Table of Contents

Volume 7, Issue 2

  February 2012

  Pages 113 - 207

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EDITOR'S PREVIEW

Open Access Article

In this issue of Epigenetics

Barbara P. Rattner
Pages 113 - 114
http://dx.doi.org/10.4161/epi.7.2.19326
PDF




REVIEWS

Non-catalytic functions of DNMT1

Jesús Espada
Pages 115 - 118
http://dx.doi.org/10.4161/epi.7.2.18756
Abstract | PDF

Mammalian DNA methyltransferase 1 (DNMT1) is essential during early embryo development. Consistent with its key role in embryogenesis, depletion of this protein in adult somatic cells promotes severe cellular dysfunctions and cell death. DNMT1 contains a highly evolutionary conserved C-terminal catalytic DNA methyltransferase domain that is thought to be the responsible for the maintenance of CpG methylation patterns in the genome. DNMT1 has also a large N-terminal region with different functional protein-protein and protein-DNA binding domains. The multi-domain N-terminal region and the abundant molecular binding patterns suggest potential non-catalytic functions for DNMT1. However, this hypothesis remains controversial and conflicting results can be found in the literature. Here, recent results presenting a functional role for DNMT1 independent of its catalytic domain are discussed.

Basic concepts of epigenetics: Impact of environmental signals on gene expression

Elizabeth A. Mazzio and Karam F.A. Soliman
Pages 119 - 130
http://dx.doi.org/10.4161/epi.7.2.18764
Abstract | PDF

Through epigenetic modifications, specific long-term phenotypic consequences can arise from environmental influence on slowly evolving genomic DNA. Heritable epigenetic information regulates nucleosomal arrangement around DNA and determines patterns of gene silencing or active transcription. One of the greatest challenges in the study of epigenetics as it relates to disease is the enormous diversity of proteins, histone modifications and DNA methylation patterns associated with each unique maladaptive phenotype. This is further complicated by a limitless combination of environmental cues that could alter the epigenome of specific cell types, tissues, organs and systems. In addition, complexities arise from the interpretation of studies describing analogous but not identical processes in flies, plants, worms, yeast, ciliated protozoans, tumor cells and mammals. This review integrates fundamental basic concepts of epigenetics with specific focus on how the epigenetic machinery interacts and operates in continuity to silence or activate gene expression. Topics covered include the connection between DNA methylation, methyl-CpG-binding proteins, transcriptional repression complexes, histone residues, histone modifications that mediate gene repression or relaxation, histone core variant stability, H1 histone linker flexibility, FACT complex, nucleosomal remodeling complexes, HP1 and nuclear lamins.




BRIEF REPORTS

Inhibition of MspI cleavage activity by hydroxymethylation of the CpG site: A concern for DNA modification studies using restriction endonucleases

Kenji Ichiyanagi
Pages 131 - 136
http://dx.doi.org/10.4161/epi.7.2.18909
Abstract | PDF

In mammalian genomic DNA, cytosine methylation predominantly occurs at CpG dinucleotides and provides epigenetic information. In some cells, 5-methyl-cytosine (5-mC) can be further converted to 5-hydroxymethyl-cytosine (5-hmC) by the ten-eleven translocation family of proteins. MspI restriction endonuclease has been used to analyze these modified cytosines. However, the kinetic analysis in this study revealed that MspI activity is dramatically decreased by symmetrical hydroxymethylation of its recognition sequence and partly inhibited by hemi-hydroxymethylation, whereas TaqI and HaeIII are relatively resistant to hydroxymethylation. Therefore, DNA modification studies that use MspI, for example, reduced representation bisulfite shotgun sequencing, quantitative analysis of 5-hmC, and cleavage-sensitivity analysis, should be carefully interpreted.

Open Access Article

Semi-quantitative immunohistochemical detection of 5-hydroxymethyl-cytosine reveals conservation of its tissue distribution between amphibians and mammals

Rimple D. Almeida, Virginie Sottile, Matthew Loose, Paul A. De Sousa, Andrew D. Johnson and Alexey Ruzov
Pages 137 - 140
http://dx.doi.org/10.4161/epi.7.2.18949
Abstract | PDF

5-Hydroxymethyl-cytosine (5-hmC) is a form of modified cytosine, which has recently attracted a considerable attention due to its potential role in transcriptional regulation. According to several reports 5-hydroxymethyl-cytosine distribution is tissue-specific in mammals. Thus, 5-hmC is enriched in embryonic cell populations and in adult neuronal tissue. Here, we describe a novel method of semi-quantitative immunohistochemical detection of 5-hmC and utilize it to assess the levels of this modification in amphibian tissues. We show that, similar to mammalian embryos, 5-hmC is enriched in axolotl tadpoles compared with adult tissues. Our data demonstrate that 5-hmC distribution is tissue-specific in amphibians, and that strong 5-hmC enrichment in neuronal cells is conserved between amphibians and mammals. In addition, we identify 5-hmC-enriched cell populations that are distributed in amphibian skin and connective tissue in a mosaic manner. Our results illustrate that immunochemistry can be successfully used not only for spatial identification of cells enriched with 5-hmC, but also for the semi-quantitative assessment of the levels of this epigenetic modification in single cells of different tissues.




RESEARCH PAPERS

Open Access Article

Rapid turnover of DNA methylation in human cells

Yoshiaki Yamagata, Pàl Szabó, David Szüts, Caroline Bacquet, Tamàs Arànyi and Andras Paldi
Pages 141 - 145
http://dx.doi.org/10.4161/epi.7.2.18906
Abstract | PDF

Recent studies demonstrated that cytosine methylation in the genome can be reversed without DNA replication by enzymatic mechanisms based on base excision-repair pathways. Both enzymatic methylation and demethylation mechanisms are active in the cell nucleus at the same time. One can hypothesize that the actual level of CpG methylation could be the result of a balance between the two antagonistic processes with a rapid turnover. In the present study, we used mass spectrometry to measure the total methyl-cytosine content of the genome in cultured human cells after short incubation with the known methyltransferase inhibitor 5-deoxy-azacytidine. A significant decrease of the DNA methylation was observed. Indeed, the inhibition of the methylation can only result in a rapid reduction of the overall methyl-cytosine level if the process of demethylation is simultaneous. These observations suggest that the enzymatic mechanisms responsible of the opposing reactions of DNA methylation and demethylation act simultaneously and may result in a continuous and rapid turnover of methylated cytosines. This conclusion is supported by the observation that 5-deoxy-azacytidine was incorporated in the genomic DNA of non-dividing cells and could be detected as soon as after two hours of incubation, hence providing a mechanistic explanation to the inhibition of methyltransferases. The observations are compatible with the idea that the enzymatic mechanisms that bring together of the opposing reactions of DNA methylation and demethylation act simultaneously and may result in a continuous and unsuspected rapid turnover of DNA methylation. This conclusion is at odds with the generally accepted view of high stability of cytosine methylation where the role of enzymatic demethylation is considered as limited to some special situations such as transcription. It places DNA methylation in the same category as other epigenetic modifications with covalent modifications dynamically added to and removed from the chromatin with high turnover rate.

Mutation of MeCP2 alters transcriptional regulation of select immediate-early genes

Dan Su, Young May Cha and Anne E. West
Pages 146 - 154
http://dx.doi.org/10.4161/epi.7.2.18907
Abstract | PDF

Loss-of-function mutations in the methyl-DNA binding protein MeCP2 are associated with neurological dysfunction and impaired neural plasticity. However, the transcriptional changes that underlie these deficits remain poorly understood. Here, we show that mice bearing a C-terminal truncating mutation in Mecp2 (Mecp2³⁰⁸) are hypersensitive to the locomotor stimulating effects of cocaine. Furthermore, these mice have gene-specific alterations in striatal immediate-early gene (IEG) induction following cocaine administration. MeCP2 mutant mice show normal levels of baseline and cocaine-induced striatal Fos expression compared with their wild-type littermates. However, the mutant mice have enhanced cocaine-induced transcription of Junb and Arc. At the chromatin level, we find increased histone H3 acetylation at gene promoters in the Mecp2 mutant mice compared with their wild-type littermates, whereas two sites of repressive histone methylation are unchanged. Interestingly, we find that MeCP2 mutant mice show increased steady-state association of elongation-competent RNA Polymerase II (RNAP II) with the Junb and Arc promoters, whereas levels of RNAP II association at the Fos promoter are unchanged. These data reveal a gene-specific effect of MeCP2 on the recruitment of RNAP II to gene promoters that may modulate the inducibility of IEGs. In addition, our findings raise the possibility that aberrant regulation of IEGs including Junb and Arc may contribute to altered cocaine-induced neuronal and behavioral plasticity in Mecp2 mutant mice.

Epigenetic and genetic variation at the IGF2/H19 imprinting control region on 11p15.5 is associated with cerebellum weight

Ruth Pidsley, Emma Dempster, Claire Troakes, Safa Al-Sarraj and Jonathan Mill
Pages 155 - 163
http://dx.doi.org/10.4161/epi.7.2.18910
Abstract | PDF

IGF2 is a paternally expressed imprinted gene with an important role in development and brain function. Allele-specific expression of IGF2 is regulated by DNA methylation at three differentially methylated regions (DMRs) spanning the IGF2/H19 domain on human 11p15.5. We have comprehensively assessed DNA methylation and genotype across the three DMRs and the H19 promoter using tissue from a unique collection of well-characterized and neuropathologically-dissected post-mortem human cerebellum samples (n = 106) and frontal cortex samples (n = 51). We show that DNA methylation, particularly in the vicinity of a key CTCF-binding site (CTCF3) in the imprinting control region (ICR) upstream of H19, is strongly correlated with cerebellum weight. DNA methylation at CTCF3 uniquely explains ~25% of the variance in cerebellum weight. In addition, we report that genetic variation in this ICR is strongly associated with cerebellum weight in a parental-origin specific manner, with maternally-inherited alleles associated with a 16% increase in cerebellum weight compared with paternally-inherited alleles. Given the link between structural brain abnormalities and neuropsychiatric disease, an understanding of the epigenetic and parent-of-origin specific genetic factors associated with brain morphology provides important clues about the etiology of disorders such as schizophrenia and autism.

Epigenetic silencing of HNF1A associates with changes in the composition of the human plasma N-glycome

Vlatka Zoldoš, Tomislav Horvat, Mislav Novokmet, Cyrille Cuenin, Ana Mužinić, Maja Pučić, Jennifer E. Huffman, Olga Gornik, Ozren Polašek, Harry Campbell, Caroline Hayward, Alan F. Wright, Igor Rudan, Katharine Owen, Mark I. McCarthy, Zdenko Herceg and Gordan Lauc
Pages 164 - 172
http://dx.doi.org/10.4161/epi.7.2.18918
Abstract | PDF

Protein glycosylation is a ubiquitous modification that affects the structure and function of proteins. Our recent genome wide association study identified transcription factor HNF1A as an important regulator of plasma protein glycosylation. To evaluate the potential impact of epigenetic regulation of HNF1A on protein glycosylation we analyzed CpG methylation in 810 individuals. The association between methylation of four CpG sites and the composition of plasma and IgG glycomes was analyzed. Several statistically significant associations were observed between HNF1A methylation and plasma glycans, while there were no significant associations with IgG glycans. The most consistent association with HNF1A methylation was observed with the increase in the proportion of highly branched glycans in the plasma N-glycome. The hypothesis that inactivation of HNF1A promotes branching of glycans was supported by the analysis of plasma N-glycomes in 61 patients with inactivating mutations in HNF1A, where the increase in plasma glycan branching was also observed. This study represents the first demonstration of epigenetic regulation of plasma glycome composition, suggesting a potential mechanism by which epigenetic deregulation of the glycome may contribute to disease development.

Trophoblast-specific DNA methylation occurs after the segregation of the trophectoderm and inner cell mass in the mouse periimplantation embryo

Momo O. Nakanishi, Koji Hayakawa, Kazuhiko Nakabayashi, Kenichiro Hata, Kunio Shiota and Satoshi Tanaka
Pages 173 - 182
http://dx.doi.org/10.4161/epi.7.2.18962
Abstract | PDF

The first cell differentiation in the mammalian development separates the trophoblast and embryonic cell lineages, resulting in the formation of the trophectoderm (TE) and inner cell mass (ICM) in blastocysts. Although a lower level of global DNA methylation in the genome of the TE compared with ICM has been suggested, the dynamics of the DNA methylation profile during TE/ICM differentiation has not been elucidated. To address this issue, first we identified tissue-dependent and differentially methylated regions (T-DMRs) between trophoblast stem (TS) and embryonic stem (ES) cells. Most of these TS–ES T-DMRs were also methylated differentially between trophoblast and embryonic tissues of embryonic day (E) 6.5 mouse embryos. Furthermore, we found that the human genomic regions homologous to mouse TS–ES T-DMRs were methylated differentially between human placental tissues and ES cells. Collectively, we defined them as cell-lineage-based T-DMRs between trophoblast and embryonic cell lineages (T–E T-DMRs). Then, we examined TE and ICM cells isolated from mouse E3.5 blastocysts. Interestingly, all T-DMRs examined, including the Elf5, Pou5f1 and Nanog loci, were in the nearly unmethylated status in both TE and ICM and exhibited no differences. The present results suggest that the establishment of DNA methylation profiles specific to each cell lineage follows the first morphological specification. Together with previous reports on asymmetry of histone modifications between TE and ICM, the results of the current study imply that histone modifications function as landmarks for setting up cell-lineage-specific differential DNA methylation profiles.

Histone deacetylase 3 (HDAC3) participates in the transcriptional repression of the p16^INK4a gene in mammary gland of the female rat offspring exposed to an early-life high-fat diet

Shasha Zheng, Qian Li, Yukun Zhang, Zachary Balluff and Yuan-Xiang Pan
Pages 183 - 190
http://dx.doi.org/10.4161/epi.7.2.18972
Abstract | PDF

Maternal exposure to environmental agents throughout pregnancy and lactation may affect offspring’s mammary gland growth and alter the epigenome. This may predispose the offspring’s mammary glands to be more susceptible to carcinogenesis. The purpose of this study was to examine the effect of a maternal high-fat diet on the regulation of p16^INK4a gene expression in the mammary gland of rat offspring. Timed-pregnant Sprague-Dawley rats were fed one of the two diets, a control (C, 16% of fat) or a high fat (HF, 45% of fat) diet, throughout gestation and lactation and sacrificed at 12 weeks of age. Compared with C, HF offspring showed a decrease of p16^INK4a gene expression in the mammary gland at both mRNA and protein levels. Chromatin immunoprecipitation (ChIP) assay demonstrated that the downregulation of p16^INK4a transcription in HF offspring was associated with reduced acetylation of histone H4 and increased recruitment of histone deacetylase 3 (HDAC3) within the p16^INK4a promoter region, but was not associated with acetylation of histone H3 or HDAC1. Methylated DNA immunoprecipitation (MeDIP) did not detect differences in methylation at different regions of the p16^INK4a gene between C and HF offspring. We conclude that maternal high fat exposure represses p16^INK4a gene expression in the mammary gland of offspring through changes of histone modifications and HDAC3 binding activity within the regulatory regions of the p16^INK4a gene.

Promoter methylation status of VEGF receptor genes: A possible epigenetic biomarker to anticipate the efficacy of intracellular-acting VEGF-targeted drugs in cancer cells

Jeeyeon Kim, Junha Hwang, Hyeseon Jeong, Hee-Jung Song, Jieun Shin, Gangmin Hur, Young Woo Park, Suk Hoon Lee and Jei Kim
Pages 191 - 200
http://dx.doi.org/10.4161/epi.7.2.18973
Abstract | PDF

We evaluated whether the inhibitory effects of vascular endothelial growth factor (VEGF)-targeted drugs on the proliferation of cancer cells differed according to VEGF receptor (VEGFR) genes, Flt1 and KDR, promoter methylation status. Five hyper-VEGFR-methylation and six no-VEGFR-methylation cancer cells were used for the present study, together with human umbilical endothelial cells (HUVECs) as a control. No-VEGFR-methylation cancer cells showed higher expression of Flt1 and KDR than hyper-VEGFR-methylation cancer cells. Hyper-VEGFR-methylation cancer cells only showed increased expression and protein levels of Flt1 and KDR after treatment with the demethylase 5-aza-2’-deoxycytidine. Two drugs (a VEGF-specific-antibody, bevacizumab, and a KDR-specific-antibody) targeting extracellular VEGF-VEGFR signaling and two VEGF-specific-tyrosine kinase inhibitors (PTK/ZK and sunitinib) targeting intracellular VEGFR signaling were used in the cell proliferation assay. HUVECs showed dose- and time-dependent proliferation decrease with all tested drugs over a 72 h incubation period. No- or hyper-VEGFR-methylation cancer cells showed no significant proliferation differences after treatment with VEGF-specific-antibody or VEGFR2-specific-antibody. After PTK/ZK or sunitinib treatment, no-VEGFR-methylation cancer cells showed dose- or time-dependent decreases in proliferation. Hyper-VEGFR-methylation cancer cells also showed proliferation inhibition by VEGF-specific-tyrosine kinase inhibitors after demethylation of Flt1 and KDR. Proliferation inhibition synergistically increased after combination of demethylation with PTK/ZK in hyper-VEGF-methylation cancer cells. We observed that intracellular targeting of VEGF-VEGFR signaling could be more effective than extracellular targeting of the pathway in the suppression of proliferation of some cancer cells. In particular, the efficacy of intracellular targeting of VEGF-specific-tyrosine kinase inhibitors might be influenced by the epigenetic alteration of VEGFRs.

Effects of TET2 mutations on DNA methylation in chronic myelomonocytic leukemia

Jumpei Yamazaki, Rodolphe Taby, Aparna Vasanthakumar, Trisha Macrae, Kelly R. Ostler, Lanlan Shen, Hagop M. Kantarjian, Marcos R. Estecio, Jaroslav Jelinek, Lucy A. Godley and Jean-Pierre J. Issa
Pages 201 - 207
http://dx.doi.org/10.4161/epi.7.2.19015
Abstract | PDF

TET2 enzymatically converts 5-methyl-cytosine to 5-hydroxymethyl-cytosine, possibly leading to loss of DNA methylation. TET2 mutations are common in myeloid leukemia and were proposed to contribute to leukemogenesis through DNA methylation. To expand on this concept, we studied chronic myelomonocytic leukemia (CMML) samples. TET2 missense or nonsense mutations were detected in 53% (16/30) of patients. In contrast, only 1/30 patient had a mutation in IDH1 or IDH2, and none of them had a mutation in DNMT3A in the sites most frequently mutated in leukemia. Using bisulfite pyrosequencing, global methylation measured by the LINE-1 assay and DNA methylation levels of 10 promoter CpG islands frequently abnormal in myeloid leukemia were not different between TET2 mutants and wild-type CMML cases. This was also true for 9 out of 11 gene promoters reported by others as differentially methylated by TET2 mutations. We found that two non-CpG island promoters, AIM2 and SP140, were hypermethylated in patients with mutant TET2. These were the only two gene promoters (out of 14,475 genes) previously found to be hypermethylated in TET2 mutant cases. However, total 5-methyl-cytosine levels in TET2 mutant cases were significantly higher than TET2 wild-type cases (median = 14.0% and 9.8%, respectively) (p = 0.016). Thus, TET2 mutations affect global methylation in CMML but most of the changes are likely to be outside gene promoters.