Table of Contents

Volume 7, Issue 5

  May 2012

  Pages 419 - 523

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EDITOR'S PREVIEW

Open Access Article

In this issue of Epigenetics

Barbara P. Rattner
Pages 419 - 420
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BRIEF REPORTS

Open Access Article

Diametrically opposite methylome-transcriptome relationships in high- and low-CpG promoter genes in postmitotic neural rat tissue

Theresa Hartung, Lu Zhang, Rahul Kanwar, Irina Khrebtukova, Matthias Reinhardt, Cheng Wang, Terry M. Therneau, Michaela S. Banck, Gary P. Schroth and Andreas S. Beutler
Pages 421 - 428
Abstract | PDF

DNA methylation can control some CpG-poor genes but unbiased studies have not found a consistent genome-wide association with gene activity outside of CpG islands or shores possibly due to use of cell lines or limited bioinformatics analyses. We performed reduced representation bisulfite sequencing (RRBS) of rat dorsal root ganglia encompassing postmitotic primary sensory neurons (n = 5, r > 0.99; orthogonal validation p < 10⁻¹⁹). The rat genome suggested a dichotomy of genes previously reported in other mammals: low CpG content (< 3.2%) promoter (LCP) genes and high CpG content (≥ 3.2%) promoter (HCP) genes. A genome-wide integrated methylome-transcriptome analysis showed that LCP genes were markedly hypermethylated when repressed, and hypomethylated when active with a 40% difference in a broad region at the 5′ of the transcription start site (p < 10⁻⁸⁷ for -6000 bp to -2000 bp, p < 10⁻⁷³ for -2000 bp to +2000 bp, no difference in gene body p = 0.42). HCP genes had minimal TSS-associated methylation regardless of transcription status, but gene body methylation appeared to be lost in repressed HCP genes. Therefore, diametrically opposite methylome-transcriptome associations characterize LCP and HCP genes in postmitotic neural tissue in vivo.

Lack of genomic imprinting of DNA primase, polypeptide 2 (PRIM2) in human term placenta and white blood cells

Jaewook Chung, Shengdar Tsai, Andra H. James, Betty H. Thames, Stephanie Shytle and Jorge A. Piedrahita
Pages 429 - 431
Abstract | PDF

PRIM2, encoding a subunit of primase involved in DNA replication and transcription, is expressed in the placenta and is crucial for mammalian development and growth. Its role in placental function is not well understood. Recently, PRIM2 was reported as imprinted in human white blood cells (WBC). We report here our failure to confirm imprinting of the PRIM2 locus in human placenta or WBC. The discordance between our results and those of others are likely due to an incorrectly annotated PRIM2 pseudogene found in the human genome database.

Smoking induces differential miRNA expression in human spermatozoa: A potential transgenerational epigenetic concern?

Emma L. Marczylo, Akwasi A. Amoako, Justin C. Konje, Timothy W. Gant and Timothy H. Marczylo
Pages 432 - 439
Abstract | PDF

Recent work has suggested that environmental chemicals, including those contained in cigarette smoke, can have adverse effects on the exposed individuals as well as their future progeny. The mechanisms underlying transmission of environmentally induced phenotypes through the germ line are not well understood. However, a predominant process appears to be the establishment of permanent heritable epigenetic alterations, and a number of studies have implicated microRNAs in such processes. Here, we show that cigarette smoke induces specific differences in the spermatozoal microRNA content of human smokers compared with non-smokers, and that these altered microRNAs appear to predominantly mediate pathways vital for healthy sperm and normal embryo development, particularly cell death and apoptosis. microRNA-mediated perturbation of such pathways may explain how harmful phenotypes can be induced in the progeny of smokers.




RESEARCH PAPERS

Alteration of DNA demethylation dynamics by in vitro culture conditions in rabbit pre-implantation embryos

Adriana R. Reis e Silva, Céline Bruno, Renaud Fleurot, Nathalie Daniel, Catherine Archilla, Nathalie Peynot, Carolina M. Lucci, Nathalie Beaujean and Veronique Duranthon
Pages 440 - 446
Abstract | PDF

Alterations to DNA methylation have been attributed to in vitro culture and may affect normal embryo development. We chose to analyze DNA methylation reprogramming in the rabbit which, of the species with delayed transcriptional activation of the embryonic genome, allows easy comparisons between in vivo-developed (IVD) and in vitro-cultured (IVC) embryos. In this species, variations in DNA methylation had not previously been quantified, even in IVD embryos. IVD and IVC embryos were recovered at the 2, 4, 8 and 16-cell, morula and blastocyst stages. Immunostaining for 5-methyl-cytidine and normalization of the quantity of methylated DNA vs. the total DNA content were then performed. Our quantitative results evidenced DNA demethylation during pre-implantation development in both IVD and IVC embryos, but with different kinetics. Demethylation occurred earlier in vitro than in vivo between the 2 and 8-cell stages in IVC embryos, reaching its lowest level, while it only started at the 4-cell stage and ended at the 16-cell stage in IVD embryos. We also showed that an absence of serum from the culture medium significantly altered the degree of DNA demethylation. Finally, at the blastocyst stage, ICM was more methylated than the trophectoderm in all cases. Despite a morphological delay observed in in vitro cultured blastocysts, the difference in DNA methylation between ICM and trophectoderm cells appeared at the same time post-fertilization in IVD and IVC embryos, which may reflect another difference in the dynamics of DNA methylation during blastocyst formation. Our data thus clearly establish an effect of embryonic environment on DNA methylation reprogramming during pre-implantation development in a non-rodent species.

SFRP1 CpG island methylation locus is associated with renal cell cancer susceptibility and disease recurrence

Faranaz Atschekzei, Jörg Hennenlotter, Stefanie Jänisch, Annika Großhennig, Wolfgang Tränkenschuh, Sandra Waalkes, Inga Peters, Thilo Dörk, Axel S. Merseburger, Arnulf Stenzl, Markus A. Kuczyk and Jürgen Serth
Pages 447 - 457
Abstract | PDF

Loss of the secreted Fzd-related protein 1 (SFRP1) and concurrent alteration of the SFRP1/WNT pathway are frequently observed in human cancers such as in renal cell cancer (RCC). Whether methylation of a SFRP1 CpG island locus in normal human solid tissues is associated with increased tissue specific cancer risk has not been determined to date. Here we measure the cancer risk attributable to SFRP1 DNA methylation in renal tissue. Pyrosequencing of bisulfite treated DNA was used for a case-control study including 120 normal-appearing renal tissues of autopsy specimens and 72 normal-appearing tissues obtained from tumor adjacent areas, and a cross sectional study of 96 RCCs. Association of methylation with demographic risk factor age, clinicopathological parameters and course of patients was investigated. We show significant hypermethylation of a SFRP1 CpG island locus in normal-appearing renal tissues from RCC patients compared with normal-appearing autopsy kidney tissues. Inter quartile analysis revealed a 6-, 13- and 11-fold increased cancer risk for the second, third and fourth quartiles of methylation in the age matched subgroup of tissues (p = 0.001, p = 1.3E-6, p = 6.9E-6). Methylation in autopsy tissues increased with age and methylation in tumors was an independent predictor of recurrence free survival. SFRP1 DNA methylation, accumulates with age in normal-appearing kidney tissues and is associated with increased renal cancer risk, suggesting this CGI sub region as an epigenetic susceptibility locus for RCC. Our data underline the need to further analyze the tissue specific risks conferred by methylated loci for the development of human cancers.

Prenatal antiepileptic exposure associates with neonatal DNA methylation differences

Alicia K. Smith, Karen N. Conneely, D. Jeffrey Newport, Varun Kilaru, James W. Schroeder, Page B. Pennell, Bettina T. Knight, Joseph F. Cubells, Zachary N. Stowe and Patricia A. Brennan
Pages 458 - 463
Abstract | PDF

Antiepileptic drugs (AEDs) are used to treat a variety of neuropsychiatric illnesses commonly encountered in women during their reproductive years, including epilepsy and bipolar disorder. Despite their widespread use, the impact of prenatal exposure on fetal development remains obscure. To evaluate whether AEDs taken by pregnant mothers influence DNA methylation patterns in their neonates, DNA was extracted from the umbilical cord blood of 201 neonates whose mothers were treated for neuropsychiatric illness during pregnancy and interrogated across 27,578 CpG sites using the Illumina HumanMethylation27 BeadChip. The association of each methylation value with the cumulative duration of prenatal AED exposure was examined using a linear mixed model. The average methylation level across all CpG sites was calculated for each subject, and this global methylation measure was evaluated similarly. Neonates with a longer duration of AED exposure in pregnancy showed a decrease in average global methylation (p = 0.0045). Further, DNA methylation of CpG sites in 14 genes significantly decreased with the duration of prenatal AED exposure even after adjusting for multiple comparisons (FDR < 0.05). For a small subset (n = 19) of these neonates, a second tissue, placenta, was available, in addition to cord blood. Methylation of 3 of these 14 CpG sites was also significantly decreased in placental tissue. These novel data suggest decreased DNA methylation in neonates of mothers who took AEDs during pregnancy. The long-term stability and potential impact of these changes warrant further attention, and caution may be warranted before prescribing AEDs to pregnant women.

ABCA1 gene promoter DNA methylation is associated with HDL particle profile and coronary artery disease in familial hypercholesterolemia

Simon-Pierre Guay, Diane Brisson, Johannie Munger, Benoit Lamarche, Daniel Gaudet and Luigi Bouchard
Pages 464 - 472
Abstract | PDF

High-density lipoproteins cholesterol (HDL-C), a strong coronary artery disease (CAD) clinical biomarker, shows significant interindividual variability. However, the molecular mechanisms involved remain mostly unknown. ATP-binding cassette A1 (ABCA1) catalyzes the cholesterol transfer from peripheral cells to nascent HDL particles. Recently, a differentially methylation region was identified in ABCA1 gene promoter locus, near the first exon. Therefore, we hypothesized that DNA methylation changes at ABCA1 gene locus is one of the molecular mechanisms involved in HDL-C interindividual variability. The study was conducted in familial hypercholesterolemia (FH), a monogenic disorder associated with a high risk of CAD . Ninety-seven FH patients (all p.W66G for the LDLR gene mutation and not under lipid-lowering treatment) were recruited and finely phenotyped for DNA methylation analyses at ABCA1 gene locus. ABCA1 DNA methylation levels were found negatively correlated with circulating HDL-C (r = -0.20; p = 0.05), HDL2-phospholipid levels (r = -0.43; p = 0.04), and with a trend for association with HDL peak particle size (r = -0.38; p = 0.08). ABCA1 DNA methylation levels were also found associated with prior history of CAD (CAD = 40.2% vs. without CAD = 34.3%; p = 0.003). These results suggest that epigenetic changes within the ABCA1 gene promoter contribute to the interindividual variability in plasma HDL-C concentrations and are associated with CAD expression. These findings could change our understanding of the molecular mechanisms involved in the pathophysiological processes leading to CAD.

Use of multilocus methylation-specific single nucleotide primer extension (MS-SNuPE) technology in diagnostic testing for human imprinted loci

Matthias Begemann, Isabelle Leisten, Lukas Soellner, Klaus Zerres, Thomas Eggermann and Sabrina Spengler
Pages 473 - 481
Abstract | PDF

A number of diseases have been found to be linked to aberrant methylation of specific genes. However, most of the routine diagnostic techniques to detect epigenetic disturbances are restricted to single loci. Additionally, a precise quantification of the methylation status is often hampered. A considerable fraction of patients with Silver-Russell syndrome, Beckwith-Wiedemann syndrome and transient neonatal diabetes mellitus exhibit loss of methylation at further imprinted loci in addition to the disease specific ones (multilocus methylation defects, MLMD). As the currently available tests are mainly focused on single imprinted loci on different chromosomes and thereby make the detection of multilocus methylation defects time-consuming and expensive, we established methylation-specific single nucleotide primer extension (MS-SNuPE) assays for a simultaneous quantification of methylation at multiple methylated loci. We chose loci generally affected in patients with MLMD. The method was validated by screening 66 individuals with known (epi)genetic disturbances. In comparison to other methylation-specific techniques, multilocus methylation-specific single nucleotide primer extension allows the quantitative analysis of numerous CpG islands of different loci in one assay and is, therefore, suitable for the simultaneous diagnostic testing for different congenital imprinting disorders in parallel, as well as for MLMD.

A human Polycomb isoform lacking the Pc box does not participate to PRC1 complexes but forms protein assemblies and represses transcription

Pamela Völkel, Perrine Le Faou, Julien Vandamme, Dorcas Pira and Pierre-Olivier Angrand
Pages 482 - 491
Abstract | PDF

Polycomb repression controls the expression of hundreds of genes involved in development and is mediated by essentially two classes of chromatin-associated protein complexes. The Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27, an epigenetic mark that serves as a docking site for the PRC1 protein complex. Drosophila core PRC1 is composed of four subunits: Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph) and Sex combs extra (Sce). Each of these proteins has multiple orthologs in vertebrates, thus generating an enormous scope for potential combinatorial diversity. In particular, mammalian genomes encode five Pc family members: CBX2, CBX4, CBX6, CBX7 and CBX8. To complicate matters further, distinct isoforms might arise from single genes. Here, we address the functional role of the two human CBX2 isoforms. Owing to different polyadenylation sites and alternative splicing events, the human CBX2 locus produces two transcripts: a 5-exon transcript that encodes the 532-amino acid CBX2–1 isoform that contains the conserved chromodomain and Pc box and a 4-exon transcript encoding a shorter isoform, CBX2–2, lacking the Pc box but still possessing a chromodomain. Using biochemical approaches and a novel in vivo imaging assay, we show that the short CBX2–2 isoform lacking the Pc box, does not participate in PRC1 protein complexes, but self-associates in vivo and forms complexes of high molecular weight. Furthermore, the CBX2 short isoform is still able to repress transcription, suggesting that Polycomb repression might occur in the absence of PRC1 formation.

Histone H3K14 and H4K8 hyperacetylation is associated with Escherichia coli induced mastitis in mice

Rahul Modak, Susweta Das Mitra, P. Krishnamoorthy, Akhsay Bhat, Apala Banerjee, B.R. Gowsica, M. Bhuvana, Velu Dhanikachalam, Krithiga Natesan, Rajeswari Shome, Bibek R. Shome and Tapas K. Kundu
Pages 492 - 501
Abstract | PDF

Mastitis is a multietiological complex disease, defined as inflammation of parenchyma of mammary glands. Bacterial infection is the predominant cause of mastitis, though fungal, viral and mycoplasma infections also have been reported. Based on the severity of the disease, mastitis can be classified into subclinical, clinical and chronic forms. Bacterial pathogens from fresh cow milk were isolated and classified by standard microbiological tests and multiplex PCR. Epidemiological studies have shown that Escherichia coli is the second largest mastitis pathogen after Staphylococcus aureus in India. Based on Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR profile and presence of virulence genes, a field isolate of E. coli was used for intramammary inoculation in lactating mice. Histopathological examination of hematoxylin and eosin stained sections showed severe infiltration of polymorphonuclear neutrophils, mononuclear inflammatory cells in the alveolar lumen and also in interstitial space, and necrosis of alveolar epithelial cells after 24 h. Western blot and immunohistochemical analysis of mice mammary tissues showed significant hyperacetylation at histone H3K14 residue of both mammary epithelial cells and migrated inflammatory cells. Quantitative real-time PCR and genome-wide gene expression profile in E. coli infected mice mammary tissue revealed differential expression of genes related to inflammation, immunity, antimicrobial peptide expression, acute phase response and oxidative stress response. Expression of milk proteins was also suppressed. ChIP assay from paraffinized tissues showed selective enrichment of acetylated histone H3K14 and H4K8 at the promoters of overexpressed genes. These data suggest that E. coli infection in mice mammary tissue leads to histone hyperacetylation at the promoter of immune genes, which is a pre-requisite for the expression of inflammatory genes in order to mount a drastic immune response.

Genetic and epigenetic analysis of non-small cell lung cancer with NotI-microarrays

Alexey A. Dmitriev, Vladimir I. Kashuba, Klas Haraldson, Vera N. Senchenko, Tatiana V. Pavlova, Anna V. Kudryavtseva, Ekaterina A. Anedchenko, George S. Krasnov, Irina V. Pronina, Vitalij I. Loginov, Tatiana T. Kondratieva, Tatiana P. Kazubskaya, Eleonora A. Braga, Surya P. Yenamandra, Ilya Ignatjev, Ingemar Ernberg, George Klein, Michael I. Lerman and Eugene R. Zabarovsky
Pages 502 - 513
Abstract | PDF

This study aimed to clarify genetic and epigenetic alterations that occur during lung carcinogenesis and to design perspective sets of newly identified biomarkers. The original method includes chromosome 3 specific NotI-microarrays containing 180 NotI clones associated with genes for hybridization with 40 paired normal/tumor DNA samples of primary lung tumors: 28 squamous cell carcinomas (SCC) and 12 adenocarcinomas (ADC). The NotI-microarray data were confirmed by qPCR and bisulfite sequencing analyses. Forty-four genes showed methylation and/or deletions in more than 15% of non–small cell lung cancer (NSCLC) samples. In general, SCC samples were more frequently methylated/deleted than ADC. Moreover, the SCC alterations were observed already at stage I of tumor development, whereas in ADC many genes showed tumor progression specific methylation/deletions. Among genes frequently methylated/deleted in NSCLC, only a few were already known tumor suppressor genes: RBSP3 (CTDSPL), VHL and THRB. The RPL32, LOC285205, FGD5 and other genes were previously not shown to be involved in lung carcinogenesis. Ten methylated genes, i.e., IQSEC1, RBSP3, ITGA9, FOXP1, LRRN1, GNAI2, VHL, FGD5, ALDH1L1 and BCL6 were tested for expression by qPCR and were found downregulated in the majority of cases. Three genes (RBSP3, FBLN2 and ITGA9) demonstrated strong cell growth inhibition activity. A comprehensive statistical analysis suggested the set of 19 gene markers, ANKRD28, BHLHE40, CGGBP1, RBSP3, EPHB1, FGD5, FOXP1, GORASP1/TTC21, IQSEC1, ITGA9, LOC285375, LRRC3B, LRRN1, MITF, NKIRAS1/RPL15, TRH, UBE2E2, VHL, WNT7A, to allow early detection, tumor progression, metastases and to discriminate between SCC and ADC with sensitivity and specificity of 80–100%.

Epigenetic regulation of glucocorticoid receptor expression in aorta from mice with hyperhomocysteinemia

Dian C. Sulistyoningrum, Ranji Singh and Angela M. Devlin
Pages 514 - 521
Abstract | PDF

Hyperhomocysteinemia (HHcy) is a risk factor for vascular disease but its underlying molecular pathology is not understood. Homocysteine is metabolically linked to the epigenetic process of DNA methylation. Tissue-specific changes in DNA methylation have been observed in HHcy but little is known about vascular tissue. The objective of this study was to determine if changes in the epigenetic regulation of glucocorticoid receptor (GR) expression (encoded by Nr3c1) in aorta are associated with HHcy. C57BL/6 mice heterozygous for disruption of the cystathionine-β-synthase gene (Cbs +/-) and controls (Cbs +/+) were fed a control or high methionine/low folate (HH) diet to induce HHcy. Cbs +/- and Cbs +/+ fed the HH diet had higher plasma total homocysteine levels (19.9 ± 3.2 and 7.0 ± 0.9 μM, respectively) than Cbs +/+ mice fed the control diet (2.7 ± 0.2 μM), and this was accompanied by lower Nr3c1 mRNA and lower GR protein in aorta. The Nr3c1 gene contains multiple first exons producing heterogeneous RNA transcripts expressed in a tissue-specific manner. We identified expression of two transcripts in aorta. Bisulfite pyrosequencing found increased methylation of the promoter regions for these transcripts at sites corresponding to Sp1 and Nrf1 binding sites. Chromatin immunoprecipitation found lower binding of Nrf1 to the Nr3c1 promoter but higher expression of Nrf1 protein in aorta from mice with HHcy. These findings show methylation and silencing of vascular Nr3c1 expression and suggest a role for epigenetic regulation of gene expression in HHcy.




MEETING REPORT

Open Access Article

Keystone symposia on epigenomics and chromatin dynamics: Keystone resort, CO, January 17–22, 2012

Kim Ravnskjaer
Pages 522 - 523
Abstract | PDF

Keystone Symposia kicked off the start of 2012 with two joint meetings on Epigenomics and Chromatin Dynamics and a star-studded list of speakers. Held in Keystone, CO, January 17–22, and organized by Steven Jacobsen and Steven Henikoff and by Bradley Cairns and Geneviève Almouzni, respectively, there was plenty happening in these sessions that it did not seem to matter that the ski-slope conditions were not ideal.