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Biomedical subjects

T H Bestor

Publications and source records attributed to T H Bestor.

At least 19 recordsLinked to original sources
PubMed2007

The decatenation checkpoint.

The decatenation checkpoint delays entry into mitosis until the chromosomes have been disentangled. Deficiency in or bypass of the decatenation checkpoint can cause chromosome breakage and nondisjunction during mitosis, which results in aneuploidy and chromosome rearrangements in the daughter cells. A deficiency in the decatenation checkpoint has been reported in lung and bladder cancer cell lines and may contribute to the accumulation of chromosome aberrations that commonly occur during tumour progression. A checkpoint deficiency has also been documented in cultured stem and progenitor cells, and cancer stem cells are likely to be derived from stem and progenitor cells that lack an effective decatenation checkpoint. An inefficient decatenation checkpoint is likely to be a source of the chromosome aberrations that are common features of most tumours, but an inefficient decatenation checkpoint in cancer stem cells could also provide a potential target for chemotherapy.

Cell Line, Tumor↗
PubMed2006

Sex-specific promoters regulate Dnmt3L expression in mouse germ cells.

BACKGROUND: Dnmt3L, a member of the DNA methyltransferase 3 family, lacks enzymatic activity but is required for de-novo methylation of imprinted genes in oocytes and for transposon repression in male germ cells. METHODS: We used northern blots, RT-PCR, 5' rapid amplification of complementary DNA (cDNA) ends (RACE), RNase H mapping, real-time/quantitative RT-PCR and in situ hybridization to identify and characterize Dnmt3L transcripts produced during germ cell development. RESULTS: Mouse Dnmt3L uses three sex-specific promoters, not the single promoter previously thought. A promoter active in prospermatogonia drives transcription of an mRNA encoding the full-length protein in perinatal testis, where de-novo methylation occurs. Late pachytene spermatocytes activate a second promoter in intron 9 of the Dnmt3L gene. After this stage, the predominant transcripts are three truncated mRNAs, which appear to be non-coding. We could also detect similar adult testis transcripts in humans. In the mouse ovary, an oocyte-specific promoter located in an intron of the neighbouring autoimmune regulator (Aire) gene produces a transcript with the full open reading frame (ORF). This is the only Dnmt3L transcript found in growing oocytes and is absent in the oocytes of Dnmt3L-/- females. CONCLUSIONS: Sex-specific promoters control Dnmt3L expression in the mouse germ line, mirroring the situation at the Dnmt1 and Dnmt3A loci.

Animals↗
PubMed2006

Origins of extreme sexual dimorphism in genomic imprinting.

Roughly equal numbers of imprinted genes are subject to repression from alleles of maternal and of paternal origin. This masks the strong sexual dimorphism that underlies major aspects of imprinted gene regulation. First, imprints are established very early in the male germ line and persist for the reproductive life of the organism, while maternal genomic imprints are established shortly prior to ovulation and are erased soon thereafter in the primordial germ cells of the next generation. Second, many CpG island-associated promoters are subject to maternal methylation but no known promoters are subject to paternal-specific germline methylation. The few known paternal methylation marks are kilobases distant from the affected genes and have a low CpG density. Third, Dnmt3L is required for imprint establishment but not transposon methylation in female germ cells, while Dnmt3L is required for transposon methylation and has only a minor role in de novo methylation at imprinted loci in male germ cells. Fourth, maternally expressed genes are commonly repressed on the paternal allele by paternally expressed imprinted genes produced in cis and encoding nontranslated RNAs. It is here suggested that rapid loss of highly mutable methylated CpG sites has led to the depletion of methylation target sites in paternally repressed imprinted genes, and that an imprinting mechanism based on RNAs or local inhibitory influences of ongoing transcription of regulatory loci has evolved to counter the erosion of paternally methylated regulatory regions. This mutability model is based on the fact that paternally methylated sequences are maintained in the methylated state for a much longer time than are maternally methylated sequences, and are therefore lost at a correspondingly faster rate. The difference in timing of imprint establishment is likely to underlie the increasing sexual dimorphism of other aspects of imprinted gene expression.

Animals↗
PubMed2004

Meiotic defects in a man with non-obstructive azoospermia: case report.

Infertile men have an increased frequency of aneuploid sperm. We have determined that decreased recombination is associated with the production of aneuploid sperm in humans. The aim of this study was to determine whether some cases of infertility are associated with decreased meiotic recombination. Analysis of the early stages of meiosis was performed in a 33-year-old man with non-obstructive azoospermia. Newly developed immunocytogenetic techniques were used to identify the synaptonemal complex (SC) in various stages of prophase. Antibodies to meiotic proteins identified the SC (SYN1/SCP3), the centromere (CREST) and recombination sites (MLH1). Only 36 meiotic spreads were recovered from the infertile man, compared with hundreds available from controls. One-third of the cells were in zygotene compared with 4% in controls, demonstrating an inability of bivalents to synapse and progress to pachytene. The infertile man had a greatly reduced frequency of recombination, with a mean of only 32.7 MLH1 foci/cell (range 1-60) compared with 46.0 (range 21-62) in control donors. A high proportion of cells (73%) contained at least one autosomal bivalent with zero MLH1 foci, compared with only 4.5% in control donors. Discontinuities in the SC were also more prevalent (68% of cells versus 26% in controls). This is the first demonstration of dramatic pachytene-stage abnormalities in an infertile man using these powerful new immunocytogenetic techniques.

Adult↗
PubMed2001

Dnmt3L and the establishment of maternal genomic imprints.

Complementary sets of genes are epigenetically silenced in male and female gametes in a process termed genomic imprinting. The Dnmt3L gene is expressed during gametogenesis at stages where genomic imprints are established. Targeted disruption of Dnmt3L caused azoospermia in homozygous males, and heterozygous progeny of homozygous females died before midgestation. Bisulfite genomic sequencing of DNA from oocytes and embryos showed that removal of Dnmt3L prevented methylation of sequences that are normally maternally methylated. The defect was specific to imprinted regions, and global genome methylation levels were not affected. Lack of maternal methylation imprints in heterozygous embryos derived from homozygous mutant oocytes caused biallelic expression of genes that are normally expressed only from the allele of paternal origin. The key catalytic motifs characteristic of DNA cytosine methyltransferases have been lost from Dnmt3L, and the protein is more likely to act as a regulator of imprint establishment than as a DNA methyltransferase.

Alleles↗
PubMed2001

Genomic imprinting disrupted by a maternal effect mutation in the Dnmt1 gene.

Maintenance of genomic methylation patterns in mammalian somatic cells depends on DNA methyltransferase-1 (Dnmt1). Mouse oocytes and preimplantation embryos lack Dnmt1 but express a variant of this protein called Dnmt1o. We eliminated Dnmt1o by deletion of the oocyte-specific promoter and first exon from the Dnmt1 locus. Homozygous animals were normal, but most heterozygous fetuses of homozygous females died during the last third of gestation. Although genomic methylation patterns were established normally in Dnmt1o-deficient oocytes, embryos derived from such oocytes showed a loss of allele-specific expression and methylation at certain imprinted loci. Transient nuclear localization of Dnmt1o in 8-cell embryos suggests that this variant of Dnmt1 provides maintenance methyltransferase activity specifically at imprinted loci during the fourth embryonic S phase.

Animals↗
PubMed2001

Structure of human DNMT2, an enigmatic DNA methyltransferase homolog that displays denaturant-resistant binding to DNA.

DNMT2 is a human protein that displays strong sequence similarities to DNA (cytosine-5)-methyltransferases (m(5)C MTases) of both prokaryotes and eukaryotes. DNMT2 contains all 10 sequence motifs that are conserved among m(5)C MTases, including the consensus S:-adenosyl-L-methionine-binding motifs and the active site ProCys dipeptide. DNMT2 has close homologs in plants, insects and Schizosaccharomyces pombe, but no related sequence can be found in the genomes of Saccharomyces cerevisiae or Caenorhabditis elegans. The crystal structure of a deletion mutant of DNMT2 complexed with S-adenosyl-L-homocysteine (AdoHcy) has been determined at 1.8 A resolution. The structure of the large domain that contains the sequence motifs involved in catalysis is remarkably similar to that of M.HHAI, a confirmed bacterial m(5)C MTase, and the smaller target recognition domains of DNMT2 and M.HHAI are also closely related in overall structure. The small domain of DNMT2 contains three short helices that are not present in M.HHAI. DNMT2 binds AdoHcy in the same conformation as confirmed m(5)C MTases and, while DNMT2 shares all sequence and structural features with m(5)C MTases, it has failed to demonstrate detectable transmethylase activity. We show here that homologs of DNMT2, which are present in some organisms that are not known to methylate their genomes, contain a specific target-recognizing sequence motif including an invariant CysPheThr tripeptide. DNMT2 binds DNA to form a denaturant-resistant complex in vitro. While the biological function of DNMT2 is not yet known, the strong binding to DNA suggests that DNMT2 may mark specific sequences in the genome by binding to DNA through the specific target-recognizing motif.

Adenosine Triphosphate↗
PubMed2000

The DNA methyltransferases of mammals.

The biological significance of 5-methylcytosine was in doubt for many years, but is no longer. Through targeted mutagenesis in mice it has been learnt that every protein shown by biochemical tests to be involved in the establishment, maintenance or interpretation of genomic methylation patterns is encoded by an essential gene. A human genetic disorder (ICF syndrome) has recently been shown to be caused by mutations in the DNA methyltransferase 3B (DNMT3B) gene. A second human disorder (Rett syndrome) has been found to result from mutations in the MECP2 gene, which encodes a protein that binds to methylated DNA. Global genome demethylation caused by targeted mutations in the DNA methyltransferase-1 (Dnmt1) gene has shown that cytosine methylation plays essential roles in X-inactivation, genomic imprinting and genome stabilization. The majority of genomic 5-methylcytosine is now known to enforce the transcriptional silence of the enormous burden of transposons and retroviruses that have accumulated in the mammalian genome. It has also become clear that programmed changes in methylation patterns are less important in the regulation of mammalian development than was previously believed. Although a number of outstanding questions have yet to be answered (one of these questions involves the nature of the cues that designate sites for methylation at particular stages of gametogenesis and early development), studies of DNA methyltransferases are likely to provide further insights into the biological functions of genomic methylation patterns.

Animals↗
PubMed2000

Cytosine methylation and human cancer.

Abnormalities of genomic methylation patterns have been attributed a role in carcinogenesis since the early 1980s, when large-scale demethylation of the genome was thought be an early event in multistep colorectal carcinogenesis. In the 1990s, local de novo methylation (with or without global demethylation) at tumor suppressor loci was held to be involved in silencing of tumor suppressor genes. The mechanisms that might mediate methylation and demethylation in carcinogenesis remain obscure, and there are questions as to whether the methylation changes are a cause or consequence of cellular transformation and clonal expansion. It is also important to derive a set of defined criteria by which a tumor suppressor gene can be concluded to have been inactivated by DNA methylation in a manner that contributes to carcinogenesis.

Cell Transformation, Neoplastic↗
PubMed1999

Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyltransferase gene.

The recessive autosomal disorder known as ICF syndrome (for immunodeficiency, centromere instability and facial anomalies; Mendelian Inheritance in Man number 242860) is characterized by variable reductions in serum immunoglobulin levels which cause most ICF patients to succumb to infectious diseases before adulthood. Mild facial anomalies include hypertelorism, low-set ears, epicanthal folds and macroglossia. The cytogenetic abnormalities in lymphocytes are exuberant: juxtacentromeric heterochromatin is greatly elongated and thread-like in metaphase chromosomes, which is associated with the formation of complex multiradiate chromosomes. The same juxtacentromeric regions are subject to persistent interphase self-associations and are extruded into nuclear blebs or micronuclei. Abnormalities are largely confined to tracts of classical satellites 2 and 3 at juxtacentromeric regions of chromosomes 1, 9 and 16. Classical satellite DNA is normally heavily methylated at cytosine residues, but in ICF syndrome it is almost completely unmethylated in all tissues. ICF syndrome is the only genetic disorder known to involve constitutive abnormalities of genomic methylation patterns. Here we show that five unrelated ICF patients have mutations in both alleles of the gene that encodes DNA methyltransferase 3B (refs 5, 6). Cytosine methylation is essential for the organization and stabilization of a specific type of heterochromatin, and this methylation appears to be carried out by an enzyme specialized for the purpose.

Alleles↗
PubMed1999

Cytosine methylation and mammalian development.

Programmed methylation and demethylation of regulatory sequences has been proposed to play a central role in vertebrate development. We report here that the methylation status of the 5' regions of a panel of tissue-specific genes could not be correlated with expression in tissues of fetal and newborn mice. Genes reported to be regulated by reversible methylation were not expressed ectopically or precociously in Dnmt1-deficient mouse embryos under conditions where demethylation caused biallelic expression of imprinted genes and activated transcription of endogenous retroviruses of the IAP class. These and other data suggest that the numerous published expression-methylation correlations may have described not a cause but a consequence of transcriptional activation. A model is proposed under which cytosine methylation represents a biochemical specialization of large genomes that participates in specialized biological functions such as allele-specific gene expression and the heritable transcriptional silencing of parasitic sequence elements, whereas cellular differentiation is controlled by conserved regulatory networks that do not depend on covalent modification of the genome.

Alleles↗
PubMed1999

Multipoint analysis of human chromosome 11p15/mouse distal chromosome 7: inclusion of H19/IGF2 in the minimal WT2 region, gene specificity of H19 silencing in Wilms' tumorigenesis and methylation hyper-dependence of H19 imprinting.

WT2 is defined by maternal-specific loss of heterozygosity (LOH) on chromosome 11p15.5 in Wilms' tumors (WTs). The imprinted H19 gene, in this region, is silenced and hypermethylated in most WTs, and this is linked to pathological biallelic expression of IGF2. However, H19 and IGF2 lie within a larger imprinted domain, and the gene specificity of H19 epimutation has been a persistent question. To address this, we assessed LOH, gene expression and DNA methylation at multiple sites in and around the imprinted domain. LOH mapping showed that the entire domain, including IGF2/H19, is within the minimal WT2 region. Genes within the domain, including IPL/TSSC3/BWR1C, IMPT1/ORCTL2/BWR1A/TSSC5, KvLQT1/KCNA9 and TAPA1/CD81, as well as the zinc finger gene ZNF195/ZNFP104 near the centromeric border, were expressed persistently in many WTs. DNA hypermethylation was not detected with 5" upstream probes for IPL, IMPT1, KvLQT1 and ZNF195 in WTs or WT-associated kidneys. Fully developed WTs showed variable hypomethylation at an imprinted CpG island in a KvLQT1 intron, but this was only complete in the cases with LOH and was not observed in pre-neoplastic WT-associated kidneys with H19 epimutation. Analysis of the corresponding region of mouse chromosome 7 using methyltransferase-hypomorphic mice showed that the H19 imprint was fully erased, but that the allelic bias at Ipl, Impt1, p57 Kip2 and, to a lesser extent, Kvlqt1, persisted. Pre-existing massive allelic asymmetry for DNA methylation and hyper-dependence of transcription on methylation status may underlie the mechanism of gene-specific silencing of H19 in Wilms' tumorigenesis.

Animals↗
PubMed1999

Sex brings transposons and genomes into conflict.

Given that transposons are so abundant in mammalian genomes, it is natural to assume that through their maintenance the host gains some net benefit. This need not be true; sexual reproduction allows a transposon to go to fixation if the reduction in fitness of the host is anything less than two-fold. Obligate outcrossing sexual reproduction therefore favors the evolution of aggressive transposons, which in turn select for the evolution of host mechanisms that suppress transposon activity. Hosts that have asexual or self-fertilizing generations will select for transposons that are more benign and self-limiting than those of obligate sexuals, and obligate asexuals and uniparental organelle genomes will be free of active transposons if these impose any fitness penalty. We are interested in host mechanisms that suppress transposons in sexuals and have found that mammals (all of which are obligate sexuals) control their large populations of potentially active retroposons by methylating the five position of cytosine residues within promoter elements. This causes strong transcriptional repression and assembly of the affected sequences into the condensed state. Methylation also causes permanent inactivation in the germline by driving C-->T transition mutations at methylated sites. It is now known that methylation remains in place for the large majority of the life of germ cells and is essential for control of the very large transposon burden. There is pressure on transposons to evolve mechanisms that overcome host suppression, and over evolutionary time, the balance swings back and forth between parasite and host. The ability of the mammalian genome to absorb and accumulate additional transposons has caused the amount of reverse transcriptase coding sequence in the human genome to far exceed the sum total of all cellular coding sequence. While transposons could, in principle, contribute functions useful to the host, the fact that asexual species and uniparental organelle genomes lack transposons is strong evidence that transposons have a net deleterious effect even in genomes that might be thought to require an additional source of plasticity. The abundance of transposons in many genomes cannot be taken as evidence of a mutualistic relationship, and the conflict between transposons and genomes may have actually retarded rather than accelerated evolution. It is suggested that the relationship between sex and transposons is as follows: (i) Obligate sexuals will tend to harbor aggressive transposons limited largely by host suppressive mechanisms, which in mammals involve methylation of transposon promoters. (ii) The aggressiveness of transposons in facultative sexuals and self-fertilizing sexuals will be in part self-limited and will be proportional to the relative frequency of asexual and outcrossing sexual generations. (iii) Obligate asexuals arid organelles transmitted in a uniparental manner will have no active transposons if these have a net negative effect on host fitness.

Animals↗
PubMed1998

The host defence function of genomic methylation patterns.

It has long been held that reversible promoter methylation allows genes to be expressed in the appropriate cell types during development. However, no endogenous gene has been proven to be regulated in this way, and it does not appear that significant numbers of promoters are methylated in non-expressing tissues. It has recently become clear that the large majority of genomic 5-methylcytosine is actually in parasitic sequence elements (transposons and endogenous retroviruses), and the primary function of DNA methylation now appears to be defence against the large burden of parasitic sequence elements, which constitute more than 35% of the human genome. Direct transcriptional repression provides short-term control, and the tendency of 5-methylcytosine to deaminate to thymidine drives irreversible inactivation. It is suggested that intragenomic parasites are recognized by virtue of their high copy number, and that the disturbances of methylation patterns commonly seen in human cancer cells activate a host of parasitic sequence elements, which destabilize the genome and tip the cell towards the transformed state.

Animals↗