The histone demethylase KDM3A regulates the transcriptional program of the androgen receptor in prostate cancer cells

Abstract

The lysine demethylase 3A (KDM3A, JMJD1A or JHDM2A) controls transcriptional networks in a variety of biological processes such as spermatogenesis, metabolism, stem cell activity, and tumor progression. We matched transcriptomic and ChIP-Seq profiles to decipher a genome-wide regulatory network of epigenetic control by KDM3A in prostate cancer cells. ChIP-Seq experiments monitoring histone 3 lysine 9 (H3K9) methylation marks show global histone demethylation effects of KDM3A. Combined assessment of histone demethylation events and gene expression changes presented major transcriptional activation suggesting that distinct oncogenic regulators may synergize with the epigenetic patterns by KDM3A. Pathway enrichment analysis of cells with KDM3A knockdown prioritized androgen signaling indicating that KDM3A plays a key role in regulating androgen receptor activity. Matched ChIP-Seq and knockdown experiments of KDM3A in combination with ChIP-Seq of the androgen receptor resulted in a gain of H3K9 methylation marks around androgen receptor binding sites of selected transcriptional targets in androgen signaling including positive regulation of KRT19, NKX3-1, KLK3, NDRG1, MAF, CREB3L4, MYC, INPP4B, PTK2B, MAPK1, MAP2K1, IGF1, E2F1, HSP90AA1, HIF1A, and ACSL3. The cancer systems biology analysis of KDM3A-dependent genes identifies an epigenetic and transcriptional network in androgen response, hypoxia, glycolysis, and lipid metabolism. Genome-wide ChIP-Seq data highlights specific gene targets and the ability of epigenetic master regulators to control oncogenic pathways and cancer progression.

Keywords: ChIP-Seq; cancer systems biology; epigenomics; oncogene; prostate cancer.

Figure 1. ChIP-Seq experiments with matched knockdown of KDM3A show gain of histone 3 lysine 9 methylation and transcriptional deactivation

A. Western blot of prostate cancer line CWR22Rv1 with antibodies against H3K9me1, H3K9me2, KDM3A (lysine demethylase 3A; Gene ID: 55818; also referred to as JMJD1A or JHDM2A), and beta-actin. ChIP-Seq experiments using B-C. H3K9me1-antibody and D-E. H3K9me2-antibody show specific gain of signal following small hairpin RNA (shRNA) knockdown of the histone methyltransferase KDM3A in the prostate cancer line CWR22Rv1. Genomic location of C) H3K9me1 and E) H3K9me2 sites identified by ChIP-Seq. F. H3K9me1 and H3K9me2 ChIP-Seq signals are overlapped and annotated. G. The majority of histone demethylase events due to KDM3A activity is detected by both, H3K9me1 and H3K9me2, ChIP-Seq antibodies. H. Overlay of gene mapping of histone methylation events identified by ChIP-Seq and transcriptomics experiments. Using this data we defined the group of 1408 genes as positively regulated by KDM3A activity (down in the prostate cancer line CWR22Rv1 with shRNA knockdown of KDM3A), and 1002 genes as negatively regulated by KDM3A activity. I. Transcriptomic impact of KDM3A knockdown shows 58.4% of gene activation (down in the prostate cancer line CWR22Rv1 with shRNA knockdown of KDM3A), and 41.6% of gene silencing.

Figure 2. Knockdown of KDM3A results in epigenetic control and transcriptional activation of the androgen response

A. Map of transcriptional regulation of KDM3A on androgen signaling. Red indicates positive response of KDM3A on gene (down with shRNA knockdown of KDM3A); blue indicates negative response of KDM3A on gene (up with shRNA knockdown of KDM3A). B. Gene set enrichment analysis of ranked transcriptomic data upon shRNA knockdown of KDM3A indicated significant enrichment of hallmark gene set of androgen response with p-value and false discovery rate q-value below 1.0E-20.

Figure 3. Detection of histone demethylase events by ChIP-qRT-PCR due to KDM3A activity in target genes involved in the androgen response

CWR22Rv1 prostate cancer cells transfected with pLKO.1 control or KDM3A shRNA were subjected to the chromatin immunoprecipitation coupled with quantitative real time polymerase chain reaction (ChIP-qRT-PCR) assay using immunoprecipitation with A. H3K9me1-antibody and B. H3K9me2-antibody. The precipitated chromatin fragments were analyzed by qRT-PCR using oligonucleotides for identified androgen response element regions of KLK3, NKX3-1 or MYC.

Figure 4. Matched ChIP-Seq and knockdown experiments of KDM3A in combination with ChIP of the androgen receptor show synergy of KDM3A and the androgen receptor

ChIP-Seq experiments in combination with KDM3A knockdown results in A. specific loss of KDM3A ChIP-Seq signal, B. specific gain of H3K9me1 ChIP-Seq signal for 97.1% of the observed histone marks, C. specific gain of H3K9me2 ChIP-Seq signal for 95.6% of the observed histone marks, and D. specific loss of ChIP-Seq signal for the androgen receptor (AR) with less than 2.3% retained binding. Peak calling utilizing a model-based analysis of ChIP-Seq algorithm results in E. 37525 peaks for KDM3A binding, F. 77911 peaks for H3K9 demethylation events (H3K9me1/2-KDM), and G. 34614 peaks for matched AR binding including 121700 androgen response elements (AREs). Motif analysis of KDM3A ChIP-Seq signals identifies pattern of transcription factor families including the AR motivating analysis of epigenetic and transcriptional cooperation between KDM3A and AR. H. KDM3A and H3K9 methylation ChIP-Seq signals are overlapped and annotated. I. H3K9 methylation and AR ChIP-Seq signals are overlapped and annotated. 1912 genes (gene set marked with *) showed coincidence of demethylation and AR binding events. J. Overlap of KDM3A and H3K9 methylation ChIP shows strong epigenetic network of KDM3A binding and activity. Overlap of KDM3A, H3K9 methylation, and matched AR ChIP shows participation of KDM3A in transcriptional activation. K. 421 genes (gene set marked with **) showed transcriptomic change upon KDM3A/AR knockdown in addition to coactivation detected by KDM/AR ChIP-Seq. 260 genes (gene set marked with ***) were positively regulated by KDM3A or AR activity (down in the prostate cancer line CWR22Rv1 with shRNA knockdown of KDM3A) and identified by KDM/AR ChIP-Seq. 45 genes genes (gene set marked with ****) showed overlap of transcriptomic response upon KDM3A and AR knockdown as well as coactivation detected by KDM/AR ChIP-Seq. L. Transcriptomic impact of KDM3A knockout shows 60.2% of gene activation (down in the prostate cancer line CWR22Rv1 with shRNA knockdown of KDM3A), and 39.8% of gene silencing.