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Epigenetics & Chromatin 2015, 8:3  doi:10.1186/1756-8935-8-3

The electronic version of this article is the complete one and can be found online at: http://www.epigeneticsandchromatin.com/content/8/1/3


Constitutive heterochromatin formation and transcription in mammals

Nehmé Saksouk, Elisabeth Simboeck and Jérôme Déjardin*


Constitutive heterochromatin, mainly formed at the gene-poor regions of pericentromeres, is believed to ensure a condensed and transcriptionally inert chromatin conformation. Pericentromeres consist of repetitive tandem satellite repeats and are crucial chromosomal elements that are responsible for accurate chromosome segregation in mitosis. The repeat sequences are not conserved and can greatly vary between different organisms, suggesting that pericentromeric functions might be controlled epigenetically. In this review, we will discuss how constitutive heterochromatin is formed and maintained at pericentromeres in order to ensure their integrity. We will describe the biogenesis and the function of main epigenetic pathways that are involved and how they are interconnected. Interestingly, recent findings suggest that alternative pathways could substitute for well-established pathways when disrupted, suggesting that constitutive heterochromatin harbors much more plasticity than previously assumed. In addition, despite of the heterochromatic nature of pericentromeres, there is increasing evidence for active and regulated transcription at these loci, in a multitude of organisms and under various biological contexts. Thus, in the second part of this review, we will address this relatively new aspect and discuss putative functions of pericentromeric expression.


epigenetic factors; heterochromatin; histone modifying enzymes; pericentromere; transcription

2013 Jun;10(6):915-8. doi: 10.4161/rna.24711. Epub 2013 Apr 17.

Sprouts of RNA epigenetics: the discovery of mammalian RNA demethylases.

Zheng G1, Dahl JA, Niu Y, Fu Y, Klungland A, Yang YG, He C.


More than 100 structurally distinct RNA modifications have been identified in all kingdoms of life. These post-transcriptional modifications are widely present in various RNAs, including ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA), long non-coding RNA (lncRNA), etc. We have shown that the methylation of N(6)-methyladenine (m(6)A) can be reversed through the discovery of the first RNA demethylase, the human fat mass and obesity-associated protein, FTO, in 2011. (Most recently, we have identified a new mammalian RNA demethylase, ALKBH5, which is also able to remove the methyl group of m(6)A from RNA both in vitro and in vivo (Fig. 1A). The ALKBH5 protein colocalizes with nuclear speckles where pre-mRNA processing occurs. This protein is actively involved in mRNA export regulation, in which its demethylation activity seems to play an important role, as well as in RNA synthesis. A knockout of the Alkbh5 gene in mice resulted in impaired male fertility due to compromised spermatogenesis. Importantly, increased m(6)A levels were observed in mRNA isolated from the Alkbh5-knockout mouse organs compared to those from wild-type littermates. RNA-Seq results indicate aberrant gene expression in spermatogenic cells of the seminoferous tubulus of testes from Alkbh5-deficient mice, thereby showing that the loss of the m(6)A demethylase influences gene expression, which, in turn, leads to defects in spermatogenesis and increased apoptosis of meiotic cells. Thus, the discovery of FTO and this new RNA demethylase strongly suggests that the methylation of RNA, like DNA and histone modifications, is dynamically regulated and likely to play broad roles in mammalian cells.


N6- methyladenine; RNA demethylase; RNA epigenetics; reversible RNA methylation


・Stimulus-triggered fate conversion of somatic cells into pluripotency

Haruko Obokata1,2,3, TeruhikoWakayama3{, Yoshiki Sasai4, Koji Kojima1, Martin P. Vacanti1,5, Hitoshi Niwa6, Masayuki Yamato7
& Charles A. Vacanti1

Here we report a unique cellular reprogramming phenomenon, called stimulus-triggered acquisition of pluripotency
(STAP), which requires neither nuclear transfer nor the introduction of transcription factors. In STAP, strong external
stimuli such as a transient low-pH stressor reprogrammed mammalian somatic cells, resulting in the generation of pluripotent
cells. Through real-time imaging of STAP cells derived from purified lymphocytes, as well as gene rearrangement
analysis, we found that committed somatic cells give rise to STAP cells by reprogramming rather than selection.
STAP cells showed a substantial decrease in DNA methylation in the regulatory regions of pluripotency marker genes.
Blastocyst injection showed that STAP cells efficiently contribute to chimaeric embryos and to offspring via germline
transmission.Wealso demonstrate the derivation of robustly expandable pluripotent cell lines fromSTAP cells. Thus, our
findings indicate thatepigenetic fatedeterminationofmammaliancells canbemarkedly convertedin a context-dependent
manner by strong environmental cues.

Dr. Robert Waterland discusses how 3′ CpG island methylation may function as a key developmental activator of gene expression. This interview was shot at the Keystone Symposia’s meeting on Nutrition, Epigenetics and Human Disease, 2013 held in Santa Fe, New Mexico

・3′ CpG Island Methylation in Gene Activation

Another really interesting project thatI’ve been involved with is in a collaboration with Lanlan Shen at Baylor College of Medicine. And we’ve been working on addressing some fundamental questions of the role of DNA methylation during development. And in this project, that was done mostly in her lab.

We did in vitro induced differentiation. So in human embryonic stem cells, we differentiated these? random differentiation in vitro and looked for methylation changes. And so we did a genome-wide screen looking for CpG islands that change methylation.

And we identified, of course, several 5-prime CpG islands that were changing methylation, but a large group of 3-primeCpG islands that underwent methylation? increases in methylation? that coincided with differentiation. And likewise, in induced dedifferentiation, the methylation status of these regions was decreased. And in each case, the methylation at these 3′ regions actually correlated with increased expression of these genes.(from epigeie http://epigenie.com/category/headlines/)

Epigenomics October 2013, Vol. 5, No. 5, Pages 487-500 , DOI 10.2217/epi.13.49

Research Article

・Early-life lead exposure results in dose- and sex-specific effects on weight and epigenetic gene regulation in weanling mice

Christopher Faulk??1, Amanda Barks??1, Kevin Liu??1, Jaclyn M Goodrich??1 & Dana C Dolinoy??*1
*?Author for correspondence

Aims: Epidemiological and animal data suggest that the development of adult chronic conditions is influenced by early-life exposure-induced changes to the epigenome. This study investigates the effects of perinatal lead (Pb) exposure on DNA methylation and bodyweight in weanling mice. Materials & methods: Viable yellow agouti (Avy) mouse dams were exposed to 0, 2.1, 16 and 32 ppm Pb acetate before conception through weaning. Epigenetic effects were evaluated by scoring coat color of Avy/a offspring and quantitative bisulfite sequencing of two retrotransposon-driven (Avy and CDK5 activator-binding protein intracisternal A particle element) and two imprinted (Igf2 and Igf2r) loci in tail DNA. Results: Maternal blood Pb levels were below the limit of detection in controls, and 4.1, 25.1 and 32.1 ?g/dl for each dose, respectively. Pb exposure was associated with a trend of increased wean bodyweight in males (p = 0.03) and altered coat color in Avy/a offspring. DNA methylation at Avy and the CDK5 activator-binding protein intracisternal A-particle element was significantly different from controls following a cubic trend (p = 0.04; p = 0.01), with male-specific effects at the Avy locus. Imprinted genes did not shift in methylation across exposures. Conclusion: Dose- and sex-specific responses in bodyweight and DNA methylation indicate that Pb acts on the epigenome in a locus-specific fashion, dependent on the genomic feature hosting the CpG site of interest, and that sex is a factor in epigenetic response.

Science 2013 Sep 6;341(6150):1106-9. doi: 10.1126/science.1239864.

Epigenetic regulation of mouse sex determination by the histone demethylase Jmjd1a.

Kuroki S, Matoba S, Akiyoshi M, Matsumura Y, Miyachi H, Mise N, Abe K, Ogura A, Wilhelm D, Koopman P, Nozaki M, Kanai Y, Shinkai Y, Tachibana M.


Experimental Research Center for Infectious Diseases, Institute for Virus Research, Kyoto University, 53 Shogoin, Kawara-cho, Sakyo-ku, Kyoto, Japan.


Developmental gene expression is defined through cross-talk between the function of transcription factors and epigenetic status, including histone modification. Although several transcription factors play crucial roles in mammalian sex determination, how epigenetic regulation contributes to this process remains unknown. We observed male-to-female sex reversal in mice lacking the H3K9 demethylase Jmjd1a and found that Jmjd1a regulates expression of the mammalian Y chromosome sex-determining gene Sry. Jmjd1a directly and positively controls Sry expression by regulating H3K9me2 marks. These studies reveal a pivotal role of histone demethylation in mammalian sex determination.

Nature 2013 Sep 8. doi: 10.1038/nature12488. [Epub ahead of print]

Uhrf1-dependent H3K23 ubiquitylation couples maintenance DNA methylation and replication.

Nishiyama A, Yamaguchi L, Sharif J, Johmura Y, Kawamura T, Nakanishi K, Shimamura S, Arita K, Kodama T, Ishikawa F, Koseki H, Nakanishi M.


Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan.


Faithful propagation of DNA methylation patterns during DNA replication is critical for maintaining cellular phenotypes of individual differentiated cells. Although it is well established that Uhrf1 (ubiquitin-like with PHD and ring finger domains 1; also known as Np95 and ICBP90) specifically binds to hemi-methylated DNA through its SRA (SET and RING finger associated) domain and has an essential role in maintenance of DNA methylation by recruiting Dnmt1 to hemi-methylated DNA sites, the mechanism by which Uhrf1 coordinates the maintenance of DNA methylation and DNA replication is largely unknown. Here we show that Uhrf1-dependent histone H3 ubiquitylation has a prerequisite role in the maintenance DNA methylation. Using Xenopus egg extracts, we successfully reproduce maintenance DNA methylation in vitro. Dnmt1 depletion results in a marked accumulation of Uhrf1-dependent ubiquitylation of histone H3 at lysine 23. Dnmt1 preferentially associates with ubiquitylated H3 in vitro though a region previously identified as a replication foci targeting sequence. The RING finger mutant of Uhrf1 fails to recruit Dnmt1 to DNA replication sites and maintain DNA methylation in mammalian cultured cells. Our findings represent the first evidence, to our knowledge, of the mechanistic link between DNA methylation and DNA replication through histone H3 ubiquitylation.

Epigenetics & Chromatin 2013, 6:26

・Identification and systematic annotation of tissue-specific differentially methylated regions using the Illumina 450k array

Roderick C Slieker1, Steffan D Bos12, Jelle J Goeman3, Judith VMG Bov?e4, Rudolf P Talens1, Ruud van der Breggen1, H Eka D Suchiman1, Eric-Wubbo Lameijer1, Hein Putter3, Erik B van den Akker15, Yanju Zhang1, J Wouter Jukema6, P Eline Slagboom12, Ingrid Meulenbelt12 and Bastiaan T Heijmans12*



DNA methylation has been recognized as a key mechanism in cell differentiation. Various studies have compared tissues to characterize epigenetically regulated genomic regions, but due to differences in study design and focus there still is no consensus as to the annotation of genomic regions predominantly involved in tissue-specific methylation. We used a new algorithm to identify and annotate tissue-specific differentially methylated regions (tDMRs) from Illumina 450k chip data for four peripheral tissues (blood, saliva, buccal swabs and hair follicles) and six internal tissues (liver, muscle, pancreas, subcutaneous fat, omentum and spleen with matched blood samples).


The majority of tDMRs, in both relative and absolute terms, occurred in CpG-poor regions. Further analysis revealed that these regions were associated with alternative transcription events (alternative first exons, mutually exclusive exons and cassette exons). Only a minority of tDMRs mapped to gene-body CpG islands (13%) or CpG islands shores (25%) suggesting a less prominent role for these regions than indicated previously. Implementation of ENCODE annotations showed enrichment of tDMRs in DNase hypersensitive sites and transcription factor binding sites. Despite the predominance of tissue differences, inter-individual differences in DNA methylation in internal tissues were correlated with those for blood for a subset of CpG sites in a locus- and tissue-specific manner.


We conclude that tDMRs preferentially occur in CpG-poor regions and are associated with alternative transcription. Furthermore, our data suggest the utility of creating an atlas cataloguing variably methylated regions in internal tissues that correlate to DNA methylation measured in easy accessible peripheral tissues.


Differentially methylated region; Illumina 450k; Annotation; Algorithm; Tissue

Epigenetics & Chromatin 2013, 6:24

・Proteomic characterization of novel histone post-translational modifications

Anna M Arnaudo12 and Benjamin A Garcia1*


Histone post-translational modifications (PTMs) have been linked to a variety of biological processes and disease states, thus making their characterization a critical field of study. In the last 5 years, a number of novel sites and types of modifications have been discovered, greatly expanding the histone code. Mass spectrometric methods are essential for finding and validating histone PTMs. Additionally, novel proteomic, genomic and chemical biology tools have been developed to probe PTM function. In this snapshot review, proteomic tools for PTM identification and characterization will be discussed and an overview of PTMs found in the last 5 years will be provided.

Keywords: Histone post-translational modifications; Mass spectrometry; Proteomics; Epigenetics

Nature 2013 Jun 30. doi: 10.1038/nature12362. [Epub ahead of print]

Vitamin-C induces Tet-dependent DNA demethylation and a blastocyst-like state in ES cells.

Blaschke K, Ebata KT, Karimi MM, Zepeda-Mart?nez JA, Goyal P, Mahapatra S, Tam A, Laird DJ, Hirst M, Rao A, Lorincz MC, Ramalho-Santos M.


1] Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Department of Obstetrics and Gynecology and Center for Reproductive Sciences, University of California San Francisco, 35 Medical Center Way, San Francisco, California 94143, USA [2].


DNA methylation is a heritable epigenetic modification involved in gene silencing, imprinting, and the suppression of retrotransposons. Global DNA demethylation occurs in the early embryo and the germ line, and may be mediated by Tet (ten?eleven?translocation) enzymes, which convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Tet enzymes have been studied extensively in mouse embryonic stem (ES) cells, which are generally cultured in the absence of vitamin?C, a potential cofactor for Fe(ii) 2-oxoglutarate dioxygenase enzymes such as Tet enzymes. Here we report that addition of vitamin?C to mouse ES cells promotes Tet activity, leading to a rapid and global increase in 5hmC. This is followed by DNA demethylation of many gene promoters and upregulation of demethylated germline genes. Tet1 binding is enriched near the transcription start site of genes affected by vitamin?C treatment. Importantly, vitamin?C, but not other antioxidants, enhances the activity of recombinant Tet1 in a biochemical assay, and the vitamin-C-induced changes in 5hmC and 5mC are entirely suppressed in Tet1 and Tet2 double knockout ES cells. Vitamin?C has a stronger effect on regions that gain methylation in cultured ES cells compared to blastocysts, and in vivo are methylated only after implantation. In contrast, imprinted regions and intracisternal A particle retroelements, which are resistant to demethylation in the early embryo, are resistant to vitamin-C-induced DNA demethylation. Collectively, the results of this study establish vitamin?C as a direct regulator of Tet activity and DNA methylation fidelity in ES cells.

Epigenetics & Chromatin 2013, 6:20

A quantitative atlas of histone modification
signatures from human cancer cells
Gary LeRoy1, Peter A DiMaggio2, Eric Y Chan3, Barry M Zee1, M Andres Blanco1, Barbara Bryant3, Ian Z Flaniken1,
Sherry Liu4,5, Yibin Kang1, Patrick Trojer3 and Benjamin A Garcia4,5*

Background: An integral component of cancer biology is the understanding of molecular properties uniquely
distinguishing one cancer type from another. One class of such properties is histone post-translational modifications
(PTMs). Many histone PTMs are linked to the same diverse nuclear functions implicated in cancer development,
including transcriptional activation and epigenetic regulation, which are often indirectly assayed with standard
genomic technologies. Thus, there is a need for a comprehensive and quantitative profiling of cancer lines focused
on their chromatin modification states.
Results: To complement genomic expression profiles of cancer lines, we report the proteomic classification of 24
different lines, the majority of which are cancer cells, by quantifying the abundances of a large panel of single and
combinatorial histone H3 and H4 PTMs, and histone variants. Concurrent to the proteomic analysis, we performed
transcriptomic analysis on histone modifying enzyme abundances as a proxy for quantifying their activity levels.
While the transcriptomic and proteomic results were generally consistent in terms of predicting histone PTM
abundance from enzyme abundances, several PTMs were regulated independently of the modifying enzyme
expression. In addition, combinatorial PTMs containing H3K27 methylation were especially enriched in breast cell
lines. Knockdown of the predominant H3K27 methyltransferase, enhancer of zeste 2 (EZH2), in a mouse mammary
xenograft model significantly reduced tumor burden in these animals and demonstrated the predictive utility of
proteomic techniques.
Conclusions: Our proteomic and genomic characterizations of the histone modification states provide a resource
for future investigations of the epigenetic and non-epigenetic determinants for classifying and analyzing cancer

Epigenetics & Chromatin 2013, 6:19

Smchd1 regulates a subset of autosomal genes
subject to monoallelic expression in addition to
being critical for X inactivation

Arne W Mould1,2, Zhenyi Pang1, Miha Pakusch3, Ian D Tonks1, Mitchell Stark1, Dianne Carrie1,
Pamela Mukhopadhyay1, Annica Seidel1, Jonathan J Ellis1, Janine Deakin4, Matthew J Wakefield3,5, Lutz Krause1,
Marnie E Blewitt3,5,6 and Graham F Kay1*
: Smchd1 is an epigenetic modifier essential for X chromosome inactivation: female embryos lacking
Smchd1 fail during midgestational development. Male mice are less affected by Smchd1-loss, with some (but not
all) surviving to become fertile adults on the FVB/n genetic background. On other genetic backgrounds, all males
lacking Smchd1 die perinatally. This suggests that, in addition to being critical for X inactivation, Smchd1 functions
to control the expression of essential autosomal genes.
Results: Using genome-wide microarray expression profiling and RNA-seq, we have identified additional genes that
fail X inactivation in female Smchd1 mutants and have identified autosomal genes in male mice where the normal
expression pattern depends upon Smchd1. A subset of genes in the Snrpn imprinted gene cluster show an
epigenetic signature and biallelic expression consistent with loss of imprinting in the absence of Smchd1. In
addition, single nucleotide polymorphism analysis of expressed genes in the placenta shows that the Igf2r
imprinted gene cluster is also disrupted, with Slc22a3 showing biallelic expression in the absence of Smchd1.
In both cases, the disruption was not due to loss of the differential methylation that marks the imprint control
region, but affected genes remote from this primary imprint controlling element. The clustered protocadherins
(Pcdhα, Pcdhβ, and Pcdhγ) also show altered expression levels, suggesting that their unique pattern of random
combinatorial monoallelic expression might also be disrupted.
Conclusions: Smchd1 has a role in the expression of several autosomal gene clusters that are subject to
monoallelic expression, rather than being restricted to functioning uniquely in X inactivation. Our findings,
combined with the recent report implicating heterozygous mutations of SMCHD1 as a causal factor in the
digenically inherited muscular weakness syndrome facioscapulohumeral muscular dystrophy-2, highlight the
potential importance of Smchd1 in the etiology of diverse human diseases.
Keywords: Clustered protocadherins, Genomic imprinting, Monoallelic expression, Smchd1, X inactivation

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