Insights into X chromosome inactivation from studies of species variation, DNA methylation and replication, and vice versa

I am indebted to Mary Lyon as her X-inactivation hypothesis stimulated my mentor, Barton Childs, and in turn, myself, to think about the consequences of X-inactivation in heterozygous females. I often reread her original papers setting forth the single active X hypothesis, and still marvel at the concise and compelling exposition of the hypothesis and the logical predictions which seemed prophetic at my first reading, and have survived the test of time. My contribution to this Festschrift reviews evidence derived from studies of DNA methylation, species variation and DNA replication that reveals an important role for methylated CpG islands and suggests a role for late DNA replication in propagating X inactivation from one cell to its progeny. These studies also show that X inactivation is a powerful research tool for identifying the factors which program and maintain developmental processes.     

The relationship between DNA replication and chromosome structure

Summary The results obtained by acridine orange staining of chromosomes, after BrdU treatment, during one or two cell cycles, are described. The alterations of chromosome structure do not depend only on BrdU incorporation into DNA. Some other mechanisms are necessarily involved, and it is postulated that they are disturbances of protein-DNA association, occurring in G₁ and in S- or G₂-phase. The aspect of metaphase chromosomes then appears as the result of several metabolic steps, all occurring during interphase.Presented at the Fifth Meeting of the Cytogenetics Section of the Society for Anthropology and Human Genetics, Basel, Switzerland, June 17–19, 1976.     

The relationship between DNA methylation and chromosome imprinting in the coccid Planococcus citri

The phenomenon of chromosome, or genomic, imprinting indicates the relevance of parental origin in determining functional differences between alleles, homologous chromosomes, or haploid sets. In mealybug males (Homoptera, Coccoidea), the haploid set of paternal origin undergoes heterochromatization at midcleavage and remains so in most of the tissues. This different behavior of the two haploid sets, which depends on their parental origin, represents one of the most striking examples of chromosome imprinting. In mammals, DNA methylation has been postulated as a possible molecular mechanism to differentially imprint DNA sequences during spermatogenesis or oogenesis. In the present article we addressed the role of DNA methylation in the imprinting of whole haploid sets as it occurs in Coccids. We investigated the DNA methylation patterns at both the molecular and chromosomal level in the mealybug Planococcus citri. We found that in both males and females the paternally derived haploid set is hypomethylated with respect to the maternally derived one. Therefore, in males, it is the paternally derived hypomethylated haploid set that is heterochromatized. Our data suggest that the two haploid sets are imprinted by parent-of-origin-specific DNA methylation with no correlation with the known gene-silencing properties of this base modification.     

线粒体DNA与DNA甲基化

线粒体是除细胞核之外唯一携带遗传物质的细胞器,其线粒体DNA（mitochondrial DNA,mtDNA）控制着线粒体一些最基本的性质,对细胞功能有着重要影响.DNA甲基化是调节基因表达的重要方式之一.研究表明mtDNA存在CpG位点的低甲基化,并且mtDNA基因的表达受核DNA（nuclear DNA,nDNA）及线粒体自身DNA甲基化的调控,mtDNA和nDNA协同作用参与机体代谢调节和疾病发生发展过程.就近年来mtDNA与DNA甲基化的关系作一综述.     

线粒体DNA与DNA甲基化的关系

线粒体DNA（mitochondrial DNA,mtDNA）遗传信息量虽小,却控制着线粒体一些最基本的性质,对细胞及其功能有着重要影响。mtDNA的损伤与衰老、肿瘤等疾病的发生有关。DNA甲基化是调节基因表达的重要方式之一。mtDNA基因的表达受核DNA（nuclear DNA,nDNA）的调控,mtDNA和nDNA协同作用参与机体代谢调节和发病。本文就近年来mtDNA与DNA甲基化的关系作一综述。     

Evidence for a large double-cruciform DNA structure on the X chromosome of human and chimpanzee

The human X chromosome consists of a high number of large inverted repeat (IR) DNA sequences which fulfill all requirements for formation of cruciform DNA structures. Such alternative DNA structures are suggested to have a great impact in altering the chromatin architecture and function. Our comprehensive analysis of the corresponding orthologous nucleotide sequences of an IR sequence from Homo sapiens and Pan troglodytes revealed that most of the nucleotide differences between the two species are symmetrical to the apex of the IR, and that the spacer region of the orthologous IRs are in reverse orientation. We provide evidence that this IR forms a large non-B DNA structure containing two Holliday junctions, allowing intrastrand nucleotide pairing of the arms and interstrand pairing of the spacer region of the IR. This structure would extrude into a large double-cruciform DNA structure providing the molecular basis of translocation events and regulation of gene expression. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

DNA methylation analysis from saliva samples for epidemiological studies

Saliva is a non-invasive, easily accessible tissue, which is regularly collected in large epidemiological studies to examine genetic questions. Recently, it is becoming more common to use saliva to assess DNA methylation. However, DNA extracted from saliva is a mixture of both bacterial and human DNA derived from epithelial and immune cells in the mouth. Thus, there are unique challenges to using salivary DNA in methylation studies that can influence data quality. This study assesses: (1) quantification of human DNA after extraction; (2) delineation of human and bacterial DNA; (3) bisulfite conversion (BSC); (4) quantification of BSC DNA; (5) PCR amplification of BSC DNA from saliva and; (6) quantitation of DNA methylation with a targeted assay. The framework proposed will allow saliva samples to be more widely used in targeted epigenetic studies.     

Mammalian embryonic development--insights from studies on the X chromosome

This paper reviews our early studies on the cycle of changes of X chromosome activity in different lineages of the developing female mouse embryo. The emphasis is placed on the insights gained into key developmental processes such as the temporal and spatial aspects of developmental totipotency, the timing and cell numbers involved in the origin of the germ line, the molecular mechanisms of genetic deprogramming, reprogramming and X chromosome imprinting, and transgenerational epigenetic inheritance. When viewed in this way, it is quite remarkable to see how much was learned about mammalian development from early studies on the X chromosome. Indeed several paradigm shifts occurred as a result of these studies and these are highlighted in this review.     

The somatic replication of DNA methylation

We have tested the hypothesis that DNA methylation patterns are replicated in the somatic cells of vertebrates. Using M-Hpa II, the modification enzyme from Haemophilus parainfluenzae which methylates the internal cytosine residues in the sequence 5'CCGG 3' GGCC, we methylated bacteriophage phi X174 RF DNA and the cloned chicken thymidine kinase (tk) gene in vitro and then introduced these DNAs and unmethylated controls into tk- cultured mouse cells by DNA-mediated transformation. Twenty-five cell generations later, the state of methylation of transferred DNA was examined by restriction endonuclease analysis and blot hybridization. We conclude that methylation at Hpa II sites is replicated by these cultured cells but not with 100% fidelity. We have also noted that methylation of the cloned chicken tk gene decreases its apparent transformation efficiency relative to unmethylated molecules.     

X chromosome inactivation,differentiation, and DNA methylation revisited,with a tribute to Susumu Ohno

X chromosome inactivation and DNA methylation are reviewed, with emphasis on the contributions of Susumu Ohno and the predictions made in my 1975 paper (Riggs, 1975), in which I proposed the "maintenance methylase" model for somatic inheritance of methylation patterns and suggested that DNA methylation would be involved in mammalian X chromosome inactivation and development. The maintenance methylase model is discussed and updated to consider methylation patterns in cell populations that have occasional, stochastic methylation changes by de novo methylation or demethylation, either active or passive. The "way station" model for the spread of X inactivation by LINE-1 elements is also considered, and some recent results from my laboratory are briefly reviewed.     

Evidence for a structural relationship between BRCT domains and the helicase domains of the replication initiators encoded by the Polyomaviridae and Papillomaviridae families of DNA tumor viruses

Studies of DNA tumor viruses have provided important insights into fundamental cellular processes and oncogenic transformation. They have revealed, for example, that upon expression of virally encoded proteins, cellular pathways involved in DNA repair and cell cycle control are disrupted. Herein, evidence is presented that BRCT-related regions are present in the helicase domains of the viral initiators encoded by the Polyomaviridae and Papillomaviridae viral families. Of interest, BRCT domains in cellular proteins recruit factors involved in diverse pathways, including DNA repair and the regulation of cell cycle progression. Therefore, the viral BRCT-related regions may compete with host BRCT domains for particular cellular ligands, a process that would help to explain the pleiotropic effects associated with infections with many DNA tumor viruses.     

Gene mapping studies confirm the homology between the platypus X and echidna X1 chromosomes and identify a conserved ancestral monotreme X chromosome

The identification of the sex chromosomes in the three extant species of Prototherian mammals (the monotremes) is complicated by their involvement in a multivalent translocation chain at the first division of male meiosis. The platypus X chromosome, identified by the presence of two copies in females and one in males, has been found to possess a suite of genes that have been mapped to the X chromosomes of all eutherian and metatherian mammals. We have extended gene mapping studies to a member of the only other extant monotreme family, the echidna, which has a G-band equivalent X₁ chromosome, as well as a smaller X₂. We find that the five human X-linked genes (G6PD, GDX, F9, AR and MCF2) map to the echidna X₁ chromosome in locations equivalent to those on the platypus X. These results confirm that the echidna X₁ is the original X chromosome in this species, and identify a conserved ancestral monotreme X chromosome.     

5-Methylcytosine as a marker for the monitoring of DNA methylation

The extent of the DNA methylation of genomic DNA as well as the methylation pattern of many gene-regulatory areas are important aspects with regard to the state of genetic information, especially their expression. There is growing evidence that aberrant methylation is associated with many serious pathological consequences. As genetic research advances, many different approaches have been employed to determine the overall level of DNA methylation in a genome or to reveal the methylation state of particular nucleotide residues, starting from semiquantitative methods up to new and powerful techniques. In this paper, the currently employed techniques are reviewed both from the point of view of their relevance in genomic research and of their analytical application. The methods discussed include approaches based on chromatographic separation (thin-layer chromatography, high-performance liquid chromatography, affinity chromatography), separation in an electric field (capillary electrophoresis, gel electrophoresis in combination with methylation-sensitive restriction enzymes and/or specific sequencing protocols), and some other methodological procedures (mass spectrometry, methyl accepting capacity assay and immunoassays).     

The relationship between DNA methylation and telomere length in dyskeratosis congenita

The regulation of telomere length (TL) is a complex process, requiring the telomerase enzyme complex and numerous regulatory proteins. Epigenetic regulation may also be important in telomere maintenance. Specifically, methylation at subtelomeres is associated with changes in TL in vitro and in mouse models. Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome characterized by exceedingly short telomeres and mutations in telomere biology genes. To understand the interaction between methylation and TL in humans, we measured LINE-1, pericentromeric (NBL2), and subtelomeric (D4Z4) methylation in peripheral blood DNA derived from 40 patients with DC and 51 mutation-negative relatives. Pearson's correlation coefficient and linear regression models were used to evaluate the relationship between age-standardized lymphocyte TL measured by flow FISH and % DNA methylation. No differences in % subtelomeric, LINE-1, or pericentromeric methylation between patients with DC and relatives were noted except for an increase in % subtelomeric methylation in DC patients with a telomerase-complex mutation (TERC, TERT, DKC1, or TCAB1) (63.0% in DC vs. 61.8% in relatives, P = 0.03). Positive correlations between TL and DNA methylation at LINE-1 (r = 0.39, P = 0.01) and subtelomeric (r = 0.32, P = 0.05) sites were present in patients with DC. The positive correlation between TL and % LINE-1 methylation was restricted to TINF2 mutations. In contrast, statistically nonsignificant inverse correlations between TL and % LINE-1 (r = -0.17), subtelomeric (r = -0.20) were present in unaffected relatives. This study suggests an interaction between TL and both subtelomeric and LINE-1 methylation, which may be altered based on mutation status of telomere biology genes.