Epigenetic Mechanisms Regulate Stem Cell Expressed Genes Pou5f1 and Gfra1 in a Male Germ Cell Line

Maren Godmann,Erin May,Sarah Kimmins,Axel Imhof

doi:10.1371/journal.pone.0012727

Abstract

Male fertility is declining and an underlying cause may be due to environment-epigenetic interactions in developing sperm, yet nothing is known of how the epigenome controls gene expression in sperm development. Histone methylation and acetylation are dynamically regulated in spermatogenesis and are sensitive to the environment. Our objectives were to determine how histone H3 methylation and acetylation contribute to the regulation of key genes in spermatogenesis. A germ cell line, GC-1, was exposed to either the control, or the chromatin modifying drugs tranylcypromine (T), an inhibitor of the histone H3 demethylase KDM1 (lysine specific demethylase 1), or trichostatin (TSA), an inhibitor of histone deacetylases, (HDAC). Quantitative PCR (qPCR) was used to identify genes that were sensitive to treatment. As a control for specificity the Myod1 (myogenic differentiation 1) gene was analyzed. Chromatin immunoprecipitation (ChIP) followed by qPCR was used to measure histone H3 methylation and acetylation at the promoters of target genes and the control, Myod1. Remarkably, the chromatin modifying treatment specifically induced the expression of spermatogonia expressed genes Pou5f1 and Gfra1. ChIP-qPCR revealed that induction of gene expression was associated with a gain in gene activating histone H3 methylation and acetylation in Pou5f1 and Gfra1 promoters, whereas CpG DNA methylation was not affected. Our data implicate a critical role for histone H3 methylation and acetylation in the regulation of genes expressed by spermatogonia – here, predominantly mediated by HDAC-containing protein complexes.

Highlights

The formation of spermatozoa from spermatogonial stem cells throughout adult life is dependent on a tightly orchestrated cell differentiation process governed by unique transcriptional programs and extensive chromatin remodeling [1]
The KDM1/histone deacetylase 1 (HDAC1) complex is present in cultured GC-1 In vivo co-immunoprecipitation experiments on total GC-1 protein extracts demonstrated that in this cell line as in the testis KDM1 is associated with HDAC1 (Figure 1A)
To determine whether targeting of the KDM1/HDAC1 repressor complex by T and/or TSA altered KDM1 or HDAC1 expression or protein stability, Western blot analysis was performed on total protein extracts isolated from GC-1 cells grown in regular culture medium, or culture medium supplemented with either T, TSA, DMSO, or T and TSA

Summary

Introduction

The formation of spermatozoa from spermatogonial stem cells throughout adult life is dependent on a tightly orchestrated cell differentiation process governed by unique transcriptional programs and extensive chromatin remodeling [1]. While current information indicates that male germ cell differentiation is driven by tight transcriptional regulation [2,3,4,5,6], the epigenetic modifications and chromatin remodelers underlying the control of this gene expression are unknown. The epigenetic layer includes modification of histones such as methylation, acetylation, phosphorylation among others, and DNA methylation [7]. Overall histone acetylation is associated to an open chromatin state and active gene transcription [10,11,12,13,14]. There is evidence that epigenetic mechanisms such as DNA methylation underlie the control of expression of key genes such as Pou5f1 (POU domain class 5 transcription factor 1, known as Oct4) and Nanog [16,17]

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Conclusion