Table of Contents

Volume 7, Issue 7

  July 2012

  Pages 669 - 796

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EDITOR'S PREVIEW

Open Access Article

In this issue of Epigenetics

Barbara P. Rattner
Pages 669 - 671
http://dx.doi.org/10.4161/epi.21053
Full Text | PDF




POINTS-OF-VIEW

Formation of a centromere-specific chromatin structure

Tatsuo Fukagawa
Pages 672 - 675
http://dx.doi.org/10.4161/epi.20389
Abstract | Full Text | PDF

The kinetochore is formed on centromeric DNA as a key interface with microtubules from the mitotic spindle to achieve accurate chromosome segregation during mitosis. However, in contrast to other regions of the chromosome, the position of the kinetochore is specified by sequence-independent epigenetic mechanisms. Most recent work on kinetochore specification has focused on the centromere-specific histone H3-variant CENP-A. Whereas CENP-A is an important epigenetic marker for the kinetochore specification, it is unclear how centromeric chromatin structure is organized. To understand centromeric chromatin structure, we focused on additional centromere proteins that have an intrinsic DNA binding activity and identified the DNA binding CENP-T-W-S-X complex. Tetramer formation of CENP-T-W-S-X is essential for functional kinetochore assembly in vertebrate cells. Our structural and biochemical analysis reveals that the CENP-T-W-S-X complex is composed of four histone-fold domains with structural similarity to nucleosomes and displays DNA supercoiling activity. These results suggest that the CENP-T-W-S-X complex forms a unique nucleosome-like structure at centromeric chromatin. In addition, CENP-S and CENP-X function at non-centromeric sites. The intriguing histone-like properties of these proteins suggest that they may form nucleosome-like structures at various genome loci, extending the chromatin code beyond classical histone variants.

Modulating epigenetic mechanisms: The diverse functions of Ski during cortical development

Constanze Baranek and Suzana Atanasoski
Pages 676 - 679
http://dx.doi.org/10.4161/epi.20590
Abstract | Full Text | PDF

In the developing forebrain, neural stem and progenitor cells generate a large variety of neurons with specific functions in the mature cortex. A central issue is to understand the roles of transcriptional networks and regulatory pathways that control these complex developmental processes. The proto-oncogene Ski is a transcriptional regulator linked to the human 1p36 deletion syndrome, which involves a set of phenotypes including nervous system defects. Ski shows a dynamic expression pattern during cortical development and, accordingly, the phenotype of Ski-deficient cortices is complex, involving altered cell cycle characteristics of neural progenitors, disturbed timing of neurogenesis and misspecification of projection neurons. Ski is likely to play a role in various pathways by virtue of its ability to interact with a range of signaling molecules, thereby modulating transcriptional activity of corresponding target genes. Ski regulates proliferation and differentiation of various cell types, and more recent data from my laboratory demonstrates that Ski is also involved in the specification of cortical projection neurons. This Point-of-View elucidates the role of Ski as an essential linker between sequence-specific transcription factors and non-DNA binding cofactors with chromatin modifying activities. In particular, it puts forward the hypothesis that the diverse functions of Ski as a co-repressor might be related to its association with distinct HDAC-complexes.

Global heterochromatin loss: A unifying theory of aging?

Amy Tsurumi and Willis Li
Pages 680 - 688
http://dx.doi.org/10.4161/epi.20540
Abstract | Full Text | PDF

The aging field is replete with theories. Over the past years, many distinct, yet overlapping mechanisms have been proposed to explain organismal aging. These include free radicals, loss of heterochromatin, genetically programmed senescence, telomere shortening, genomic instability, nutritional intake and growth signaling, to name a few. The objective of this Point-of-View is to highlight recent progress on the “loss of heterochromatin” model of aging and to propose that epigenetic changes contributing to global heterochromatin loss may underlie the various cellular processes associated with aging.




BRIEF REPORT

Supra-physiological folic acid concentrations induce aberrant DNA methylation in normal human cells in vitro

Michelle A. Charles, Ian T. Johnson and Nigel J. Belshaw
Pages 689 - 694
http://dx.doi.org/10.4161/epi.20461
Abstract | Full Text | PDF

The micronutrients folate and selenium may modulate DNA methylation patterns by affecting intracellular levels of the methyl donor S-adenosylmethionine (SAM) and/or the product of methylation reactions S-adenosylhomocysteine (SAH). WI-38 fibroblasts and FHC colon epithelial cells were cultured in the presence of two forms of folate or four forms of selenium at physiologically-relevant doses, and their effects on LINE-1 methylation, gene-specific CpG island (CGI) methylation and intracellular SAM:SAH were determined. At physiologically-relevant doses the forms of folate or selenium had no effect on LINE-1 or CGI methylation, nor on intracellular SAM:SAH. However the commercial cell culture media used for the selenium studies, containing supra-physiological concentrations of folic acid, induced LINE-1 hypomethylation, CGI hypermethylation and decreased intracellular SAM:SAH in both cell lines. We conclude that the exposure of normal human cells to supra-physiological folic acid concentrations present in commercial cell culture media perturbs the intracellular SAM:SAH ratio and induces aberrant DNA methylation.




RESEARCH PAPERS

Open Access Article

SIRT1-mediated deacetylation of MeCP2 contributes to BDNF expression

Loredana Zocchi and Paolo Sassone-Corsi
Pages 695 - 700
http://dx.doi.org/10.4161/epi.20733
Abstract | Full Text | PDF

Methyl-CpG binding protein 2 (MeCP2) binds methylated cytosines at CpG sites on DNA and it is thought to function as a critical epigenetic regulator. Mutations in the MeCP2 gene have been associated to Rett syndrome, a human neurodevelopmental disorder. Here we show that MeCP2 is acetylated by p300 and that SIRT1 mediates its deacetylation. SIRT1, the mammalian homologue of Sir2 in yeast, is a nicotinamide-adenine dinucleotide (NAD⁺)-dependent histone deacetylase that belongs to the family of HDAC class III sirtuins. Importantly, SIRT1 has been shown to play a critical role in synaptic plasticity and memory formation. This study reveals a functional interplay between two critical epigenetic regulators, MeCP2 and SIRT1, which controls MeCP2 binding activity to the brain-derived neurotrophic factor (BDNF) promoter in a specific region of the brain.

Biomarkers for detection and prognosis of breast cancer identified by a functional hypermethylome screen

Jana Jeschke, Leander Van Neste, Sabine C. Glöckner, Mashaal Dhir, Marilia Freitas Calmon, Valérie Deregowski, Wim Van Criekinge, Ilse Vlassenbroeck, Alexander Koch, Timothy A. Chan, Leslie Cope, Craig M. Hooker, Kornel E. Schuebel, Edward Gabrielson, Andreas Winterpacht, Stephen B. Baylin, James G. Herman and Nita Ahuja
Pages 701 - 709
http://dx.doi.org/10.4161/epi.20445
Abstract | Full Text | PDF

Breast cancer (BC) is a disease with diverse tumor heterogeneity, which challenges conventional approaches to develop biomarkers for early detection and prognosis. To identify effective biomarkers, we performed a genome-wide screen for functional methylation changes in BC, i.e., genes silenced by promoter hypermethylation, using a functionally proven gene expression approach. A subset of candidate hypermethylated genes were validated in primary BCs and tested as markers for detection and prognosis prediction of BC. We identified 33 cancer specific methylated genes and, among these, two categories of genes: (1) highly frequent methylated genes that detect early stages of BC. Within that category, we have identified the combination of NDRG2 and HOXD1 as the most sensitive (94%) and specific (90%) gene combination for detection of BC; (2) genes that show stage dependent methylation frequency pattern, which are candidates to help delineate BC prognostic signatures. For this category, we found that methylation of CDO1, CKM, CRIP1, KL and TAC1 correlated with clinical prognostic variables and was a significant prognosticator for poor overall survival in BC patients. CKM [Hazard ratio (HR) = 2.68] and TAC1 (HR = 7.73) were the strongest single markers and the combination of both (TAC1 and CKM) was associated with poor overall survival independent of age and stage in our training (HR = 1.92) and validation cohort (HR = 2.87). Our study demonstrates an efficient method to utilize functional methylation changes in BC for the development of effective biomarkers for detection and prognosis prediction of BC.

Correlation between BRAF mutation and promoter methylation of TIMP3, RARβ2 and RASSF1A in thyroid cancer

Mariana Brait, Myriam Loyo, Eli Rosenbaum, Kimberly L. Ostrow, Alina Markova, Silvana Papagerakis, Marianna Zahurak, Steven M. Goodman, Martha Zeiger, David Sidransky, Christopher B. Umbricht and Mohammad O. Hoque
Pages 710 - 719
http://dx.doi.org/10.4161/epi.20524
Abstract | Full Text | PDF

Our aim was to comprehensively analyze promoter hypermethylation of a panel of novel and known methylation markers for thyroid neoplasms and to establish their relationship with BRAF mutation and clinicopathologic parameters of thyroid cancer. A cohort of thyroid tumors, consisting of 44 cancers and 44 benign thyroid lesions, as well as 15 samples of adjacent normal thyroid tissue, was evaluated for BRAF mutation and promoter hypermethylation. Genes for quantitative methylation specific PCR (QMSP) were selected by a candidate gene approach. Twenty-two genes were tested: TSHR, RASSF1A, RARβ2, DAPK, hMLH1, ATM, S100, p16, CTNNB1, GSTP1, CALCA, TIMP3, TGFßR2, THBS1, MINT1, CTNNB1, MT1G, PAK3, NISCH, DCC, AIM1 and KIF1A. The PCR-based “mutector assay” was used to detect BRAF mutation. All p values reported are two sided. Considerable overlap was seen in the methylation markers among the different tissue groups. Significantly higher methylation frequency and level were observed for KIF1A and RARß2 in cancer samples compared with benign tumors. A negative correlation between BRAF mutation and RASSF1A methylation, and a positive correlation with RARß2 methylation were observed in accordance with previous results. In addition, positive correlation with TIMP3 and a marginal correlation with DCC methylation were observed. The present study constitutes a comprehensive promoter methylation profile of thyroid neoplasia and shows that results must be analyzed in a tissue-specific manner to identify clinically useful methylation markers. Integration of genetic and epigenetic changes in thyroid cancer will help identify relevant biologic pathways that drive its development.

Association of cigarette smoking and CRP levels with DNA methylation in α-1 antitrypsin deficiency

Mateusz Siedlinski, Barbara Klanderman, Robert A. Sandhaus, Alan F. Barker, Mark L. Brantly, Edward Eden, N. Gerard McElvaney, Stephen I. Rennard, James M. Stocks, James K. Stoller, Charlie Strange, Gerard M. Turino, Edward J. Campbell and Dawn L. DeMeo
Pages 720 - 728
http://dx.doi.org/10.4161/epi.20319
Abstract | Full Text | PDF

Alpha-1 antitrypsin (AAT) deficiency and tobacco smoking are confirmed risk factors for Chronic Obstructive Pulmonary Disease. We hypothesized that variable DNA methylation would be associated with smoking and inflammation, as reflected by the level of C-Reactive Protein (CRP) in AAT-deficient subjects. Methylation levels of 1,411 autosomal CpG sites from the Illumina GoldenGate Methylation Cancer Panel I were analyzed in 316 subjects. Associations of five smoking behaviors and CRP levels with individual CpG sites and average methylation levels were assessed using non-parametric testing, linear regression and linear mixed effect models, with and without adjustment for age and gender. Univariate linear regression analysis revealed that methylation levels of 16 CpG sites significantly associated with ever-smoking status. A CpG site in the TGFBI gene was the only site associated with ever-smoking after adjustment for age and gender. No highly significant associations existed between age at smoking initiation, pack-years smoked, duration of smoking, and time since quitting smoking as predictors of individual CpG site methylation levels. However, ever-smoking and younger age at smoking initiation associated with lower methylation level averaged across all sites. DNA methylation at CpG sites in the RUNX3, JAK3 and KRT1 genes associated with CRP levels. The most significantly associated CpG sites with gender and age mapped to the CASP6 and FZD9 genes, respectively. In summary, this study identified multiple potential candidate CpG sites associated with ever-smoking and CRP level in AAT-deficient subjects. Phenotypic variability in Mendelian diseases may be due to epigenetic factors.

Normal early pregnancy: A transient state of epigenetic change favoring hypomethylation

Wendy M. White, Brian C. Brost, Zhifu Sun, Carl Rose, Iasmina Craici, Steven J. Wagner, Stephen Turner and Vesna D. Garovic
Pages 729 - 734
http://dx.doi.org/10.4161/epi.20388
Abstract | Full Text | PDF

The objective of this study was to analyze genome-wide differential methylation patterns in maternal leukocyte DNA in early pregnant and non-pregnant states. This is an age and body mass index matched case-control study comparing the methylation patterns of 27,578 cytosine-guanine (CpG) sites in 14,495 genes in maternal leukocyte DNA in early pregnancy (n = 14), in the same women postpartum (n = 14), and in nulligravid women (n = 14) on a BeadChip platform. Transient widespread hypomethylation was found in early pregnancy as compared with the non-pregnant states. Methylation of nine genes was significantly different in early pregnancy compared with both postpartum and nulligravid states (< 10% False Discovery Rate). Early pregnancy may be characterized by widespread hypomethylation compared with non-pregnant states; there is no apparent permanent methylation imprint after a normal term gestation. Nine potential candidate genes were identified as differentially methylated in early pregnancy and may play a role in the maternal adaptation to pregnancy.

Open Access Article

Depression in pregnancy, infant birth weight and DNA methylation of imprint regulatory elements

Ying Liu, Susan K. Murphy, Amy P. Murtha, Bernard F. Fuemmeler, Joellen Schildkraut, Zhiqing Huang, Francine Overcash, Joanne Kurtzberg, Randy Jirtle, Edwin S. Iversen, Michele R. Forman and Cathrine Hoyo
Pages 735 - 746
http://dx.doi.org/10.4161/epi.20734
Abstract | Full Text | PDF

Depressed mood in pregnancy has been linked to low birth weight (LBW, < 2,500 g), a risk factor for adult-onset chronic diseases in offspring. We examined maternal depressed mood in relation to birth weight and evaluated the role of DNA methylation at regulatory sequences of imprinted genes in this association. We measured depressed mood among 922 pregnant women using the CES-D scale and obtained birth weight data from hospital records. Using bisulfite pyrosequencing of cord blood DNA from 508 infants, we measured methylation at differentially methylated regions (DMRs) regulating imprinted genes IGF2/H19, DLK1/MEG3, MEST, PEG3, PEG10/SGCE, NNAT and PLAGL1. Multiple regression models were used to examine the relationship between depressed mood, birth weight and DMR methylation levels. Depressed mood was associated with a more that 3-fold higher risk of LBW, after adjusting for delivery mode, parity, education, cigarette smoking, folic acid use and preterm birth. The association may be more pronounced in offspring of black women and female infants. Compared with infants of women without depressed mood, infants born to women with severe depressed mood had a 2.4% higher methylation at the MEG3 DMR. Whereas LBW infants had 1.6% lower methylation at the IGF2 DMR, high birth weight (> 4,500 g) infants had 5.9% higher methylation at the PLAGL1 DMR compared with normal birth weight infants. Our findings confirm that severe maternal depressed mood in pregnancy is associated with LBW, and that MEG3 and IGF2 plasticity may play important roles.

Open Access Article

Analysis of active chromatin modifications in early mammalian embryos reveals uncoupling of H2A.Z acetylation and H3K36 trimethylation from embryonic genome activation

Ana Bošković, Ambre Bender, Laurence Gall, Celine Ziegler-Birling, Nathalie Beaujean and Maria-Elena Torres-Padilla
Pages 747 - 757
http://dx.doi.org/10.4161/epi.20584
Abstract | Full Text | PDF

Early embryonic development is characterized by dramatic changes in cell potency and chromatin organization. The role of histone variants in the context of chromatin remodeling during embryogenesis remains under investigated. In particular, the nuclear distribution of the histone variant H2A.Z and its modifications have not been examined. Here we investigated the dynamics of acetylation of H2A.Z and two other active chromatin marks, H3K9ac and H3K36me3, throughout murine and bovine pre-implantation development. We show that H2A.Z distribution is dynamic during the earliest stages of mouse development, with protein levels significantly varying across stages and lowest at the 2-cell stage. When present, H2A.Z localizes preferentially to euchromatin at all stages analyzed. H2A.Z is acetylated in pre-implantation blastomeres and is preferentially localized to euchromatin, in line with the known role of H2A.Zac in transcriptional activation. Interestingly, however, H2A.Zac is undetectable in mouse embryos at the 2-cell stage, the time of major embryonic genome activation (EGA). Similarly, H3K36me3 is present exclusively in the maternal chromatin immediately after fertilization but becomes undetectable in interphase nuclei at the 2-cell stage, suggesting uncoupling of these active marks with global embryonic transcription activation. In bovine embryos, which undergo EGA at the 8-cell stage, H2A.Zac can be detected in zygotes, 4-, 8- and 16-cell stage embryos as well as in blastocysts, indicating that the dynamics of H2A.Zac is not conserved in mammals. In contrast, H3K36me3 displays mostly undetectable and heterogeneous localization pattern throughout bovine pre-implantation development. Thus, our results suggest that 'canonical' active chromatin marks exhibit a dynamic behavior in embryonic nuclei, which is both stage- and species-specific. We hypothesize that chromatin of early embryonic nuclei is subject to fine-tuning through differential acquisition of histone marks, allowing for proper chromatin remodeling and developmental progression in a species-specific fashion.

Naturally occurring endo-siRNA silences LINE-1 retrotransposons in human cells through DNA methylation

Long Chen, Jane E. Dahlstrom, Sung-Hun Lee and Danny Rangasamy
Pages 758 - 771
http://dx.doi.org/10.4161/epi.20706
Abstract | Full Text | PDF

Long interspersed nuclear element 1 (LINE-1) retrotransposons are mutagens that are capable of generating deleterious mutations by inserting themselves into genes and affecting gene function in the human genome. In normal cells, the activity of LINE-1 retrotransposon is mostly repressed, maintaining a stable genome structure. In contrast, cancer cells are characterized by aberrant expression of LINE-1 retrotransposons, which, in principle, have the potential to contribute to genomic instability. The mechanistic pathways that regulate LINE-1 expression remain unclear. Using deep-sequencing small RNA analysis, we identified a subset of differentially expressed endo-siRNAs that directly regulate LINE-1 expression. Detailed analyses suggest that these endo-siRNAs are significantly depleted in human breast cancer cells compared with normal breast cells. The overexpression of these endo-siRNAs in cancer cells markedly silences endogenous LINE-1 expression through increased DNA methylation of the LINE-1 5′-UTR promoter. The finding that endo-siRNAs can silence LINE-1 activity through DNA methylation suggests that a functional link exists between the expression of endo-siRNAs and LINE-1 retrotransposons in human cells.

A systematic comparison of quantitative high-resolution DNA methylation analysis and methylation-specific PCR

Rainer Claus, Stefan Wilop, Thomas Hielscher, Miriam Sonnet, Edgar Dahl, Oliver Galm, Edgar Jost and Christoph Plass
Pages 772 - 780
http://dx.doi.org/10.4161/epi.20299
Abstract | Full Text | PDF

Assessment of DNA methylation has become a critical factor for the identification, development and application of methylation based biomarkers. Here we describe a systematic comparison of a quantitative high-resolution mass spectrometry-based approach (MassARRAY), pyrosequencing and the broadly used methylation-specific PCR (MSP) technique analyzing clinically relevant epigenetically silenced genes in acute myeloid leukemia (AML). By MassARRAY and pyrosequencing, we identified significant DNA methylation differences at the ID4 gene promoter and in the 5′ region of members of the SFRP gene family in 62 AML patients compared with healthy controls. We found a good correlation between data obtained by MassARRAY and pyrosequencing (correlation coefficient R² = 0.88). MSP-based assessment of the identical samples showed less pronounced differences between AML patients and controls. By direct comparison of MSP-derived and MassARRAY-based methylation data as well as pyrosequencing, we could determine overestimation of DNA methylation data by MSP. We found sequence-context dependent highly variable cut-off values of quantitative DNA methylation values serving as discriminator for the two MSP methylation categories. Moreover, good agreements between quantitative methods and MSP could not be achieved for all investigated loci. Significant correlation of the quantitative assessment but not of MSP-derived methylation data with clinically important characteristics in our patient cohort demonstrated clinical relevance of quantitative DNA methylation assessment. Taken together, while MSP is still the most commonly applied technique for DNA methylation assessment, our data highlight advantages of quantitative approaches for precise characterization and reliable biomarker use of aberrant DNA methylation in primary patient samples, particularly.

Open Access Article

RNA polymerase V-dependent small RNAs in Arabidopsis originate from small, intergenic loci including most SINE repeats

Tzuu-fen Lee, Sai Guna Ranjan Gurazada, Jixian Zhai, Shengben Li, Stacey A. Simon, Marjori A. Matzke, Xuemei Chen and Blake C. Meyers
Pages 781 - 795
http://dx.doi.org/10.4161/epi.20290
Abstract | Full Text | PDF

In plants, heterochromatin is maintained by a small RNA-based gene silencing mechanism known as RNA-directed DNA methylation (RdDM). RdDM requires the non-redundant functions of two plant-specific DNA-dependent RNA polymerases (RNAP), RNAP IV and RNAP V. RNAP IV plays a major role in siRNA biogenesis, while RNAP V may recruit DNA methylation machinery to target endogenous loci for silencing. Although small RNA-generating regions that are dependent on both RNAP IV and RNAP V have been identified previously, the genomic loci targeted by RNAP V for siRNA accumulation and silencing have not been described extensively. To characterize the RNAP V-dependent, heterochromatic siRNA-generating regions in the Arabidopsis genome, we deeply sequenced the small RNA populations of wild-type and RNAP V null mutant (nrpe1) plants. Our results showed that RNAP V-dependent siRNA-generating loci are associated predominately with short repetitive sequences in intergenic regions. Suppression of small RNA production from short repetitive sequences was also prominent in RdDM mutants including dms4, drd1, dms3 and rdm1, reflecting the known association of these RdDM effectors with RNAP V. The genomic regions targeted by RNAP V were small, with an estimated average length of 238 bp. Our results suggest that RNAP V affects siRNA production from genomic loci with features dissimilar to known RNAP IV-dependent loci. RNAP V, along with RNAP IV and DRM1/2, may target and silence a set of small, intergenic transposable elements located in dispersed genomic regions for silencing. Silencing at these loci may be actively reinforced by RdDM.




LETTER TO THE EDITOR

Response to “Different measures of ‘genome-wide’ DNA methylation exhibit unique properties in placental and somatic tissues”

Weiwei Li
Page 796
http://dx.doi.org/10.4161/epi.21099
Abstract | PDF

I found the research paper “Different measures of ‘genome-wide’ DNA methylation exhibit unique properties in placental and somatic tissues” by Price ME and colleagues in the June 2012 issue of Epigenetics to be an interesting read, but it contains errors in regards to the use of the MethylFlash Methylated DNA Quantification Kit. The text contained in the “Global DNA Methylation” paragraph under the “Methods” section of the paper claims that the kit was used by “following the manufacturer’s protocol.” I find that this is quite misleading to the reader as we have identified, based on the original electronic publication ahead of print, that the steps had not been correctly carried out.

The original text was the following: “DNA was hybridized to wells treated to have a high affinity for DNA. The wells were washed with a 5-mC-specific capture antibody followed by a detection antibody. The absorbance (or optical density) of each well was measured using a microplate spectrophotometer. Correlation of technical replicates was poor (r = 0.09, p = 0.72) using this kit.” However, the user guide of this kit clearly states that DNA is bound, not hybridized, to the strip wells and that the wells should be washed with the included Wash Buffer rather than a “5-mC-specific capture antibody.”

Therefore, it is not surprising that the result or correlation of replicates was poor and that the authors’ “results were variable” due to improper use of the kit. Based on our quality control tests and feedback from the vast amount of users of this popular kit, the variation between replicates should be less than 10% with R value > 0.9 (p < 0.01), assuming proper user performance according to the product manual.

I hope this helps to correct the misinformation presented in the paper as I feel that it is important to promote accuracy on behalf of the epigenetic research community. I also kindly encourage any users of an Epigentek product to work with our very knowledgeable technical support team should they have any difficulties.