A potentially critical Hpa II site of the X chromosome-linked PGK1 gene is unmethylated prior to the onset of meiosis of human oogenic cells.

Abstract

Hpa II site H8 is in the CpG-rich 5' untranslated region of the human X chromosome-linked gene for phosphoglycerate kinase 1 (PGKI). It is the only Hpa II site in the CpG "island" whose methylation pattern is perfectly correlated with transcriptional silence of this gene. We measured DNA methylation at site H8 in fetal oogonia and oocytes and found, using a quantitative assay based on the polymerase chain reaction, that purified germ cells isolated by micromanipulation were unmethylated in 47-day to 110-day fetuses, whereas ovaries depleted of germ cells and non-ovary tissues were methylated. We conclude that site H8 is unmethylated in germ cells prior to the onset of meiosis and reactivation of the X chromosome.Female primordial germ cells of mammals resemble somatic cells in having both an active and an inactive X chromosome. Evidence for inactivity of one X chromosome of oogonia has been found both cytologically and by studies on glucose-6-phosphate dehydrogenase heterodimer formation (1, 2). After migration of the oogonia to the genital ridge at, or shortly before, the time of entry into meiosis, reactivation of the previously inactive X chromosome occurs (1-6). DNA methylation has been correlated with X chromosome inactivation in many studies (reviewed in refs. 7-9) and it is of interest to determine whether a loss of methylation is concomitant with X chromosome reactivation during oogenesis. Data on DNA methylation of oogenic cells are sparse. Several years ago evidence was obtained for undermethylation of some repeated sequences in female germ-line DNA (10, 11), and more recently, Driscoll and Migeon (12) obtained evidence for lack of methylation of several X-linked and autosomal genes in oogenic and spermatogenic cell fractions.As the latter study was done on tissue fractions rather than preparations free of somatic cells, we thought it was important to measure DNA methylation of highly purified germ cells. As such cells are isc. table only in small quantities, we used a polymerase chain raction (PCR)-based assay previously used to measure DNA methylation at a CCGG site in individual mouse embryos (13).The site we chose to assay was Hpa II site H8 in the 5' untranslated coding region of the human X-linked PGKI gene, 20 base pairs (bp) downstream of the major transcription start site (14). The 5' region of the PGKI gene is now the most highly characterized X-linked promoter region, and the relationship between methylation, transcription, and transcription factors has been studied both in normal human lymphocytes and in cultured cell lines (15)(16)(17)(18)(19). Recently, ligation-mediated genomic sequencing has been used to establish that the promoter region of the PGKI gene on the inactive X chromosome is methylated at 60 of 61 CpG sites, whereas the active promoter has no methylated sites (19). Prior to these studies, methylation-sensitive restriction enzymes had been used to investigate methylation at the 8 Hpa II sites in the PGKI 5' region in lymphocytes (15) and in cell lines before and after...

Multiple spatially distinct types of facultative heterochromatin on the human inactive X chromosome

“…These findings raise several important questions regarding X inactivation and its relationship to other examples of epigenetic silencing, both in mammals and other systems. In light of previous observations of the Barr body and its colocalization with XIST RNA, macroH2A, and various modifications of histone H3 (5,(8)(9)(10)(11)40), it is important to reconcile our findings with models that assume a uniform type of facultative heterochromatin, with particular emphasis on the redundant nature of X inactivation (27,28,30,47,48). Such models have interpreted the stability of the silenced state as a suggestion that the loss of one feature would be compensated by additional epigenetic features.…”

Association between the abnormal expression of matrix‐degrading enzymes by human osteoarthritic chondrocytes and demethylation of specific CpG sites in the promoter regions

“…In the X chromosomelinked phosphoglycerate kinase 1 gene, an Hpa II site at -21 bp was the only Hpa II site in the promoter whose methylation pattern was found to correlate fully with altered expression (43). Similarly, 5-azacytidine-induced transcriptional activation of the Epstein-Barr virus latency C promoter was found to be the result of demethylation at a single CpG site (61).…”

“…Typically, ϳ1-2 g of DNA per OA patient and ϳ2-5 g of DNA per control patient were extracted. There are 2 methods of analyzing methylation status: bisulfite modification (41) and cleavage with specific methylation-sensitive restriction enzymes (42,43). The bisulfite modification method is essential for examination of methylation in CpG islands, because information can be obtained on the methylation status of all CpG sites within the region of interest.…”

Lack of expression from a retroviral vector after transduction of murine hematopoietic stem cells is associated with methylation in vivo.

“…We have observed that this same Sma I site is completely methylated in vector-transduced embryonic stem cell lines, which also do not show expression from the MoMuLV-LTR (data not shown). Moreover, studies done by Singer-Sam et al (18) on the phosphoglycerate kinase promoter present on the inactive X chromosome have shown that methylation of a similar site, a Hpa II site (CCGG) at position +20, correlates with lack of transcription from the promoter. Nevertheless, the observed association between proviral methylation and expression inactivity does not show whether methylation plays a causal role in suppressing expression or is merely a secondary event after failure of expression has occurred.…”