DNA Methylation-mediated Tissue-specific Gene Expression Of DAZ Family Genes And Their Relevance To Human Infertility And Cancer

The highly conserved DAZ family consists of Y-linked DAZ and autosomal homologs Boule and Dazl. All three genes encode RNA-binding proteins that are nearly exclusively expressed in germ cells. Dazl has functions during PGC evelopment and migration, spermatogonia differentiation and the onset and progression of meiosis. Dazl homologs are expressed in both males and females in all species among vertebrates, and a common pattern of continuous Dazl expression from ESCs through haploid early round spermatids has been proved. We are highly interested in the question of what mechanism controls Dazl’s tissue specific expression. Epigenetic control may be involved in the regulation of the germ cell specific expression of Dazl.The establishment and maintenance of the germline requires precise orchestration of epigenetic modulation, and temporal and regional changes in the germ cell DNA methylation state serve as developmental triggers for gametogenesis. Accordingly, it can be hypothesized that the disruption of the epigenetic regulation of germ cell differentiation genes represents a risk factor or a causative agent of spermatogenic failure. A group of germline genes, called Cancer-testis genes (CT genes), or Cancer-germline genes (CG genes), have been found to be abberantly expressed in multiple human malignant tumors. Understanding how germ line genes are regulated and what type of germ lines genes are CG genes could provide insights into the mechanisms in tumorigenesis inducing aberrant activation of those germline genes in cancer.Differential DNA Methylation profile in the promoters of mouse Dazl between the testis and somatic tissueswe performed quantitative real-time PCR of RNA from various tissues of adult male mice, and found that mouse Dazl exhibit a unique tissue-specific expression pattern, being highly expressed in the testis but not detectable in somatic tissues. After prediction of CpG island in promoter of Dazl gene using " Methyl Primer Expresss V1.0" software, the methylation status of CpG islands in the promoter of Dazl gene was analyzed using bisulfite sequencing PCR(BSP) and sequence assay. The results revealed that the CpG islands in the promoter of Dazl were hyper-methylated in somatic tissues but were hypo-methylated in testis and epididymial spermatozoa. There was a negative correlation between the methylation status of CpG islands in promoter of Dazl gene and the Dazl expression. Results of this study provide a hint for the relationship of the methylation status of CpG islands in promoter of Dazl gene and its tissue specific expression.Developmental profiles of DNA Methylation in Dazl promoters during postnatal testicular developmentWe next investigate the relationship of DNA methylation and Dazl postnatal testicular expression. Dazl mRNA starts its expression early from postnatal day 3 testis and increase gradually, reach its peak at day 18 testes. Then Dazl expression decreased at day 20 testis. Similarly, we found that methylation status of Dazl promoter at day 6-8 and day 10-12 are hypo-methylated but at day 20-24 day testis, it become hypermethylated, consistent with decreased expression of Dazl in round spermatid and later stages. Such postnatal DNA methylation change in parallel to expression change of Dazl suggests that DNA methylation pattern of Dazl are also developmentally regulated and further support a role of DNA methylation in DAZ family protein gene expression control.Demethylation of somatic cells via aza treatment activates Dazl expressionTo determine whether methylation regulate Dazl tissue specific expression, we performed 5 aza-dC experiment, which could demethylate the CpGs of Dazl. Then we tested the mRNA level of Dazl by qRT-PCR, the results showed that after 5 aza-dC treatment, Dazl mRNA level rise significantly.Remarkable correlation of promoter differential methylation pattern and tissue-specific gene expression for Dazl genes in human, pig, chicken and zebrafishWe first analyze the promoter regions of Dazl, and determine the existence of CpG islands in both promoter regions. RNAs from various human tissues (testis, colon, prostate, stomach, gallbladder and spleen) are collected and assayed for Dazl expression level by quantitative real-time PCR. Dazl are highly expressed in the testis but barely or not detectable in somatic tissues confirming human Dazl are specifically highly expressed in the gonad but not in somatic tissues. DNA methylation study indicated that human Dazl CpG island exhibited a similar differential methylation pattern to that of mouse. Somatic tissues (spleen and prostate) exhibit high DNA methylation, sperm and testis DNA exhibit hypo-methylation. We found that not only pig Dazl contains a similar CpG island upstream of its exonl, but also porcine Dazl promoter exhibit remarkably similar differential methylation between testis and somatic tissues.Given Dazl is known to be present from fish to mammals, and its tissue-specific expression pattern is also conserved beyond mammals, to the fish. The results showed that Dazl is highly expressed in the testis but not detectable in all the somatic tissues assayed, confirming the gonad-specific expression of Chicken Dazl. Dazl CpG island in chicken testis is clearly hypo-methylated but hyper-methylated in liver and lung. We use zebra fish (Danio rerio) tissues to extract RNA and genomic DNA. Realtime PCR confirmed that Dazl is clearly highly expressed in the testis but not detectable in somatic tissues (muscle, skin, gill and guts). Zebra fish Dazl promoter again exhibits similar differential DNA methylation pattern between the testis and somatic tissues (muscle and gill). The presence of such mechanism in select species from fish, chicken to mammals including humans argues strongly for differential DNA methylation of Dazl promoter as an ancient mechanism involved in tissue-specific regulation.Conserved cis-regulatory elements in the promoters of Boule and Dazl in mammalsWe searched for conserved sequence elements in the promoter region of Dazl by PROMO. Since the promoter for Dazl is clearly defined in mouse, human as well as rat based on sequence alignments, we compare 3.5kb sequences immediately upstream the first exon of Dazl from human, mouse, pig and rat, and search for sequence elements with seven bases or longer length present in all four species. To our surprise, we have identified 12 7-nucleotide sequence elements conserved in the promoter region of all four species (human, mouse, pig and rat).Further analysis shows some of them are indeed transcription factors binding sites including Hoxa5, YY1, C/EBPalpha, C/EBPbeta.Abnormal DNA methylation pattern in DAZL is found in sperm from infertile menWe collected sperm from men with infertility in the reproductive medicine clinic and performed DNA methylation assay on their DAZL promoter. We first collected sperm from 10 men with normal sperm count and normal motility as our control group. We found that Dazl promoters in the control are almost uniformly free of DNA methylation, with the average methylation residues under 1%.We then investigated sperm DNA samples from 15 oligoasthenoteratozoospermia patients. We found that DAZL promoter DNA is significantly more methylated than those from control (8.34% vs 0.98%, P<0.05).DAZL genes expression are increased in some cancersSince proper genomic methylation pattern becomes profoundly altered in cancer cells, DNA hypomethylation in malignant tumor has affected a particular group of genes, called Cancer-Testis genes or Cancer-germline genes (CG genes). We performed search for DAZL expression on TGCA database between tumors and normal tissues. Although the expression of DAZL in the tumors are not high, but there is a significant increase relative to control tissues in terms of their expression level in head and neck tumor, lung carcinoma and breast cancer. Such increase in expression in tumors is evident for DAZL in breast cancer and lung carcinoma. This suggests that Dazl, despite correlation with DNA methylation, is different from typical CG genes in that the expression is relatively low in comparison to other CG genes in the same tumors.