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Genome-wide identification of estrogen receptor binding sites reveals novel estrogen-responsive pathways in adult male germ cells

ABSTRACT: Spermatogenesis occurs in the seminiferous epithelium that shows presence of estrogen receptors alpha (ERα) and beta (ERβ), both of which regulate gene transcription by binding to the DNA. Hormone responsive phases of spermatogenesis are well documented; yet, the genes regulated remain inexplicit. To study the regulation of genes by estrogen in male germ cells, we performed chromatin immunoprecipitation (ChIP) sequencing for ERα and ERβ under normal physiological conditions. We observed a total of 27,221 peaks in ERα and 20,926 peaks in ERβ. Majority of the peaks were present in the intronic regions and located 20kb upstream or downstream from the transcription start site (TSS). Upon pathway analysis, the genes enriched showed involvement in several biological pathways. Genes involved in pathways whose role in spermatogenesis is unexplored were validated; these included prolactin, GnRH and oxytocin signalling. All the genes showed presence of estrogen response elements (EREs) and majority of them showed significant enrichment by ChIP-qPCR. Functional validation using seminiferous tubule culture after treatment with receptor specific agonist and antagonist confirmed the regulation of these genes by estrogen through its receptors. This study provides new insights about the estrogen regulated genes during spermatogenesis.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE147079 | GEO | 2020/03/17

REPOSITORIES: GEO

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Project description:The closely related transcription factors (TFs), estrogen receptors ERα and ERβ, regulate divergent gene expression programs and proliferative outcomes in breast cancer. Utilizing MCF-7 breast cancer cells with ERα, ERβ, or both receptors as a model system to define the basis of differing response specification by related TFs, we show that these TFs and their key coregulators, SRC3 and RIP140, generate overlapping as well as unique chromatin-binding and transcription-regulating modules. Cistrome and transcriptome analyses and use of clustering algorithms delineated 11 clusters representing different chromatin-bound receptor and coregulator assemblies that could be functionally associated through enrichment analysis with distinct patterns of gene regulation and preferential coregulator usage, RIP140 with ERβ and SRC3 with ERα. The receptors modified each other’s transcriptional effect, and ERβ countered the proliferative drive of ERα through several novel mechanisms associated with specific binding site clusters. Our findings delineate distinct TF-coregulator assemblies that function as control nodes specifying precise patterns of gene regulation, proliferation, and metabolism, as exemplified by two of the most important nuclear hormone receptors in human breast cancer. MCF-7 cells expressing endogenous ERalpha were infected with adenovirus carrying either estrogen receptor beta (AdERb) or no insert (Ad) at multiplicity of infection (moi) of 50. ERβ only cells were generated from these cells by knockdown of ERα in parental cells using the following siERα sequences from Dharmacon: forward, 5’-UCAUCGCAUUCCUUGCAAAdTdT-3’, and reverse, 5’-UUUGCAAGGAAUGCGAUGAdTdT-3’. siRNA experiments were performed as previously described, and resulted in knocknown of ERα mRNA and protein by greater than 95% (GSE4006, PMID 16809442). Briefly, cells were transfected with 20 nM siCtrl [GSE4006] or siERα for 48 h after infection. Then cells were treated with 0.1% EtOH (Veh) or 10 nM E2 (Sigma-Aldrich) for 24h. All experiments were conducted with two replicates. key words; Adenovirus infection,siRNA knock-down, ligand treatment

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Genome-wide identification of estrogen receptor binding sites reveals novel estrogen-responsive pathways in adult male germ cells

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