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Timothy H Bestor

Publications and source records attributed to Timothy H Bestor.

At least 19 recordsLinked to original sources
PubMed2006

Computational prediction of methylation status in human genomic sequences.

Epigenetic effects in mammals depend largely on heritable genomic methylation patterns. We describe a computational pattern recognition method that is used to predict the methylation landscape of human brain DNA. This method can be applied both to CpG islands and to non-CpG island regions. It computes the methylation propensity for an 800-bp region centered on a CpG dinucleotide based on specific sequence features within the region. We tested several classifiers for classification performance, including K means clustering, linear discriminant analysis, logistic regression, and support vector machine. The best performing classifier used the support vector machine approach. Our program (called hdfinder) presently has a prediction accuracy of 86%, as validated with CpG regions for which methylation status has been experimentally determined. Using hdfinder, we have depicted the entire genomic methylation patterns for all 22 human autosomes.

Algorithms↗
PubMed2006

Methylation of tRNAAsp by the DNA methyltransferase homolog Dnmt2.

The sequence and the structure of DNA methyltransferase-2 (Dnmt2) bear close affinities to authentic DNA cytosine methyltransferases. A combined genetic and biochemical approach revealed that human DNMT2 did not methylate DNA but instead methylated a small RNA; mass spectrometry showed that this RNA is aspartic acid transfer RNA (tRNA(Asp)) and that DNMT2 specifically methylated cytosine 38 in the anticodon loop. The function of DNMT2 is highly conserved, and human DNMT2 protein restored methylation in vitro to tRNA(Asp) from Dnmt2-deficient strains of mouse, Arabidopsis thaliana, and Drosophila melanogaster in a manner that was dependent on preexisting patterns of modified nucleosides. Indirect sequence recognition is also a feature of eukaryotic DNA methyltransferases, which may have arisen from a Dnmt2-like RNA methyltransferase.

Animals↗
PubMed2005

Large-scale structure of genomic methylation patterns.

The mammalian genome depends on patterns of methylated cytosines for normal function, but the relationship between genomic methylation patterns and the underlying sequence is unclear. We have characterized the methylation landscape of the human genome by global analysis of patterns of CpG depletion and by direct sequencing of 3073 unmethylated domains and 2565 methylated domains from human brain DNA. The genome was found to consist of short (<4 kb) unmethylated domains embedded in a matrix of long methylated domains. Unmethylated domains were enriched in promoters, CpG islands, and first exons, while methylated domains comprised interspersed and tandem-repeated sequences, exons other than first exons, and non-annotated single-copy sequences that are depleted in the CpG dinucleotide. The enrichment of regulatory sequences in the relatively small unmethylated compartment suggests that cytosine methylation constrains the effective size of the genome through the selective exposure of regulatory sequences. This buffers regulatory networks against changes in total genome size and provides an explanation for the C value paradox, which concerns the wide variations in genome size that scale independently of gene number. This suggestion is compatible with the finding that cytosine methylation is universal among large-genome eukaryotes, while many eukaryotes with genome sizes <5 x 10(8) bp do not methylate their DNA.

Animals↗
PubMed2005

Transposons reanimated in mice.

Transposons have proved valuable in the genetic modification of many organisms but not mammals. New work reported by Ding et al. (2005) in this issue of Cell and by Collier et al. (2005) and Dupuy et al. (2005) in a recent issue of Nature now reveals that insertional mutagenesis in mammalian cells is possible thanks to modified derivatives of the piggyBac and Sleeping Beauty DNA transposons. However, the slow rate of proliferation of DNA transposons suggests that derivatives of the L1 retrotransposon might be more powerful for mutagenesis of germline cells. In either case, the future of transposon-driven genetic analyses of mice and other mammals looks promising.

Animals↗
PubMed2005

Identification of the control region for tissue-specific imprinting of the stimulatory G protein alpha-subunit.

Gnas is a complex gene with multiple imprinted promoters. The upstream Nesp and Nespas/Gnasxl promoters are paternally and maternally methylated, respectively. The downstream promoter for the stimulatory G protein alpha-subunit (G(s)alpha) is unmethylated, although in some tissues (e.g., renal proximal tubules), G(s)alpha is poorly expressed from the paternal allele. Just upstream of the G(s)alpha promoter is a primary imprint mark (1A region) where maternal-specific methylation is established during oogenesis. Pseudohypoparathyroidism type 1B, a disorder of renal parathyroid hormone resistance, is associated with loss of 1A methylation. Analysis of embryos of Dnmt3L(-/-) mothers (which cannot methylate maternal imprint marks) showed that Nesp, Nespas/Gnasxl, and 1A imprinting depend on one or more maternal primary imprint marks. We generated mice with deletion of the 1A differentially methylated region. These mice had normal Nesp-Nespas/Gnasxl imprinting, indicating that the Gnas locus contains two independent imprinting domains (Nespas-Nespas/Gnasxl and 1A-G(s)alpha) controlled by distinct maternal primary imprint marks. Paternal, but not maternal, 1A deletion resulted in G(s)alpha overexpression in proximal tubules and evidence for increased parathyroid hormone sensitivity but had no effect on G(s)alpha expression in other tissues where G(s)alpha is normally not imprinted. The 1A region is a maternal imprint mark that contains one or more methylation-sensitive cis-acting elements that suppress G(s)alpha expression from the paternal allele in a tissue-specific manner.

Animals↗
PubMed2005

Decatenation checkpoint deficiency in stem and progenitor cells.

The decatenation checkpoint normally delays entry into mitosis until chromosomes have been disentangled through the action of topoisomerase II. We have found that the decatenation checkpoint is highly inefficient in mouse embryonic stem cells, mouse neural progenitor cells, and human CD34+ hematopoietic progenitor cells. Checkpoint efficiency increased when embryonic stem cells were induced to differentiate, which suggests that the deficiency is a feature of the undifferentiated state. Embryonic stem cells completed cell division in the presence of entangled chromosomes, which resulted in severe aneuploidy in the daughter cells. The decatenation checkpoint deficiency is likely to increase the rates of chromosome aberrations in progenitor cells, stem cells, and cancer stem cells.

Animals↗
PubMed2005

Eukaryotic cytosine methyltransferases.

Large-genome eukaryotes use heritable cytosine methylation to silence promoters, especially those associated with transposons and imprinted genes. Cytosine methylation does not reinforce or replace ancestral gene regulation pathways but instead endows methylated genomes with the ability to repress specific promoters in a manner that is buffered against changes in the internal and external environment. Recent studies have shown that the targeting of de novo methylation depends on multiple inputs; these include the interaction of repeated sequences, local states of histone lysine methylation, small RNAs and components of the RNAi pathway, and divergent and catalytically inert cytosine methyltransferase homologues that have acquired regulatory roles. There are multiple families of DNA (cytosine-5) methyltransferases in eukaryotes, and each family appears to be controlled by different regulatory inputs. Sequence-specific DNA-binding proteins, which regulate most aspects of gene expression, do not appear to be involved in the establishment or maintenance of genomic methylation patterns.

Animals↗
PubMed2004

Meiotic catastrophe and retrotransposon reactivation in male germ cells lacking Dnmt3L.

Mammalian genomes employ heritable cytosine methylation in the long-term silencing of retrotransposons and genes subject to genomic imprinting and X chromosome inactivation. Little is known of the mechanisms that direct cytosine methylation to specific sequences. Here we show that DNA methyltransferase 3-like (Dnmt3L (ref. 1)) is expressed in testes during a brief perinatal period in the non-dividing precursors of spermatogonial stem cells at a stage where retrotransposons undergo de novo methylation. Deletion of the Dnmt3L gene prevented the de novo methylation of both long-terminal-repeat (LTR) and non-LTR retrotransposons, which were transcribed at high levels in spermatogonia and spermatocytes. Loss of Dnmt3L from early germ cells also caused meiotic failure in spermatocytes, which do not express Dnmt3L. Whereas dispersed repeated sequences were demethylated in mutant germ cells, tandem repeats in pericentric regions were methylated normally. This result indicates that the Dnmt3L protein might have a function in the de novo methylation of dispersed repeated sequences in a premeiotic genome scanning process that occurs in male germ cells at about the time of birth.

Animals↗
PubMed2004

Windows for sex-specific methylation marked by DNA methyltransferase expression profiles in mouse germ cells.

The acquisition of genomic methylation in the male germ line is initiated prenatally in diploid gonocytes, while DNA methylation in the female germ line is initiated postnatally in growing oocytes. We compared the temporal expression patterns of the DNA methyltransferases, DNMT1, DNMT3a, DNMT3b, and DNMT3l in the male and female germ lines. DNMT1 expression was examined by immunocytochemistry and Northerns with an emphasis on the prenatal period. In the female, there is a gradual down-regulation of DNMT1 protein in prenatal meiotic prophase I oocytes that is not associated with the production of an untranslated transcript, as it is in the male; these results suggest that the mechanism of meiotic down-regulation differs between the sexes. In the male, DNMT1 is unlikely to play a role in the prenatal acquisition of germ line methylation patterns since it is down-regulated in gonocytes between 14.5 and 18.5 days of gestation and is absent at the time of initiation of DNA methylation. To search for candidate DNMTs that could be involved in establishing methylation patterns in both germ lines, real-time RT-PCR was used to simultaneously study the expression profiles of the three DNMT3 enzymes in developing testes and ovaries; DNMT1 expression was included as a control. Expression profiles of DNMT3a and DNMT3l provide support for an interaction of the two enzymes during prenatal germ cell development and de novo methylation in the male. DNMT3l is the predominant DNMT3 enzyme expressed at high levels in the postnatal female germ line at the time of acquisition of DNA methylation patterns. DNMT1 and DNMT3b expression levels peak concomitantly, shortly after birth in the male, consistent with a role in the maintenance of methylation patterns in proliferating spermatogonia. Together, the results provide clues to specific roles for the different DNMT family members in de novo and maintenance methylation in the developing testis and ovary.

Animals↗
PubMed2003

Imprinting errors and developmental asymmetry.

There are, in the broadest sense, two mechanisms by which gene expression can be extinguished in vertebrates. The first of these is based on mass action effects of positive and negative regulatory factors and is termed activation and repression; the second is independent of positive regulatory factors but is based on the history of the affected gene and is termed silencing. It can be said, again in the broadest sense, that imprinted genes, genes subject to X inactivation, and transposon promoters are subject to silencing, while the promoters of tissue-specific genes in non-expressing tissues are controlled by activation and repression. The escape of imprinted genes from silencing through unknown mechanisms can cause developmental abnormalities and can predispose to the formation of embryonal tumours. One developmental disorder caused by loss of imprinting of genes on chromosome 11p15.5 is Beckwith-Wiedemann syndrome (BWS). This syndrome has long been known to be inexplicably common in monozygotic twins; the twins are nearly always discordant for BWS, and nearly all twins are female. A loss of imprinting model based on stochastic errors in the nucleocytoplasmic trafficking of the DNA methyltransferase DNMT1, or a paternally expressed function that opposes maintenance methylation of maternally repressed growth-enhancing genes, is proposed to explain the perplexing genetics of BWS in monozygotic twins.

Beckwith-Wiedemann Syndrome↗
PubMed2003

Cytosine methylation mediates sexual conflict.

Transposons are infectious agents in sexual populations and can go to fixation even if they reduce the fitness of the host. Sexual hosts are therefore under selective pressure to evolve defensive functions that relieve the fitness penalty imposed by transposons. In mammals, this defensive function depends on transcriptional silencing through targeted cytosine methylation of transposon promoters in both germ-line and somatic tissues (with the probable involvement of some post-transcriptional gene silencing by pathways related to RNA interference). New families of transposons evolve in response to selective pressures imposed by host silencing mechanisms, with the result that the mammalian genome has suffered waves of transposon bombardment that have contributed the majority of the total sequence. Cytosine methylation defends the sexual genome against transposon damage even as it enforces a requirement for sexual reproduction through the phenomenon of genomic imprinting.

Animals↗
PubMed2003

Unanswered questions about the role of promoter methylation in carcinogenesis.

It has been proposed that tumor suppressor genes can be silenced by ectopic de novo methylation during tumor progression and that this epigenetic silencing is an alternative to mutation in tumor suppressor inactivation during oncogenic transformation. However, methylation may follow inactivation and may not directly participate in tumor progression. There are no genetic data that implicate ectopic de novo methylation in cancer, and no DNA methyltransferase gene has been shown to be mutated in any cancer. Promoter methylation at tumor suppressor loci may be a consequence of transcriptional inactivity imposed by mutations in upstream components of the transcriptional machinery or signal transduction pathways. Current estimates of the importance of epigenetic changes in the etiology of cancer may be inflated, and consequences may have been mistaken for causes in some cases.

Cell Transformation, Neoplastic↗
PubMed2002

Reactivation of a silenced H19 gene in human rhabdomyosarcoma by demethylation of DNA but not by histone hyperacetylation.

BACKGROUND: The active copy of the imprinted gene H19 is turned off by inappropriate methylation in several pediatric tumors including Wilms' Tumour and embryonal rhabdomyosarcoma. H19 controls in cis the linked Insulin-like Growth Factor 2 (IGF2) gene, encoding an important growth factor. Recent work has suggested that methylation of a gene may lead to deacetylation of its associated histones and that silenced genes can be reactivated by increasing histone acetylation levels. RESULTS: Treatment of a rhabdomyosarcoma cell line which has a silent, methylated H19 gene with histone deacetylase (HDAC) inhibitors under conditions which gave maximal hyperacetylation of histone 4, both globally and at the H19 gene itself could not reactivate H19 or affect the active Insulin-like Growth Factor 2 (IGF2) gene, but caused clear up-regulation of the Tissue-type Plasminogen Activator (TPA) gene, a non-imprinted gene known to respond to changes in histone acetylation. In contrast, mild treatment of the cells with the methylation inhibitor 5-AzaC-2'-deoxycytidine (AzaC) on its own was able to reactivate H19. Combining AzaC treatment with HDAC inhibitors gave a reduced rather than enhanced reactivation. These findings were confirmed in mouse primary liver and kidney explants which maintain normal imprinting, where we also found that the silent Igf2 gene could not be reactivated by HDAC inhibitors. CONCLUSION: These results suggest that DNA methylation rather than histone acetylation is the primary determinant of silencing of H19 in rhabdomyosarcoma.

Acetylation↗
PubMed2002

Dynamics of Dnmt1 methyltransferase expression and intracellular localization during oogenesis and preimplantation development.

The imprinting of mammalian genes depends on the maintenance of DNA methylation patterns during pre- and postimplantation development. Dnmt1o is a variant form of the somatically expressed Dnmt1 cytosine methyltransferase that is synthesized and stored in the oocyte cytoplasm and trafficks to the eight-cell nucleus during preimplantation development, where it maintains DNA methylation patterns on alleles of imprinted genes. Transcripts encoding Dnmt1 are present in preimplantation embryos, suggesting that Dnmt1 protein is also expressed in the preimplantation embryo, and may account for maintenance methylation at preimplantation stages other than the eight-cell embryo. However, using an antibody that detects Dnmt1, but not Dnmt1o, no Dnmt1 protein was detected on immunoblots or by immunocytochemical staining in wildtype preimplantation embryos. Moreover, Dnmt1 protein produced in the oocyte from a modified Dnmt1 allele, Dnmt1(1s/1o), trafficked to nuclei of eight-cell embryos, but not to nuclei of other stages. The highly restricted nuclear localization patterns of oocyte-derived Dnmt1o and Dnmt1 during preimplantation development add further support to the notion that DNA methyltransferases other than Dnmt1 are required for maintaining imprints during preimplantation development.

Alternative Splicing↗