Grant, Mark James; (1993) X-chromosome inactivation, DNA methylation and imprinting. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The X chromosome in placental mammals is subject to a unique system of developmental regulation which involves the coordinate activation and inactivation of all or most of an entire chromosome in a cis-limited fashion. Females begin life with two active X chromosomes but, in the course of their development, genes on one of these X chromosomes become transcriptionally silent and late replicating. X-inactivation results in dosage compensation whereby female (XX) somatic cells become equivalent to male (XY) cells in terms of their X-linked gene products. The inactivation event in the embryonic lineages is random with respect to the parental origin of X chromosomes such that the developing embryo and resulting adult is a mosaic of cells with one or the other X chromosome active. However, X-inactivation is non-random in female marsupials and in the extraembryonic tissues of developing female rodents; in these cases, the paternally-inherited X chromosome is preferentially inactivated. Paternal X-inactivation is one of the best known examples of imprinting, i.e., the differential expression of the genetic material dependent on its gamete of origin. It has been suggested that imprinting of the X chromosome and of autosomal regions are manifestations of the same phenomenon and that evidence from X-inactivation studies may help to elucidate factors responsible for the imprinting of autosomal genes. Although the molecular mechanisms which underly the phenomena of X-inactivation and imprinting have remained elusive, there is growing evidence that DNA modification, in the form of cytosine methylation, may be involved. There is considerable evidence that DNA methylation is associated with changes in chromatin structure and potential for gene expression and critical methylation changes in the promoter regions and other sites correlate with X-linked gene silencing on the inactive X chromosome. The main subject of this thesis is the investigation of the changes in methylation of specific CpG sequences associated with X-linked gene inactivation and an imprinted transgene at different stages of embryonic development. The approach used is PCR amplification of sequences containing informative CCGG sites, 5’ to the X-linked Pgk-1, Hprt and G6pd genes and within the CAT 17 transgene. The DNA is cut with HpaII before amplification; if the site is methylated, amplification will be resistant to HpaII digestion. Primordial germ cells, oocytes, sperm, individual preimplantation embryos, dissected regions of postimplantation embryos and embryonic stem cells have been analysed. Consistent with the activity of both X chromosomes in female embryos as determined by biochemical criteria, it has been shown that oocytes, individual preimplantation embryos and embryonic stem cells are unmethylated. Methylation of the Pgk-1 gene on the inactive X chromosome occurs at the time of X-inactivation in the blastocyst, whereas methylation of the G6pd gene occurs later, but within two days of the initiation of X-inactivation (by 5.5 days’ gestation). The methylation process may be progressive in that it occurs earlier for the Pgk-1 gene which is located close to the inactivation centre. Methylation also occurs in female extraembryonic tissues (paternal X-inactivation) although the sites are unmethylated on the inactive X chromosome in sperm. Hence, X-linked gene methylation is not part of the gamete imprinting mechanism distinguishing the paternal X chromosome. In the female germ cell lineage, which is derived from the epiblast after X-inactivation has occurred, these sites do not become methylated on the inactive X chromosome. Thus, it appears that the germ cell lineage remains separate and undifferentiated with respect to méthylation. HpaII-sensitive PCR analysis of specific CpG sites failed to elucidate the role of methylation in the phenomenon of imprinting associated with this transgene. It is still unclear whether or not the parent-of-origin-dependent differential methylation observed in some transgenes is a parallel system to the genomic imprinting of endogenous genes. These studies have advanced our knowledge of the role of methylation as a molecular mechanism regulating the differential activation and silencing of specific genes in different lineages in early development. The sensitive techniques devised will undoubtedly be informative when applied to an analysis of specific CpG sites associated with the X-inactivation centre gene, Xist, and to endogenous imprinted genes in mouse and human.

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