Thomas P. Yang

Folate and DNA Methylation: A Review of Molecular Mechanisms and the Evidence for Folate’s Role

DNA methylation is an epigenetic modification critical to normal genome regulation and development. The vitamin folate is a key source of the one carbon group used to methylate DNA. Because normal mammalian development is dependent on DNA methylation, there is enormous interest in assessing the potential for changes in folate intake to modulate DNA methylation both as a biomarker for folate status and as a mechanistic link to developmental disorders and chronic diseases including cancer. This review highlights the role of DNA methylation in normal genome function, how it can be altered, and the evidence of the role of folate/folic acid in these processes.

Developmentally regulated alterations in Polycomb repressive complex 1 proteins on the inactive X chromosome

Polycomb group (PcG) proteins belonging to the polycomb (Pc) repressive complexes 1 and 2 (PRC1 and PRC2) maintain homeotic gene silencing. In Drosophila, PRC2 methylates histone H3 on lysine 27, and this epigenetic mark facilitates recruitment of PRC1. Mouse PRC2 (mPRC2) has been implicated in X inactivation, as mPRC2 proteins transiently accumulate on the inactive X chromosome (Xi) at the onset of X inactivation to methylate histone H3 lysine 27 (H3-K27). In this study, we demonstrate that mPRC1 proteins localize to the Xi, and that different mPRC1 proteins accumulate on the Xi during initiation and maintenance of X inactivation in embryonic cells. The Xi accumulation of mPRC1 proteins requires Xist RNA and is not solely regulated by the presence of H3-K27 methylation, as not all cells that exhibit this epigenetic mark on the Xi show Xi enrichment of mPRC1 proteins. Our results implicate mPRC1 in X inactivation and suggest that the regulated assembly of PcG protein complexes on the Xi contributes to this multistep process.

Analysis of expressed sequence tags from Plasmodium falciparum

An initiative was undertaken to sequence all genes of the human malaria parasite Plasmodium falciparum in an effort to gain a better understanding at the molecular level of the parasite that inflicts much suffering in the developing world. 550 random complimentary DNA clones were partially sequenced from the intraerythrocytic form of the parasite as one of the approaches to analyze the transcribed sequences of its genome. The sequences, after editing, generated 389 expressed sequence tag sites and over 105 kb of DNA sequences. About 32% of these clones showed significant homology with other genes in the database. These clones represent 340 new Plasmodium falciparum expressed sequence tags.

MTHFR 677C→T genotype is associated with folate and homocysteine concentrations in a large, population-based, double-blind trial of folic acid supplementation

BACKGROUND The methylenetetrahydrofolate reductase (MTHFR) genotype is associated with modification of disease and risk of neural tube defects. Plasma and red blood cell (RBC) folate and plasma homocysteine concentrations change in response to daily intakes of folic acid supplements, but no large-scale or population-based randomized trials have examined whether the MTHFR genotype modifies the observed response. OBJECTIVE We sought to determine whether the MTHFR 677C→T genotype modifies the response to folic acid supplementation during and 3 mo after discontinuation of supplementation. DESIGN Northern Chinese women of childbearing age were enrolled in a 6-mo supplementation trial of different folic acid doses: 100, 400, and 4000 μg/d and 4000 μg/wk. Plasma and RBC folate and plasma homocysteine concentrations were measured at baseline; after 1, 3, and 6 mo of supplementation; and 3 mo after discontinuation of supplementation. MTHFR genotyping was performed to identify a C→T mutation at position 677 (n = 932). RESULTS Plasma and RBC folate and homocysteine concentrations were associated with MTHFR genotype throughout the supplementation trial, regardless of folic acid dose. MTHFR TT was associated with lower folate concentrations, and the trend of TT < CC was maintained at even the highest doses. Folic acid doses of 100 μg/d or 4000 μg/wk did not reduce high homocysteine concentrations in those with the MTHFR TT genotype. CONCLUSION MTHFR genotype was an independent predictor of plasma and RBC folate and plasma homocysteine concentrations and did not have a significant interaction with folic acid dose during supplementation. This trial was registered at clinicaltrials.gov as NCT00207558.

Evidence That Silencing of the HPRT Promoter by DNA Methylation Is Mediated by Critical CpG Sites

The strong correlation between promoter hypermethylation and gene silencing suggests that promoter methylation represses transcription. To identify methylation sites that may be critical for maintaining repression of the human HPRT gene, we treated human/hamster hybrid cells containing an inactive human X chromosome with the DNA demethylating agent 5-azadeoxycytidine (5aCdr), and we then examined the high resolution methylation pattern of theHPRT promoter in single cell-derived lines. Reactivation ofHPRT correlated with complete promoter demethylation. In contrast, the 61 5aCdr-treated clones that failed to reactivateHPRT exhibited sporadic promoter demethylation. However, three specific CpG sites remained methylated in all unreactivated clones, suggesting these sites may be critical for maintaining transcriptional silencing of the HPRT gene. Re-treatment of partially demethylated (and unreactivated) clones with a second round of 5aCdr did not increase the frequency of HPRTreactivation. This is consistent with mechanisms of methylation-mediated repression requiring methylation at specific critical sites and argues against models invoking overall levels or a threshold of promoter methylation. Treatment of cells with the histone deacetylase inhibitor, trichostatin A, failed to reactivateHPRT on the inactive X chromosome, even when the promoter was partially demethylated by 5aCdr treatment, suggesting that transcriptional repression by DNA methylation is unlikely to depend upon a trichostatin A-sensitive histone deacetylase.

Genomic DNA Methylation Changes in Response to Folic Acid Supplementation in a Population-Based Intervention Study among Women of Reproductive Age

Folate is a source of one-carbons necessary for DNA methylation, a critical epigenetic modification necessary for genomic structure and function. The use of supplemental folic acid is widespread however; the potential influence on DNA methylation is unclear. We measured global DNA methylation using DNA extracted from samples from a population-based, double-blind randomized trial of folic acid supplementation (100, 400, 4000 µg per day) taken for 6 months; including a 3 month post-supplementation sample. We observed no changes in global DNA methylation in response to up to 4,000 µg/day for 6 months supplementation in DNA extracted from uncoagulated blood (approximates circulating blood). However, when DNA methylation was determined in coagulated samples from the same individuals at the same time, significant time, dose, and MTHFR genotype-dependent changes were observed. The baseline level of DNA methylation was the same for uncoagulated and coagulated samples; marked differences between sample types were observed only after intervention. In DNA from coagulated blood, DNA methylation decreased (−14%; P<0.001) after 1 month of supplementation and 3 months after supplement withdrawal, methylation decreased an additional 23% (P<0.001) with significant variation among individuals (max+17%; min-94%). Decreases in methylation of ≥25% (vs. <25%) after discontinuation of supplementation were strongly associated with genotype: MTHFR CC vs. TT (adjusted odds ratio [aOR] 12.9, 95%CI 6.4, 26.0). The unexpected difference in DNA methylation between DNA extracted from coagulated and uncoagulated samples in response to folic acid supplementation is an important finding for evaluating use of folic acid and investigating the potential effects of folic acid supplementation on coagulation.

Characterization of cis- and trans-acting elements in the imprinted human SNURF-SNRPN locus

The imprinted SNRPN locus is a complex transcriptional unit that encodes the SNURF and SmN polypeptides as well as multiple non-coding RNAs. SNRPN is located within the Prader-Willi and Angelman syndrome (PWS/AS) region that contains multiple imprinted genes, which are coordinately regulated by a bipartite imprinting center (IC). The SNRPN 5′ region co-localizes with the PWS-IC and contains two DNase I hypersensitive sites, DHS1 at the SNRPN promoter, and DHS2 within intron 1, exclusively on the paternally inherited chromosome. We have examined DHS1 and DHS2 to identify cis- and trans-acting regulatory elements within the endogenous SNRPN 5′ region. Analysis of DHS1 by in vivo footprinting and chromatin immunoprecipitation identified allele-specific interaction with multiple regulatory proteins, including NRF-1, which regulates genes involved in mitochondrial and metabolic functions. DHS2 acted as an enhancer of the SNRPN promoter and contained a highly conserved region that showed allele-specific interaction with unphosphorylated RNA polymerase II, YY1, Sp1 and NRF-1, further suggesting a key role for NRF-1 in regulation of the SNRPN locus. We propose that one or more of the regulatory elements identified in this study may also contribute to PWS-IC function.

Ontogeny of a Demethylation Domain and Its Relationship to Activation of Tissue-Specific Transcription

Abstract We examined DNA methylation throughout the endogenous murine testis-specific phosphoglycerate kinase (Pgk2) gene and in human PGK2 promoter/CAT reporter transgenes in mouse spermatogenic cells before, during, and following the period of active transcription of this gene. We observed the gradual development of a domain of demethylation beginning over the promoter and then expanding approximately 1 kilobase in each direction within the endogenous Pgk2 gene. This demethylation domain develops in the absence of DNA replication and precedes other molecular changes that potentiate tissue-specific activation of this gene. Studies with transgenes show that a signal residing in the Pgk2 core promoter directs this gene-, cell type-, and stage-specific demethylation process. These results are consistent with a model in which regulated, tissue- and gene-specific demethylation initiates a cascade of subsequent molecular events required for tissue-specific activation of transcription during spermatogenesis in vivo.

Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter

ABSTRACT Differential chromatin structure is one of the hallmarks distinguishing active and inactive genes. For the X-linked human hypoxanthine phosphoribosyltransferase gene (HPRT), this difference in chromatin structure is evident in the differential general DNase I sensitivity and hypersensitivity of the promoter regions on active versus inactive X chromosomes. Here we characterize the nucleosomal organization responsible for the differential chromatin structure of the active and inactive HPRT promoters. The micrococcal nuclease digestion pattern of chromatin from the active allele in permeabilized cells reveals an ordered array of translationally positioned nucleosomes in the promoter region except over a 350-bp region that is either nucleosome free or contains structurally altered nucleosomes. This 350-bp region includes the entire minimal promoter and all of the multiple transcription initiation sites of the HPRT gene. It also encompasses all of the transcription factor binding sites identified by either dimethyl sulfate or DNase I in vivo footprinting of the active allele. In contrast, analysis of the inactive HPRT promoter reveals no hypersensitivity to either DNase I or a micrococcal nuclease and no translational positioning of nucleosomes. Although nucleosomes on the inactive promoter are not translationally positioned, high-resolution DNase I cleavage analysis of permeabilized cells indicates that nucleosomes are rotationally positioned over a region of at least 210 bp on the inactive promoter, which coincides with the 350-bp nuclease-hypersensitive region on the active allele, including the entire minimal promoter. This rotational positioning of nucleosomes is not observed on the active promoter. These results suggest a model in which the silencing of the HPRT promoter during X chromosome inactivation involves remodeling a transcriptionally competent, translationally positioned nucleosomal array into a transcriptionally repressed architecture consisting of rotationally but not translationally positioned nucleosomal arrays.

A new deletion refines the boundaries of the murine Prader-Willi syndrome imprinting center

The human chromosomal 15q11-15q13 region is subject to both maternal and paternal genomic imprinting. Absence of paternal gene expression from this region results in Prader-Willi syndrome (PWS), while absence of maternal gene expression leads to Angelman syndrome. Transcription of paternally expressed genes in the region depends upon an imprinting center termed the PWS-IC. Imprinting defects in PWS can be caused by microdeletions and the smallest commonly deleted region indicates that the PWS-IC lies within a region of 4.3 kb. The function and location of the PWS-IC is evolutionarily conserved, but delineation of the PWS-IC in mouse has proven difficult. The first targeted mutation of the PWS-IC, a deletion of 35 kb spanning Snrpn exon 1, exhibited a complete PWS-IC deletion phenotype. Pups inheriting this mutation paternally showed a complete loss of paternal gene expression and died neonatally. A reported deletion of 4.8 kb showed only a reduction in paternal gene expression and incomplete penetrance of neonatal lethality, suggesting that some PWS-IC function had been retained. Here, we report that a 6 kb deletion spanning Snrpn exon 1 exhibits a complete PWS-IC deletion phenotype. Pups inheriting this mutation paternally lack detectable expression of all PWS genes and paternal silencing of Ube3a, exhibit maternal DNA methylation imprints at Ndn and Mkrn3 and suffer failure to thrive leading to a fully penetrant neonatal lethality.