Fig. 5: USP35 enhances ERα transcriptional activity by binding to estrogen responsive element containing region in ERα target genes.

USP35 overexpression increases (a) and USP35 knockdown decreases (b) mRNA levels of the estrogen induced genes. Indicated MCF-7 cell lines were treated with vehicle or E₂ (10 nM) for 6 h before subjected to qRT-PCR assay. c USP35 overexpression enhances ERE-luciferase reporter activity. Indicated cell lines were cotransfected with C3-ERE-luc reporter and TK-renilla plasmids, treated with E₂ (10 nM) for 24 h, and subjected to luciferase activity assay. TK renilla luciferase was used to normalize transfection efficiency. d Estrogen enhances USP35 binding to ERE-containing region in the ERα target genes. MCF-7 cells stimulated with E₂ (10 nM) were subjected to ChIP assay using USP35 or ERα antibody followed by qPCR of the pS2 and GREB1 DNA regions containing ERE. The quantification of fold enrichment relative to input levels was shown. e, f USP35 promotes ERα binding to ERE-containing region in pS2 and GREB1 in MCF-7 cells. MCF-7 cells stably expressing vector and USP35 (e) or MCF-7 cells expressing con-sh and USP35-sh#1 (f) were stimulated with E₂ (10 nM) for 15 min and subjected to ChIP assay using ERα antibody. g USP35 and ERα are recruited together to ERE-containing regions of pS2 and GREB1. MCF7 cells were ChIP and re-ChIP using USP35 and ERα antibodies sequentially. Chromatin samples were analyzed by qPCR. *, p < 0.05; ***, p < 0.001.