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Results for “epigenetic modification”

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PubMed2005

Sequence-selective 5-methylcytosine oxidation for epigenotyping.

Methylation of DNA is an epigenetic modification that can play an important role in the control of gene expression in mammalian cells. The development of a simple and convenient method for site-specific discrimination of cytosine methylation is imperative for genomic studies. Here we report a facile method for distinguishing between cytosine and 5-methylcytosine. Osmium tetroxide caused the dihydroxylation of the C5-C6 double bond of 5-methylcytosine under an appropriate reaction conditions. The oxidation of 5-methylcytosine-containing target DNA was controlled by hybridization with a guide DNA. This technique facilitates the typing of cytosine methylation at a specific site of the target DNA.

5-Methylcytosine↗
PubMedDate not supplied

Interspecies approaches for the analysis of parental imprinting during mouse development.

Genomic imprinting results in the functional specialization of the maternal and paternal genomes during development whereby offspring inherit only one active copy of a gene. Although the reason for its evolutionary genesis remains speculative, the consequences of genomic imprinting are evidenced by the failure of parthenogenetic and androgenetic development in mammals, and parental-specific effects in the etiology of a number of human diseases. While the precise molecular mechanism of imprinting is unknown, evidence suggests that the specialization of the parental genomes is established during gametogenesis when the parental genomes are epigenetically modified to reflect the parent of origin. To examine the epigenetic modification of specific imprinted genes and subsequent differential expression, an assay is required that can distinguish between the maternal and paternal alleles and their respective transcripts. During the past 5 years several strategies have been used to identify imprinted genes, ranging from the fortuitous disruption of specific parental alleles to subtraction hybridization between cDNAs from normal and parthenogenetic embryos. To study the developmental regulation of these imprinted genes during mammalian development, we describe a relatively simple interspecies "mRNA phenotyping" approach applicable to the analysis of allele-specific expression as well as the identification of candidate imprinted genes.

Animals↗
PubMed2002

Genomic DNA methylation-demethylation during aging and reinvigoration of Pinus radiata.

In animals, DNA methylation is related to gene silencing during ontogenic development. Little is known about DNA methylation in plants, although occasional changes in the DNA methylation state of specific gene promoters have been reported in angiosperms during some developmental processes. We found large differences in the extent of DNA methylation between meristematic areas of juvenile and mature Pinus radiata D. Don. trees, whereas differences in the extent of DNA methylation between differentiated tissues of juvenile and mature trees were small. In meristematic areas, there was a gradual decrease in extent of DNA methylation as the degree of reinvigoration increased. The observed changes in extent of DNA methylation during aging and reinvigoration indicate that reinvigoration could be a consequence of epigenetic modifications opposite in direction to those that occur during aging.

DNA Methylation↗
PubMed2003

Nuclear-cytoplasmic interactions affect in utero developmental capacity, phenotype, and cellular metabolism of bovine nuclear transfer fetuses.

We generated a clone of bovine somatic cell nuclear transfer embryos using oocyte pools from defined maternal sources to study nuclear-cytoplasmic interactions. Nucleocytoplasmic hybrids were reconstructed with Bos taurus (Brown Swiss) granulosa cells and oocytes that contained B. taurus A (Simmental), B. taurus B (Simmental), or Bos indicus (Dwarf Zebu) cytoplasm. Another set of embryos was reconstructed with randomly selected Brown Swiss (B. taurus R) oocytes. Embryo transfer resulted in nine (12.5%), nine (13.8%), three (50%), and 11 (16.7%) Day 80 fetuses, of which eight (11.1%), three (4.6%), three (50%), and 10 (15.2%) were viable, respectively. The proportion of viable fetuses was affected by cytoplasm (likelihood ratio test, P < 0.02) and was higher for embryos with B. indicus cytoplasm than for the B. taurus A (P < 0.05) and B (P < 0.01) groups. Furthermore, the proportion of surviving Day 80 fetuses was reduced for B. taurus B as compared with B. taurus A and B. taurus R cytoplasm (P < 0.05 and P < 0.02). Body weight of nucleocytoplasmic hybrid fetuses was not significantly different from Brown Swiss control fetuses produced by artificial insemination (AI), but fetuses reconstructed with random cytoplasts of the same breed as the nuclear donor exhibited overgrowth (P < 0.01) and a higher coefficient of variation in weight. Furthermore, body weight, crown rump length, thorax circumference (P < 0.05), and femur length (P < 0.01) of fetuses with B. taurus A cytoplasm differed from fetuses with B. taurus R cytoplasms. Fetal skin, heart, and liver cells with B. indicus cytoplasm showed a greater increase in number per time period (P < 0.001) and oxygen consumption rate per cell (skin and liver, P < 0.001; heart, P < 0.08) in comparison with their counterparts with B. taurus A cytoplasm. These data point to complex oocyte cytoplasm-dependent epigenetic modifications and/or nuclear DNA-mitochondrial DNA interactions with relevance to nuclear transfer and other reproductive technologies such as ooplasmic transfer in human assisted reproduction.

Animals↗
PubMed2004

Epigenetic and genomic imprinting analysis in nuclear transfer derived Bos gaurus/Bos taurus hybrid fetuses.

Somatic cell nuclear transfer (NT) in cattle is an inefficient process, whereby the production of calves is hindered by low pregnancy rates as well as fetal and placental abnormalities. Interspecies models have been previously used to facilitate the identification of single nucleotide polymorphisms (SNPs) within coding regions of genes to discriminate between parental alleles in the offspring. Here we report the use of a bovine interspecies model (Bos gaurus x Bos taurus) for the assessment and characterization of epigenetic modifications and genomic imprinting in Day 40-old female NT-derived fetuses and placenta. Analysis of NT and control pregnancies indicated disruption of genomic imprinting at the X inactivation-specific transcript (XIST) locus in the chorion, but not the fetus of clones, whereas proper allelic expression of the insulin-like growth factor II (IGF2) and gene trap locus 2 (GTL2) loci was maintained in both the fetus and placenta. Analysis of the XIST differentially methylated region (DMR) in clones indicated normal patterns of methylation; however, bisulfite sequencing of the satellite I repeat element and epidermal cytokeratin promoter indicated hypermethylation in the chorion of clones when compared with controls. No differences were detected in methylation levels in the fetus proper. These results indicate that the nuclear transfer process affects gene expression patterns in the trophectoderm- and inner cell mass-derived tissues to different extents.

Animals↗
PubMed2006

Bovine SNRPN methylation imprint in oocytes and day 17 in vitro-produced and somatic cell nuclear transfer embryos.

Findings from recent studies have suggested that the low survival rate of animals derived via somatic cell nuclear transfer (SCNT) may be in part due to epigenetic abnormalities brought about by this procedure. DNA methylation is an epigenetic modification of DNA that is implicated in the regulation of imprinted genes. Genes subject to genomic imprinting are expressed monoallelically in a parent of origin-dependent manner and are important for embryo growth, placental function, and neurobehavioral processes. The vast majority of imprinted genes have been studied in mice and humans. Herein, our objectives were to characterize the bovine SNRPN gene in gametes and to compare its methylation profile in in vivo-produced, in vitro-produced, and SCNT-derived Day 17 elongating embryos. A CpG island within the 5' region of SNRPN was identified and examined using bisulfite sequencing. SNRPN alleles were unmethylated in sperm, methylated in oocytes, and approximately 50% methylated in somatic samples. The examined SNRPN region appeared for the most part to be normally methylated in three in vivo-produced Day 17 embryos and in eight in vitro-produced Day 17 embryos examined, while alleles from Day 17 SCNT embryos were severely hypomethylated in seven of eight embryos. In this study, we showed that the SNRPN methylation profiles previously observed in mouse and human studies are also conserved in cattle. Moreover, SCNT-derived Day 17 elongating embryos were abnormally hypomethylated compared with in vivo-produced and in vitro-produced embryos, which in turn suggests that SCNT may lead to faulty reprogramming or maintenance of methylation imprints at this locus.

Animals↗
PubMed2002

Phenylketonuria: an update.

Phenylketonuria is a flagship inborn error of metabolism and has been at the forefront of our growing understanding, diagnosis, and treatment of this family of disorders. In this article, the current understanding of its diagnosis, treatment, and complex molecular biology and physiology is reviewed. Recent papers exploring newer and less well-delineated areas of cofactor supplementation and genetic and epigenetic modification of the genotypic expression are presented. The excitement surrounding the continued exploration of the hyperphenylalaninemias is emphasized.

Animals↗
PubMed2004

Oligoclonality in bladder cancer: the implication for molecular therapies.

PURPOSE: There is conflicting evidence in the published literature regarding the clonal or oligoclonal origin of bladder cancer. MATERIALS AND METHODS: A MEDLINE search of articles on the clonality, genetic, epigenetic and tumor microenvironment of bladder cancer cells was done. Laboratory and clinical studies were included and relevant articles were selected if tumor cell clonality was part of the study. We reviewed this published evidence. RESULTS: Current thinking proposes 2 main theories. 1) In the clonogenic theory multifocal and recurrent tumors evolve from a single transformed cell and, hence, all progeny share a number of identical genetic mutations. 2) The field change theory assumes a global change in the urothelium with multiple transformed cells evolving into mature tumors independently. The evidence for and against each theory is compelling. Of equal importance are the parallel epigenetic modifications and changes in the cellular microenvironment that permit tumor evolution. CONCLUSIONS: The presence of oligoclonality has implications for the potential efficacy of novel molecular therapeutic agents for bladder cancer. The molecular targets for such therapies must be widely sampled in a tumor population to assess expression in separate clones.

Carcinoma, Transitional Cell↗
PubMed2006

Gene expression during terminal erythroid differentiation.

PURPOSE OF REVIEW: Expression profiling is a powerful technique to sample cell state. This review shows how expression profiling is being applied to the study of erythroid differentiation. RECENT FINDINGS: Expression-based studies of multipotential hematopoietic progenitor cells has shown that these cells express lineage-restricted genes from multiple lineages at low levels, and that they are in effect 'primed' to develop into all hematopoietic cell types. Expression profiling of oligopotent and committed progenitor cells has further shown that commitment to the erythroid lineage is associated with a progressive decline in the number of expressed genes. Lineage commitment is regulated by lineage-restricted transcription factors, and studies show that the erythroid transcription factor GATA1, in addition to activating a subset of genes, has global repressive effects on gene expression. Terminal erythroid differentiation is associated with further reduction in the number of expressed genes. The erythroid program is defined by those genes that are still expressed, and their high-level expression depends on specific epigenetic modifications, recruitment of transcription factors, and posttranscriptional effects. SUMMARY: Expression profiling provides the means to identify novel targets for the therapy of erythrocytes disorders, and to obtain insights into the mechanisms of cellular differentiation.

Animals↗
PubMed1993

The effect of DNA methylation on gene regulation of human papillomaviruses.

Integration of human papillomaviruses (HPVs) into the host genome is considered to be an early and important event in HPV-linked cervical carcinogenesis. Consequently, the viral DNA potentially becomes a target for cellular control mechanisms normally acting on the corresponding integration site. Besides resulting position effects, host-specific DNA methylation may play a functional role in HPV gene regulation. To elucidate the influence of such a kind of epigenetic modification on viral transcription, in vitro methylation studies on HPV-18 upstream regulatory region (URR)-controlled reporter plasmids were carried out. Selective methylation of the viral URR results in a down-regulation of the transcriptional activity, which can be attributed to nonrandom distribution of methyl-acceptor sites clustered within the constitutive enhancer region. In vivo competition experiments show that suppression is not directly mediated by steric hindrance of methyl residues with transcription factors, but rather is due to the association with methyl-CpG DNA-binding proteins. Using a restriction enzyme accessibility assay on both the DNA and chromatin levels, it could be demonstrated that, in vivo, extensively methylated viral DNA is nucleosomally organized, characteristic of transcriptionally inactive chromatin. These data suggest that DNA methylation is an important regulatory pathway in the modulation of HPV expression and as a consequence the proliferation rate of virus-infected cells.

Binding, Competitive↗
PubMed2023

Inferring Metabolic States from Single Cell Transcriptomic Data via Geometric Deep Learning.

The ability to measure gene expression at single-cell resolution has elevated our understanding of how biological features emerge from complex and interdependent networks at molecular, cellular, and tissue scales. As technologies have evolved that complement scRNAseq measurements with things like single-cell proteomic, epigenomic, and genomic information, it becomes increasingly apparent how much biology exists as a product of multimodal regulation. Biological processes such as transcription, translation, and post-translational or epigenetic modification impose both energetic and specific molecular demands on a cell and are therefore implicitly constrained by the metabolic state of the cell. While metabolomics is crucial for defining a holistic model of any biological process, the chemical heterogeneity of the metabolome makes it particularly difficult to measure, and technologies capable of doing this at single-cell resolution are far behind other multiomics modalities. To address these challenges, we present GEFMAP (Gene Expression-based Flux Mapping and Metabolic Pathway Prediction), a method based on geometric deep learning for predicting flux through reactions in a global metabolic network using transcriptomics data, which we ultimately apply to scRNAseq. GEFMAP leverages the natural graph structure of metabolic networks to learn both a biological objective for each cell and estimate a mass-balanced relative flux rate for each reaction in each cell using novel deep learning models.

Preprint↗
PubMed2003

Asynchronous replication timing of imprinted loci is independent of DNA methylation, but consistent with differential subnuclear localization.

Genomic imprinting in mammals marks the two parental alleles resulting in differential gene expression. Imprinted loci are characterized by distinct epigenetic modifications such as differential DNA methylation and asynchronous replication timing. To determine the role of DNA methylation in replication timing of imprinted loci, we analyzed replication timing in Dnmt1- and Dnmt3L-deficient embryonic stem (ES) cells, which lack differential DNA methylation and imprinted gene expression. Asynchronous replication is maintained in these ES cells, indicating that asynchronous replication is parent-specific without the requirement for differential DNA methylation. Imprinting centers are required for regional control of imprinted gene expression. Analysis of replication fork movement and three-dimensional RNA and DNA fluorescent in situ hybridization (FISH) analysis of the Igf2-H19 locus in various cell types indicate that the Igf2-H19 imprinting center differentially regulates replication timing of nearby replicons and subnuclear localization. Based on these observations, we suggest a model in which cis elements containing nonmethylation imprints are responsible for the movement of parental imprinted loci to distinct nuclear compartments with different replication characteristics resulting in asynchronous replication timing.

Alleles↗
PubMed2006

Jmjd2b antagonizes H3K9 trimethylation at pericentric heterochromatin in mammalian cells.

Histone lysine trimethyl states represent some of the most robust epigenetic modifications in eukaryotic chromatin. Using a candidate approach, we identified the subgroup of murine Jmjd2 proteins to antagonize H3K9me3 at pericentric heterochromatin. H3K27me3 and H4K20me3 marks are not impaired in inducible Jmjd2b-GFP cell lines, but Jmjd2b also reduces H3K36 methylation. Since recombinant Jmjd2b appears as a very poor enzyme, we applied metabolic labeling with heavy methyl groups to demonstrate Jmjd2b-mediated removal of chromosomal H3K9me3 as an active process that occurs well before replication of chromatin. These data reveal that certain members of the jmjC class of hydroxylases can work in a pathway that actively antagonizes a histone lysine trimethyl state.

Animals↗
PubMed1992

Parental imprinting: potentially active chromatin of the repressed maternal allele of the mouse insulin-like growth factor II (Igf2) gene.

The mouse insulin-like growth factor II (Igf2) gene, which is located on distal chromosome 7 (Chr7), has been shown previously to undergo tissue-specific parental imprinting. This imprinting results in expression of Igf2 from the paternally inherited chromosome and repression of the maternally inherited allele in most tissues of the developing embryo. We are using embryos with the maternal duplication and paternal deficiency of distal Chr7 to characterize the mechanism that underlies repression of the maternal allele. We show that the chromatin of the 5' region of the repressed Igf2 allele is potentially active for transcription rather than heterochromatic. In particular, a CpG island that comprises one of the two strong promoters is unmethylated at both parental alleles, and DNase I hypersensitive sites in and around the strong promoters are consistently present on both parental chromosomes. In agreement with the chromatin state, primary transcripts from the maternal Igf2 allele have been detected at low but significant levels. These findings differ from observations in other instances of imprinting, namely, X-chromosome inactivation and transgene imprinting in mice. Although no parent-specific differences were detected in either DNA methylation or sensitivity to nucleases at these promoters, we have observed parental methylation differences in a region several kilobases upstream of the first exon. The differential activity of the parental Igf2 alleles could be achieved through epigenetic modifications situated outside the promoters or by subtle and yet unidentified modifications at the promoters.

Alleles↗
PubMed2007

Serum response factor binding sites differ in three human cell types.

The serum response factor (SRF) is essential for embryonic development and maintenance of muscle cells and neurons. The mechanism by which this factor controls these divergent pathways is unclear. Here we present a genome-wide view of occupancy of SRF at its binding sites with a focus on those that vary with cell type. We used chromatin immunoprecipitation (ChIP) in combination with human promoter microarrays to identify 216 putative SRF binding sites in the human genome. We performed independent quantitative PCR validation at over half of these sites that resulted in 146 sites we assert to be true binding sites at over 90% confidence. Nearly half of the sites are bound by SRF in only one of the three cell types we tested, providing strong evidence for the diverse roles for SRF in different cell types. We also explore possible mechanisms controlling differential binding of SRF in these cell types by assaying cofactor binding, DNA methylation, histone methylation, and histone acetylation at a subset of sites bound preferentially in smooth muscle cells. Although we did not see a strong correlation between SRF binding and epigenetics modifications, at these sites, we propose that SRF cofactors may play an important role in determining cell-dependent SRF binding sites. ELK4 (previously known as SAP-1 [SRF-associated protein-1]) is ubiquitously expressed. Therefore, we expected it to occupy sites where SRF binding is common in all cell types. Indeed, 90% of SRF sites also bound by ELK4 were common to all three cell types. Together, our data provide a more complete understanding of the regulatory network controlled by SRF.

Binding Sites↗
PubMed2006

Regulation of seed size by hypomethylation of maternal and paternal genomes.

DNA methylation is an epigenetic modification of cytosine that is important for silencing gene transcription and transposons, gene imprinting, development, and seed viability. DNA METHYLTRANSFERASE1 (MET1) is the primary maintenance DNA methyltransferase in Arabidopsis (Arabidopsis thaliana). Reciprocal crosses between antisense MET1 transgenic and wild-type plants show that DNA hypomethylation has a parent-of-origin effect on seed size. However, due to the dominant nature of the antisense MET1 transgene, the parent with a hypomethylated genome, its gametophyte, and both the maternal and paternal genomes of the F(1) seed become hypomethylated. Thus, the distinct role played by hypomethylation at each generation is not known. To address this issue, we examined F(1) seed from reciprocal crosses using a loss-of-function recessive null allele, met1-6. Crosses between wild-type and homozygous met1-6 parents show that hypomethylated maternal and paternal genomes result in significantly larger and smaller F(1) seeds, respectively. Our analysis of crosses between wild-type and heterozygous MET1/met1-6 parents revealed that hypomethylation in the female or male gametophytic generation was sufficient to influence F(1) seed size. A recessive mutation in another gene that dramatically reduces DNA methylation, DECREASE IN DNA METHYLATION1, also causes parent-of-origin effects on F(1) seed size. By contrast, recessive mutations in genes that regulate a smaller subset of DNA methylation (CHROMOMETHYLASE3 and DOMAINS REARRANGED METHYLTRANSFERASES1 and 2) had little effect on seed size. Collectively, these results show that maternal and paternal genomes play distinct roles in the regulation of seed size in Arabidopsis.

Arabidopsis↗
PubMed2005

A two-edged role for the transposable element Kiddo in the rice ubiquitin2 promoter.

Miniature inverted repeat transposable elements (MITEs) are thought to be a driving force for genome evolution. Although numerous MITEs are found associated with genes, little is known about their function in gene regulation. Whereas the rice ubiquitin2 (rubq2) promoter in rice (Oryza sativa) line IR24 contains two nested MITEs (Kiddo and MDM1), that in line T309 has lost Kiddo, providing an opportunity to understand the role of MITEs in promoter function. No difference in endogenous rubq2 transcript levels between T309 and IR24 was evident using RT-PCR. However, promoter analysis using both transient and stably transformed calli revealed that Kiddo contributed some 20% of the total expression. Bisulfite genomic sequencing of the rubq2 promoters revealed specific DNA methylation at both symmetric and asymmetric cytosine residues on the MITE sequences, possibly induced by low levels of homologous transcripts. When methylation of the MITEs was blocked by 5-azacytidine treatment, a threefold increase in the endogenous rubq2 transcript level was detected in IR24 compared with that in T309. Together with the observed MITE methylation pattern, the detection of low levels of transcripts, but not small RNAs, corresponding to Kiddo and MDM1 suggested that RNA-dependent DNA methylation is induced by MITE transcripts. We conclude that, although Kiddo enhances transcription from the rubq2 promoter, this effect is mitigated by sequence-specific epigenetic modification.

Base Sequence↗
PubMed2005

DNA deamination in immunity.

A functional immune system is one of the prerequisites for the survival of a species. Humans have one of the most complicated immune systems, with the ability to learn from and adapt to pathogens. At first, a primary repertoire of antibodies is generated, which, upon antigen encounter, will diversify and adapt to produce a highly specific and potent secondary response, part of which is kept in memory to fight off future infections. In this review, the mechanism as well as the specificities of the key protein in the secondary immune response, activation-induced cytidine deaminase (AID), are highlighted, as well as its role in the DNA deamination model of immunoglobulin diversification. The review also highlights aspects of AID's regulation on both the transcriptional as well as post-translational level and its potential molecular mechanism and specificity. Furthermore, it expands outside the involvement of AID in somatic hypermutation, class switching, and gene conversion to discuss the implications of DNA deamination in epigenetic modifications of DNA (as a potential demethylase), the induction of mutations during oncogenesis, and includes an evolutionary comparison to the DNA deaminase family member APOBEC3G, a key protein in human immunodeficiency virus pathogenesis.

B-Lymphocytes↗