Fig. 3: Binding of SIN3/HDAC to DREAM and cell-cycle gene promoters.

a HDACI/II activity of samples immunoprecipitated with the indicated antibodies from HCT116 wild-type and knockout cells treated with 5 µM Idasanutlin for 48 hours. Each data point contains four technical replicates ( ± SEM) of a representative experiment. Two biological replicates produced comparable results. b Protein expression and immunoprecipitation efficiency of the samples analyzed in (a) were evaluated by Western blotting. Two biological replicates produced comparable results. c ChIP-qPCR was performed to analyze the 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. d HCT116 clonal cell lines containing a non-functional CHR element in the BUB1 or CCNB2 promoter on both alleles were created by CRISPR/Cas9-mediated knock-in. Sanger sequencing confirmed the mutation of the elements. e The binding of SIN3A, SIN3B, and HDAC1 to the BUB1 or CCNB2 promoter in the cell lines described in (d) was studied by ChIP-qPCR. Loss of DREAM binding upon mutation of the CHR was verified by analyzing the binding of the DREAM components LIN37 and p130. Note the BUB1 CHR wild-type cell line in this experiment is the CCNB2 CHR mutant line and vice versa. f mRNA expression of BUB1, CCNB2, and NEK2 was analyzed by RT-qPCR in the HCT116 clones carrying mutated CHR sites in the BUB1 or CCNB2 promoter after 48 hours treatment with 5 µM Idasanutlin. The log2 fold change between untreated and treated cells is shown. For experiments in (c), (e), and (f), averages of three independent experiments measured with two technical replicates are shown and presented as mean values ± SEM. Significances were calculated with the two-tailed Student’s T-Test (ns – not significant, * p ≤.05, ** p ≤ .01, *** p ≤ .001). Source data are provided as a Source Data file.