Figure 7.

KDM5C and ZMYND8 interact with CDK9 and CCNT1 in the P‐TEFb complex, respectively, to activate estrogen/ERα‐target genes. A) The number of peptide spectrum matches (PSMs), unique peptides, and the abundance ratio (estrogen vs control) for CDK9 and CCNT1 proteins identified to be associated with KDM5C in the nucleus as described in Figure 5A was shown. B) Nuclear extracts from MCF7 cells were subjected to immunoprecipitation (IP) with anti‐KDM5C antibody followed by immunoblotting (IB) analysis as indicated. C) Nuclear extracts from HEK293T cells transfected with HA‐tagged KDM5C and Flag‐tagged CDK9 or CCNT1 were subjected to immunoprecipitation (IP) with anti‐Flag antibody followed by immunoblotting (IB) analysis as indicated. IgG H.C: IgG heavy chain. D) Nuclear extracts from HEK293T cells transfected with HA‐tagged ZYMND8 and Flag‐tagged CDK9 or CCNT1 were subjected to immunoprecipitation (IP) with anti‐Flag antibody followed by immunoblotting (IB) analysis as indicated. IgG H.C: IgG heavy chain. E) In vitro pull‐down assay was performed by mixing in vitro purified Flag‐tagged CDK9 and HA‐tagged KDM5C or ZYMND8 proteins from HEK293T cells overexpressing these proteins, followed by immunoprecipitation (IP) with anti‐HA antibody and immunoblotting (IB) analysis with anti‐Flag antibody. F) In vitro pull‐down assay was performed by mixing in vitro purified Flag‐tagged CCNT1 and HA‐tagged KDM5C or ZYMND8 proteins from HEK293T cells overexpressing these proteins, followed by immunoprecipitation (IP) with anti‐HA antibody and immunoblotting (IB) analysis with anti‐Flag antibody. G) In vitro purified HA‐tagged KDM5C and ZYMND8, and Flag‐tagged CDK9 and CCNT1 proteins were examined by coomassie blue staining (C.B.S) and indicated by white arrows. Non‐specific band was indicated by asterisk. H) MCF7 cells were transfected with control siRNA (siCTL) or siRNA specific against KDM5C (siKDM5C) or ZYMND8 (siZYMND8) in stripping medium for three days, and treated with or without estrogen (E₂, 10⁻⁷ m, 1 h) followed by CDK9 ChIP‐seq analysis. Metagenes of CDK9 ChIP‐seq tag density across those genes induced by estrogen and dependent on KDM5C and ZYMND8 for expression (n = 784) were shown. Units are mean tags per bin for 500 bins across the transcribed region of each gene with 3 kb upstream (300 bins of 10 bp each) and 3 kb downstream flanking regions (300 bins of 10 bp each). I) Heat map representation of CDK9 ChIP‐seq tag density as shown in (H).