 promoter. SIRT1 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.

FIG. 1.

HIC1 interacts with MTA1 and recruits it to the SIRT1 promoter in vivo. (A) Scheme of the human HIC1 and MTA1 proteins. The BTB/POZ domain, the central region (CR), containing the CtBP-interaction domain (CID) and the acetylation/SUMOylation switch motif (ψK³¹⁴XEP), and the five C₂H₂ zinc fingers are shown. The bait included the two autonomous repression domains of HIC1 (amino acids 1 to 422). The domains in MTA1 include BAH, ELM, SANT, and the GATA-like zinc finger. The two first cysteine of the zinc finger were not present in the isolated prey (underlined lowercase letters). (B) In vitro, HIC1 interacts with MTA1. HEK293T transfected as indicated were lysed, immunoprecipitated, and immunoblotted with the indicated antibodies to detect MTA1 and HIC1. Totals of 2% of the whole-cell extracts were similarly analyzed (Input). (C) Co-occupancy of HIC1 and MTA1 on the SIRT1 promoter. Human WI38 fibroblasts were analyzed by single ChIP with the indicated antibodies. PCR amplifications were performed with primers flanking the functional HiREs identified in SIRT1. PCR with the 5′ promoter of GAPDH was used as an internal nonbinding control.

FIG. 2.

HIC1 interacts with the NuRD complex. (A) Scheme of human NuRD complexes. The core components (HDAC1/2, RBBP7/RbAp46, and RBBP4/RbAp48) shared with the Sin3 complex are shown in black, whereas NuRD-specific subunits (MTA1/2, p66, ATPase Mi2, and MBD3) are shown in light gray. (B) In vitro, HIC1 interacts with MBD3 and MBD2. HEK 293T were transfected as indicated, immunoprecipitated and analyzed by Western blotting. Totals of 2% of each whole-cell extract were similarly analyzed (Inputs). (C) HIC1 interacts with the NuRD complex. Total extracts from HEK 293T transfected with the FLAG-HIC1 or a control vector (FLAG) were immunoprecipitated by anti-FLAG antibodies coupled to agarose beads. Western blot analyses showed the coimmunoprecipitation of endogenous NuRD subunits with HIC1. (D) In vivo, endogenous HIC1 interacts with MBD3. DAOY cells, a medulloblastoma cell line expressing HIC1, were lysed, immunoprecipitated, and analyzed by immunoblotting. A shorter exposure of the input lane is shown on the right. Arrows indicate the MBD3 bands. (E) MBD3 binds the SIRT1 promoter. Human WI38 fibroblasts were analyzed by ChIP as described for Fig. 1D.

FIG. 3.

SUMOylation on HIC1 K314 favors the interaction with MTA1. (A and B) HEK 293T were transfected with the indicated HIC1 mutants. The consequences on interaction with CtBP, as well as on potential PTMs, are shown. HIC1 was immunoprecipitated, and MTA1 coprecipitation was detected by Western blotting.

FIG. 4.

The acetylation/SUMOylation switch on HIC1 K314 regulates the interaction with two components of the NuRD complex (MTA1 and RBBP4) but has no effect on the recruitment of CtBP2. (A) A mutant protein, FLAG-HIC1 K314Q, mimicking a constitutively acetylated isoform of HIC1 is shown. The P317A mutant has been previously described. HEK 293T cells were transfected as indicated. HIC1 was immunoprecipitated and MTA1 coprecipitation was detected by Western blotting. (B) A similar experiment was performed with wt HIC1, the K314R and K314Q point mutants, and HA-RBBP4. (C) wt HIC1 and the constitutively acetylated K314Q mutant were similarly tested for interaction with CtBP2. (D) HEK 293T cells were transfected with wt, E316A, and K314R HIC1 with (+) or without (−) P300. Whole-cell extracts were analyzed by immunoblotting with the specific Ac-HIC1 K314, with FLAG used as a loading control, and with P300 antibodies to detect its overexpression. (E) Overexpression of Gal4-P300, which acetylates HIC1 on K314, impairs the interaction with MTA1. HIC1 was immunoprecipitated and MTA1 coprecipitation was detected by Western blotting.

FIG. 5.

Promoter association analyses through sequential ChIP assays. (A) Preparation of quiescent G₀ and growing mid-G₁ WI38 cell populations. Subconfluent WI38 cells were maintained in medium without serum (Quiescent Cells) for 72 h. The cells were cultured for 10 h in medium with 10% serum to obtain growing cells synchronized in mid-G₁ phase. Whole-cell extracts were analyzed by Western blotting for PCNA, cyclin D1, and P57KIP2. In the latter case, the antibody detected the full-length P57KIP2 protein (arrow), as well as an ∼40-kDa product (asterisk). HSP60 was used as a loading control. (B) HIC1, SUMOylated proteins, MTA1, SIRT1, and HDAC4 are present on SIRT1 and ATOH1. Sequential ChIP were carried out on mid-G₁ WI38 cells with the indicated antibodies. PCR amplifications were performed as described for Fig. 1D. (C) Differential regulation of known (SIRT1, ATOH1, CXCR7, and E2F1) and newly identified (Cyclin D1 and P57KIP2) HIC1 target genes by CtBP and NuRD complexes in quiescent versus growing WI38 cells. Sequential ChIP analyses were performed to analyze the co-occupancy of HIC1 and MTA1 and of HIC1 and CtBP1 on the indicated target genes. As a control, sequential MTA1/HIC1 ChIP was also performed in quiescent cells.

FIG. 6.

The K314Q point mutant mimicking constitutive acetylation is unable to repress Cyclin D1 and P57KIP2 in NIH 3T3 cells. (A) HIC1 but not the K314Q mutant markedly repressed the expression of P57KIP2 and cyclin D1 proteins. NIH 3T3 cells were transfected with the pcDNA3 FLAG-HIC1 (wt), FLAG-HIC1 L225A, and FLAG-HIC1 K314Q expression vectors containing a neomycin resistance gene. After 10 days in medium containing 1 mg of G418/ml, whole cells extracts were analyzed by SDS-PAGE. HIC1 overexpression and upregulation of endogenous P57KIP2 and cyclin D1 proteins were analyzed by immunoblotting. The antibody directed against P57KIP2 detected the full-length protein (arrow) and an ∼40-kDa product (asterisk), exactly as in WI38 cells (Fig. 5A). Actin was used as a loading control. (B) SUMOylation-dependent association of HIC1 and MTA1 on P57KIP2. Human SKNMC cells were transfected with an expression vector for the de-SUMOylase SSP3 and analyzed by single ChIP. PCR amplifications were performed on P57KIP2 and on GAPDH, as a control.

FIG. 7.

HIC1 recruits the CtBP and NuRD complexes on mutually exclusive sites of the SIRT1 promoter in quiescent versus mid-G₁ growing WI38 cells. (A) Identification of a new, evolutionarily conserved, HIC1 binding site on the SIRT1 promoter. In silico analyses demonstrated that the two previously described “distal” HiRE (black boxes) are not well conserved and identified a new proximal HiRE conserved in the three genomes (gray box). The transcription start site (+1, bent arrow) and the ATG initiation codon (+54) are indicated. The “proximal” HIC1 site is adjacent to a conserved E2F1 binding site (plain oval), whereas a site not found in the murine genome is shown as a dotted oval. (B) HIC1 occupies the distal and proximal sites on the SIRT1 promoter in a mutually exclusive way. ChIP was performed on WI38 cells growing in complete medium (0 h) or after 24, 48, or 72 h of serum starvation. (C) Differential binding of HIC1/MTA1 and HIC1/CtBP complexes on the SIRT1 distal and proximal promoters. Sequential ChIP analyses were performed on quiescent and mid-G₁ WI38 cells, followed by PCR with primers amplifying the two sites in SIRT1.

FIG. 8.

Epigenetic marks deposited on the two mutually exclusive HiRE of SIRT1 in quiescent versus mid-G₁ cells. (A) Changes in enrichment of silencing epigenetic marks on the mutually exclusive HiRE of the SIRT1 promoter in quiescent versus mid-G₁ WI38 cells. ChIP were performed with antibodies directed against various histone posttranslational modifications. PCR amplifications were performed with primers amplifying the two types of HiREs in SIRT1 or the −1623 HiRE in Cyclin D1. (B) Whole-cell extracts from quiescent or mid-G₁ WI38 cells were analyzed by Western blotting for the expression of SIRT1 and HIC1. In each case, HSP60 was used as a loading control. (C) Expression levels of SIRT1 and Cyclin D1 in quiescent versus mid-G₁ WI38 fibroblasts were assessed by qPCR, and the values were normalized to 18S RNA. The P values are indicated (*, P < 0.05). (D) Knockdown of endogenous MTA1 in mid-G₁ growing WI38 cells increases the level of acetylated H3 at the SIRT1 distal promoter and upregulates SIRT1 expression. WI38 cells were transfected with nontargeting or MTA1 siRNA and synchronized in mid-G₁ phase (see Fig. 5A). MTA1 and SIRT1 levels were analyzed by immunoblotting with actin as a loading control. ChIP analyses were performed with total acetylated H3 and H3 antibodies, followed by quantitative PCR with primers amplifying the two distal HiREs.

FIG. 9.

Model. (A) HIC1 repressive complexes and epigenetic marks found on the distal and proximal SIRT1 promoter in quiescent versus mid-G₁ WI38 fibroblasts. The brown circle represents a yet-undefined component of the NuRD complex that could directly interact with SUMO covalently linked to HIC1 K314. RBBP4 interacts directly with the MTA1 GATA zinc finger. (B) Dynamic binding of the HIC1/CtBP and HIC1/NuRD complexes on the two mutually exclusive HiRE on SIRT1. (a) In asynchronously growing WI38 fibroblasts, HIC1 recruits CtBP and NuRD complexes on the previously described distal HiRE (8). (b) After 24 h of serum starvation, when SIRT1 mRNAs and proteins levels increase (42, 71), HIC1 is no more detected on the SIRT1 promoter. (c) After 48 h, HIC1 is detected again on SIRT1 but on the proximal site identified herein. The presence of the NuRD and CtBP complexes was not investigated. (d) However, as shown by sequential ChIP, both complexes are present on the conserved proximal site after 72 h in starvation medium. (e) After serum replenishment for 10 h, the HIC1/CtBP and HIC1/NuRD complexes are relocalized on the distal sites.