Elsevier

Differentiation

Volume 77, Issue 4, April 2009, Pages 335-349
Differentiation

An epigenetic mechanism regulates germ cell-specific expression of the porcine Deleted in Azoospermia-Like (DAZL) gene

https://doi.org/10.1016/j.diff.2008.08.001Get rights and content

Abstract

The Deleted in Azoospermia-Like (DAZL) gene is specifically expressed in fetal and adult gonads. While DAZL is known to play a role during gametogenesis, the mechanisms governing its germ cell-specific expression remain unclear. We identified the 5′ untranslated region (UTR) of the porcine DAZL gene and cloned and characterized 2 kilobase pairs of its TATA-less 5′ flanking region, identifying CpG-rich regions within the proximal promoter. Nine of 18 CpG sites in proximity to one region were largely unmethylated in germ cells but hypermethylated in somatic cells, suggesting that DNA methylation may regulate DAZL promoter activity. Furthermore, DAZL expression was induced in fibroblasts treated with a demethylating agent. Deletion analyses revealed that the minimal 149 base pair promoter region was sufficient to activate transcription. In vitro methylation of a reporter construct corresponding to these 149 base pairs resulted in complete suppression of DAZL promoter activity in primordial germ cells, further supporting a role for methylation in regulating DAZL expression. Interestingly, the differentially methylated region was shown to harbor several putative Sp1-binding sites. Mutation of only the most highly conserved site significantly reduced promoter activity in a reporter assay. Furthermore, gel shift assays revealed that Sp1 was able to specifically bind to this site, and that complex formation was inhibited when CpG dinucleotides within this region were methylated. Chromatin immunoprecipitation (ChIP) assays revealed that in vivo Sp1 binding to the core DAZL promoter region was enriched in germ cells but not in fibroblasts. Our data suggests that DNA methylation may suppress DAZL expression in somatic cells by interfering with Sp1 binding. This study provides insights into the potential mechanisms underlying the regulation of germ cell-specific gene expression.

Introduction

In mammals, early embryonic development and gametogenesis require tightly regulated mechanisms to orchestrate spatial and temporal coordination of gene expression. Although its exact function remains unclear, Deleted in Azoospermia-Like (DAZL), which belongs to a set of genes encoding highly conserved RNA-binding proteins, is essential during gametogenesis. Neither its mRNA nor protein are present in the ovaries of menopausal women (Nishi et al., 1999), and the levels of its transcripts are significantly lower in men presenting with spermatogenic failure (Lin et al., 2001). Furthermore, in both male and female DAZL knock-out mice, a significant loss of late embryonic germ cells and a complete disruption in gametogenesis has been reported (Ruggiu et al., 1997; Saunders et al., 2003), demonstrating that DAZL plays an essential role during germ cell development and differentiation. DAZL is expressed throughout spermatogenesis (Lin et al., 2001; Reijo et al., 1996; Ruggiu et al., 1997), and in oogonia and oocytes of the fetal and adult ovary (Nishi et al., 1999; Brekhman et al., 2000; Tsai et al., 2000; Dorfman et al., 1999). It is not present in non-gonadal tissues (Reijo et al., 1996; Saxena et al., 1996), and has therefore been classified as a germ cell maker (Yen, 2004).

One potential mechanism by which DAZL gene expression may be restricted to germ cells is through differential DNA methylation of its regulatory region. Methylation of cytosines has been shown to result in the silencing of repetitive regions, transposable elements, and retrovirally integrated sequences within mammalian genomes (Okano et al., 1999) through formation of heterochromatin (Bird and Wolffe, 1999; Fazzari and Greally, 2004). Required for normal embryonic development during imprinting (Imamura et al., 2005; Gosden et al., 2003) and inactivation of the X chromosome, DNA methylation also serves as a means to regulate gene expression from GC-rich promoters (Jones et al., 1998), and complex changes in the pattern of DNA methylation have been associated with the onset of various cancers (Smith and Costello, 2006; Furlan et al., 2006; Staub et al., 2007). The majority of cytosine methylation occurs at CpG dinucleotides (phosphodiester-linked cytosine and guanine nucleotide pairs), which are relatively scarce in eukaryotic genomes. It has been suggested that CpGs present within the promoters of ubiquitously expressed genes remain largely unmodified, while sites within tissue-specific gene promoters are hypermethylated in all cells except those in which the transcript is normally present (Yeivin and Razin, 1993). Indeed, mammalian genomes include a growing number of non-imprinted genes with 5′ flanking regions that harbor tissue-dependent and differentially methylated regions (T-DMRs) (Ohgane et al., 1998; Imamura et al., 2001; Shiota et al., 2002; Nishino et al., 2004), and DNA methylation of T-DMRs that are located proximal to coding regions has been shown to result in transcriptional silencing (Cho et al., 2001; Imamura et al., 2001; Hattori et al., 2004; Nishino et al., 2004; Tomikawa et al., 2006). Beyond controlling the expression of housekeeping and imprinted genes, methylation also orchestrates cell differentiation and contributes to the spatio-temporally restricted expression of genes that play key roles during early embryonic development and gametogenesis (Shiota et al., 2002; Tomikawa et al., 2006). In addition to DNA methylation, histone modifications are involved in the formation of heterochromatin and, alone or in combination with methylation, also influence gene regulation (Jenuwein and Allis, 2001; Li et al., 2003). Although the 5′ end of the DAZL gene has been shown to be hypomethylated in human spermatozoa and in post-migratory murine germ cells while being hypermethylated in human leukocytes and placenta or in migrating primordial germ cells (PGCs) (Chai et al., 1997; Maatouk et al., 2006), the DAZL promoter has not been cloned or systematically characterized in any species, and the precise mechanisms governing its germ cell-specific expression are unclear.

In the present study, we cloned the porcine 5′ untranslated region (UTR) of the DAZL gene as well as its putative promoter and 5′ flanking region. We then demonstrated that differential DNA methylation affects binding of the Sp1 transcription factor, thereby providing one possible means by which the spatial expression of DAZL is regulated. An in-depth understanding of how DAZL expression is restricted to gonadal cells may provide insights into the overall mechanisms that regulate other important germ cell-specific genes during gametogenesis. Our study is particularly relevant in the context of emerging evidence demonstrating that epigenetic mechanisms, including DNA methylation, play a central role in orchestrating early germ cell differentiation and subsequent development of the germ cell lineage.

Section snippets

5′ Rapid amplification of cDNA ends (RACE)

Total RNA from denuded porcine oocytes was prepared using the RNeasy kit (Qiagen, Inc.). cDNA was synthesized according to the manufacturer's protocol using the BD SMART RACE cDNA amplification kit (BD Biosciences). 2.5 μl of cDNA was included in 50 μl PCR reactions with FastStart Taq DNA polymerase (Roche), universal primers (BD Biosciences), and gene-specific primers designed based on sequence homology within exon 2 of the human and mouse DAZL genes (GSP-1: 5′-CAAACTCAACTATCTCCAGAGAGGCCAGCA-3′;

Identification of the DAZL 5′ UTR, its proximal promoter, and the 5′ flanking sequence

To identify the porcine 5′ UTR, RACE was carried out using two different gene-specific primers (GSP-1 and GSP-2, Fig. 1A) that were designed based on sequence homology within exon 2 of the human and mouse DAZL genes. Reactions with both primers gave rise to one product that overlapped with the expected region within exon 2 (based on human and mouse exon numbering). The newly identified 126 bp 5′ UTR (in italics with the putative transcriptional start site indicated by the position numbering,

Discussion

While it has been shown that DAZL is expressed in both male and female germ cells throughout gametogenesis, the mechanisms regulating its restricted expression profile have not been fully explored. The current investigation is the first to systematically characterize the DAZL promoter in any species, supporting the finding of Maatouk et al. (2006) that germ cell-specific expression of this gene may be epigenetically mediated. More specifically, we demonstrated that changes in the methylation

Acknowledgement

The authors would like to thank Jinghe Liu for kindly providing the images used for immunocytochemistry in Fig. 2B.

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