The Role of DNA Methylation in Regulating Tissue-Specific Gene Expression During Gametogenesis.John R. McCarrey, Ph.D.

Abstract

In addition to regulating processes such as genomic imprinting and X-chromosome inactivation, abundant evidence suggests that DNA methylation also contributes to regulation of tissue-specific gene expression in both germ cells and somatic cells. In mammals, the autosomal Pgk2 gene is actively transcribed in meiotic spermatocytes and postmeiotic spermatids where it is hypomethylated in the promoter region, while it is repressed in all somatic tissues wherein the Pgk2 promoter is hypermethylated. In the spermatogenic cell lineage, a domain of demethylation develops over the Pgk2 promoter several days before activation of transcription of this gene. Changes in histone modifications, chromatin remodeling, binding of transcriptional activators and initiation of transcription from the Pgk2 promoter all occur several days after demethylation of DNA in this region. We have investigated two primary questions regarding this demethylation event and its contribution to activation of transcription from the Pgk2 promoter - 1) how is this gene- and cell type-specific demethylation event regulated? and 2) to what extent is demethylation of DNA in the promoter region of this gene a prerequisite for subsequent events leading up to and including initiation of transcription of the Pgk2 gene? Studies conducted in transgenic mice indicate that a specific region of the core promoter of the Pgk2 gene acts to signal the gene- and cell type-specific demethylation event. Thus, transgenes carrying either the Pgk2 core promoter plus the upstream enhancer region, or the core promoter region alone, ligated to a reporter coding sequence (the prokaryotic chloramphenicol acetyltransferase [CAT] gene) show gene-, and spermatogenic cell type-specific demethylation in a pattern that mimics that shown by the endogenous mouse Pgk2 gene. This demethylation event also demonstrates developmental stage-specificity during spermatogenesis, occurring during the late fetal and early postnatal periods in type T1 and type T2 prospermatogonia. However, transgenes lacking the 5' portion of the Pgk2 core promoter including the CAAT-box fail to undergo demethylation in spermatogenic cells. In addition, transgenes carrying the entire Pgk2 core promoter but with the region encompassing the CAAT-box mutated to a different sequence, also fail to undergo demethylation, indicating a gene-specific demethylation signal resides in this region. Additional studies, still in progress, indicate that when this signal sequence is disrupted such that demethylation of the Pgk2 promoter region is precluded, subsequent initiation of transcription is also precluded. Although transgenes carrying the Pgk2 core promoter undergo normal demethylation, they fail to initiate transcription unless the upstream enhancer region is also included in the transgene construct. This indicates that demethylation alone is not sufficient to activate transcription. Interestingly, transgenes carrying both the Pgk2 enhancer and core promoter regions, but with the putative demethylation signal sequence mutated to inactivity also fail to initiate transcription. This suggests that demethylation may be a necessary prerequisite for activation of transcription from this promoter. Supported by NIH grant HD46637.