HDAC activity is dispensable for repression of cell-cycle genes by DREAM and E2F:RB complexes

Abstract

Histone deacetylases (HDACs) are pivotal in transcriptional regulation, and their dysregulation has been associated with various diseases including cancer. One of the critical roles of HDAC-containing complexes is the deacetylation of histone tails, which is canonically linked to transcriptional repression. Previous research has indicated that HDACs are recruited to cell-cycle gene promoters through the RB protein or the DREAM complex via SIN3B and that HDAC activity is essential for repressing G1/S and G2/M cell-cycle genes during cell-cycle arrest and exit. In this study, we sought to explore the interdependence of DREAM, RB, SIN3 proteins, and HDACs in the context of cell-cycle gene repression. We found that genetic knockout of SIN3B did not lead to derepression of cell-cycle genes in non-proliferating HCT116 and C2C12 cells. A combined loss of SIN3A and SIN3B resulted in a moderate upregulation in mRNA expression of several cell-cycle genes in arrested HCT116 cells, however, these effects appeared to be independent of DREAM or RB. Furthermore, HDAC inhibition did not induce a general upregulation of RB and DREAM target gene expression in arrested transformed or non-transformed cells. Our findings provide evidence that E2F:RB and DREAM complexes can repress cell-cycle genes without reliance on HDAC activity.

Keywords: DREAM; HDAC; RB; SIN3B; cell cycle arrest; gene expression; histone deacetylase; p53; transcriptional regulation.

Fig. 1:. In silico identification of chromatin-binding proteins enriched at the promoters of G1/S and G2/M DREAM target genes.

The TFEA.ChIP tool was utilized for screening the ReMap2022 database for chromatin-binding proteins enriched at the promoters of (A) G1/S (n=109) or (B) G2/M (n=132) DREAM target genes. The plots show Log2(Odds Ratio) versus Log10(adjusted p-value) for each protein (represented as a single dot) in all included ChIP-Seq experiments.

Fig. 2:. SIN3B is not essential for the repression of G1/S and G2/M cell-cycle genes as a response to DNA damage or p53 activation in HCT116 cells.

(A) HCT116 cell lines negative for SIN3B were generated with a CRISPR/Cas9-nickase approach. Two pairs of guide RNAs, one targeting exon 3 and one targeting exon 4, were selected. Knockout clones were confirmed with antibodies binding epitopes within amino acids 172-228 (SIN3B-H4) or amino acids 668-758 (SIN3B polyclonal). Cells negative for SIN3B and LIN37 or RB were generated based on single knockout clones that we described earlier (Uxa et al. 2019). (B) mRNA expression of G2/M (BUB1, NEK2) and G1/S (MCM5, ORC1) cell-cycle genes was analyzed by RT-qPCR in wild-type (WT) and knockout lines after 48h treatment with 0.5 μM doxorubicin. The log2 fold change between untreated and treated cells is shown. Two independent SIN3B^−/−, SIN3B^−/−;LIN37^−/−, and SIN3B^−/−;RB^−/− clones were compared with wild-type cells and one LIN37^−/−, RB^−/− and LIN37^−/−;RB^−/− clone. The data set contains two biological replicates, and each one was measured with two technical replicates. Mean values +− SD are given. (C) Same experimental setup as in (B), but gene repression was induced by treatment with 5 μM Idasanutlin for 48 h. (D) Protein expression of HCT116 wild-type and knockout cells after treatment with DMSO or 5μM Idasanutlin for 48 h was analyzed by Western blotting. One representative experiment of three replicates is shown. (E) HDACI/II activity of samples immunoprecipitated with the indicated antibodies from HCT116 wild-type and knockout cells treated with 5 μM Idasanutlin for 48 h. Each data point contains four technical replicates of a representative experiment. Two biological replicates produced comparable results. (F) Protein expression and immunoprecipitation efficiency of the samples analyzed in (E) were evaluated by Western blotting. (G) ChIP-qPCR was performed to analyze binding of SIN3B, SIN3A, HDAC1, and the DREAM component p130 to DREAM target gene promoters in wild-type and SIN3B^−/− cells. A non-promoter region in the 3’ untranslated region of the DHFR gene (DHFR 3’ UTR) was amplified as a negative control. Results from one out of four independent experiments are shown. Data in Figs. B, C, E, and G are presented as mean values ± SD. Significances were calculated with the two-tailed Student’s T-Test (ns ‒ not significant, * p ≤.05, ** p ≤ .01, *** p ≤ .001). At least two biological replicates were performed for each Western Blot experiment, and results were similar.

Fig. 3:. Loss of SIN3B does not phenocopy LIN37 deficiency in T98G cells.

(A) A CRISPR/Cas9-nickase approach to introduce mutations in exon 4 of SIN3B and exon 6 of LIN37 was applied to generate cell lines negative for SIN3B, LIN37, or both proteins. SIN3B knockout clones were confirmed with antibodies targeting amino acids 172-228 (SIN3B-H4) or amino acids 668-758 (SIN3B polyclonal). LIN37 knockout was confirmed with a polyclonal antibody raised against full-length LIN37. (B) mRNA expression of G2/M (BUB1, CCNB2, BIRC5) and G1/S (MCM5, ORC1) cell-cycle genes was analyzed by RT-qPCR in wild-type and knockout lines arrested by serum-starvation. Two independent SIN3B^−/−, LIN37^−/−, and SIN3B^−/−;LIN37^−/− clones were compared with two wild-type (WT) clones measured with two technical replicates each. Significances of differences in gene expression after 120 h of serum deprivation were analyzed with the two-tailed Student’s T-Test. (C) Protein expression of one of the wild-type and knockout clones measured in (C) was analyzed by Western blotting. Similarly, (D) mRNA expression and (E) protein levels of cell-cycle genes were analyzed in two wild-type or knockout lines treated with 10 μM Palbociclib for 24 or 48 hours. (F) Indicated wild-type and knockout lines (two clones each) were serum-starved for 96hrs with or without 10 μM Palbociclib for the final 48 h. mRNA was measured (two technical replicates each) and compared with untreated wild-type mRNA levels. (G) Samples shown in (F) were additionally analyzed for protein expression by Western blotting. (H) HDACI/II activity of samples immunoprecipitated from T98G wild-type and knockout cells serum-starved for 96 h with the indicated antibodies. Each data point contains four technical replicates of a representative experiment. Two biological replicates produced similar results. (I) Protein expression and immunoprecipitation efficiency of the samples analyzed in (H) were evaluated by Western blotting. Data in Figs. B, D, F, and H are given as mean values ± SD, and significances were calculated with the two-tailed Student’s T-Test (ns – not significant, * p ≤.05, ** p ≤ .01, *** p ≤ .001). At least two biological replicates were performed for each Western Blot experiment, and results were similar.

Fig. 4:. Sin3b knockout does not phenocopy loss of Lin37 in mouse C2C12 cells.

(A) A CRISPR/Cas9-nickase approach was applied to generate cell lines negative for Sin3b. Sin3b knockout clones were confirmed with antibodies targeting an epitope within amino acids 172-228 (SIN3B-H4). Lin37^−/− C2C12 cells were described before (Mages et al. 2017). (B) mRNA expression of cell-cycle genes was analyzed by RT-qPCR in wild-type and knockout lines arrested by serum starvation over 48 and 72h. Two wild-type, one Lin37^−/−, and three SIN3B^−/− clones were measured, with two technical replicates each. (C) EdU incorporation was analyzed in wild-type and Sin3B^−/− lines (3 clones each) after 48 h of serum deprivation. (D) mRNA expression of cell-cycle genes was analyzed by RT-qPCR in the same wild-type and knockout lines shown in (B) treated with 5 μM Idasanutlin for 24 and 48 h. (E) Protein expression of one clone analyzed in (D) was studied by Western blot. Data in (B), (C), and (D) Data are presented as mean values ± SD. Significances were calculated with the two-tailed. Student’s T-Test (ns – not significant, * p ≤.05, ** p ≤ .01, *** p ≤ .001). Western Blot experiment were performed with at least two biological replicates with similar results.

Fig. 5:. Combined depletion of SIN3A and SIN3B derepresses a subset of cell-cycle genes independently of DREAM or RB.

Transcriptome analyses were performed with HCT116 wild-type (WT) and SIN3B^−/− HCT116 cells transfected either with a non-targeting siRNA or with SIN3A siRNAs for 48 h and treated with Idasanutlin for the final 24 h. (A) Volcano plots showing up and downregulated genes in comparison to wild-type cells. Numbers of significantly regulated genes (p<0.05) with an FC ≥1.5 are shown in red. Genes identified as LIN37/DREAM targets before (Uxa et al., 2019) are highlighted in blue. The p-values indicate the probability that the respective overlap between LIN37 and SIN3-regulated genes could be observed by chance (hypergeometric test). (B) The number and overlap of LIN37 target genes identified in Uxa et al. and genes upregulated (p<0.05; FC ≥1.5) in Idasanutlin-treated HCT116 cells depleted of SIN3A, SIN3B, or both proteins. (C) GO analyses (biological processes) of significantly upregulated (p<0.05; FC ≥1.5) genes. The top ten hits based on their false discovery rate (FDR) are shown. (D) Wild-type, SIN3B^−/−, SIN3B^−/−;LIN37^−/−, and SIN3B^−/−;RB^−/− knockout lines were transfected with non-targeting or SIN3A siRNAs for 48 h, and Idasanutlin was applied for the final 24 h. Protein levels were analyzed by Western blotting. A representative blot of cells transfected with either a non-silencing control RNA (CTRL) or SIN3A siRNA 1 is shown. A biological replicate with SIN3A siRNA 4 produced similar results. (E) mRNA levels of genes identified as significantly upregulated in SIN3A/B-depleted cells in the transcriptome analysis were evaluated by RT-qPCR. Cells were treated as described in (D), but additionally transfected with SIN3A siRNA 4. Averages (mean values ± SEM) of three biological and two technical replicates are given. Significances were calculated with the two-tailed Student’s T-Test (ns – not significant, * p ≤05, ** p ≤ .01, *** p ≤ .001).

Fig. 6:. HDAC activity is not generally required for cell-cycle gene repression in arrested cells.

In all the following experiments, cells were treated with 5 μM Idasanutlin for 48 h and 4 nM Romidepsin for the final 24 h. (A) Histone modifications at cell-cycle gene promoters in HCT116 cells were analyzed by ChIP-qPCR. mRNA expression was evaluated by RT-qPCR and compared to DMSO-treated cells for each respective line. One representative experiment with two technical replicates (mean values ± SD) is shown. A biological replicate produced similar results. Data are presented as gene-set clusters (left) and individual genes (right) in: (B) HCT116, (D) A549, (F) C2C12, and (H) BJ-hTERT cells. The datasets contain two biological replicates with two technical replicates each. Mean values ± SEM are shown, and significances were calculated with the two-tailed Students T-Test (ns – not significant, * p ≤.05, ** p ≤ .01, *** p ≤ .001). Protein expression and histone acetylation were evaluated via Western blotting with the indicated antibodies for: (C) HCT116, (E) A549, (G) C2C12, and (I) BJ-hTERT cells. A biological replicate for each Western blot experiment produced similar results.