Publications by authors named "Paul A Wade"

93 Publications

DNMT1 reads heterochromatic H4K20me3 to reinforce LINE-1 DNA methylation.

Nat Commun 2021 05 3;12(1):2490. Epub 2021 May 3.

Department of Biochemistry, University of California, Riverside, CA, USA.

DNA methylation and trimethylated histone H4 Lysine 20 (H4K20me3) constitute two important heterochromatin-enriched marks that frequently cooperate in silencing repetitive elements of the mammalian genome. However, it remains elusive how these two chromatin modifications crosstalk. Here, we report that DNA methyltransferase 1 (DNMT1) specifically 'recognizes' H4K20me3 via its first bromo-adjacent-homology domain (DNMT1). Engagement of DNMT1-H4K20me3 ensures heterochromatin targeting of DNMT1 and DNA methylation at LINE-1 retrotransposons, and cooperates with the previously reported readout of histone H3 tail modifications (i.e., H3K9me3 and H3 ubiquitylation) by the RFTS domain to allosterically regulate DNMT1's activity. Interplay between RFTS and BAH1 domains of DNMT1 profoundly impacts DNA methylation at both global and focal levels and genomic resistance to radiation-induced damage. Together, our study establishes a direct link between H4K20me3 and DNA methylation, providing a mechanism in which multivalent recognition of repressive histone modifications by DNMT1 ensures appropriate DNA methylation patterning and genomic stability.
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http://dx.doi.org/10.1038/s41467-021-22665-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8093215PMC
May 2021

Interaction of the pioneer transcription factor GATA3 with nucleosomes.

Nat Commun 2020 08 18;11(1):4136. Epub 2020 Aug 18.

Laboratory of Chromatin Structure and Function, Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan.

During cellular reprogramming, the pioneer transcription factor GATA3 binds chromatin, and in a context-dependent manner directs local chromatin remodeling and enhancer formation. Here, we use high-resolution nucleosome mapping in human cells to explore the impact of the position of GATA motifs on the surface of nucleosomes on productive enhancer formation, finding productivity correlates with binding sites located near the nucleosomal dyad axis. Biochemical experiments with model nucleosomes demonstrate sufficiently stable transcription factor-nucleosome interaction to empower cryo-electron microscopy structure determination of the complex at 3.15 Å resolution. The GATA3 zinc fingers efficiently bind their target 5'-GAT-3' sequences in the nucleosome when they are located in solvent accessible, consecutive major grooves without significant changes in nucleosome structure. Analysis of genomic loci bound by GATA3 during reprogramming suggests a correlation of recognition motif sequence and spacing that may distinguish productivity of new enhancer formation.
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http://dx.doi.org/10.1038/s41467-020-17959-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7434886PMC
August 2020

Direct readout of heterochromatic H3K9me3 regulates DNMT1-mediated maintenance DNA methylation.

Proc Natl Acad Sci U S A 2020 08 16;117(31):18439-18447. Epub 2020 Jul 16.

Department of Biochemistry, University of California, Riverside, CA 92521;

In mammals, repressive histone modifications such as trimethylation of histone H3 Lys9 (H3K9me3), frequently coexist with DNA methylation, producing a more stable and silenced chromatin state. However, it remains elusive how these epigenetic modifications crosstalk. Here, through structural and biochemical characterizations, we identified the replication foci targeting sequence (RFTS) domain of maintenance DNA methyltransferase DNMT1, a module known to bind the ubiquitylated H3 (H3Ub), as a specific reader for H3K9me3/H3Ub, with the recognition mode distinct from the typical trimethyl-lysine reader. Disruption of the interaction between RFTS and the H3K9me3Ub affects the localization of DNMT1 in stem cells and profoundly impairs the global DNA methylation and genomic stability. Together, this study reveals a previously unappreciated pathway through which H3K9me3 directly reinforces DNMT1-mediated maintenance DNA methylation.
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http://dx.doi.org/10.1073/pnas.2009316117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7414182PMC
August 2020

Comprehensive structure-function characterization of DNMT3B and DNMT3A reveals distinctive de novo DNA methylation mechanisms.

Nat Commun 2020 07 3;11(1):3355. Epub 2020 Jul 3.

Environmental Toxicology Graduate Program, University of California, Riverside, CA, 92521, USA.

Mammalian DNA methylation patterns are established by two de novo DNA methyltransferases, DNMT3A and DNMT3B, which exhibit both redundant and distinctive methylation activities. However, the related molecular basis remains undetermined. Through comprehensive structural, enzymology and cellular characterization of DNMT3A and DNMT3B, we here report a multi-layered substrate-recognition mechanism underpinning their divergent genomic methylation activities. A hydrogen bond in the catalytic loop of DNMT3B causes a lower CpG specificity than DNMT3A, while the interplay of target recognition domain and homodimeric interface fine-tunes the distinct target selection between the two enzymes, with Lysine 777 of DNMT3B acting as a unique sensor of the +1 flanking base. The divergent substrate preference between DNMT3A and DNMT3B provides an explanation for site-specific epigenomic alterations seen in ICF syndrome with DNMT3B mutations. Together, this study reveals distinctive substrate-readout mechanisms of the two DNMT3 enzymes, implicative of their differential roles during development and pathogenesis.
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http://dx.doi.org/10.1038/s41467-020-17109-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7335073PMC
July 2020

Cancer-specific mutation of GATA3 disrupts the transcriptional regulatory network governed by Estrogen Receptor alpha, FOXA1 and GATA3.

Nucleic Acids Res 2020 05;48(9):4756-4768

Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.

Estrogen receptors (ER) are activated by the steroid hormone 17β-estradiol. Estrogen receptor alpha (ER-α) forms a regulatory network in mammary epithelial cells and in breast cancer with the transcription factors FOXA1 and GATA3. GATA3 is one of the most frequently mutated genes in breast cancer and is capable of specifying chromatin localization of FOXA1 and ER-α. How GATA3 mutations found in breast cancer impact genomic localization of ER-α and the transcriptional network downstream of ER-α and FOXA1 remains unclear. Here, we investigate the function of a recurrent patient-derived GATA3 mutation (R330fs) on this regulatory network. Genomic analysis indicates that the R330fs mutant can disrupt localization of ER-α and FOXA1. Loci co-bound by all three factors are enriched for genes integral to mammary gland development as well as epithelial cell biology. This gene set is differentially regulated in GATA3 mutant cells in culture and in tumors bearing similar mutations in vivo. The altered distribution of ER-α and FOXA1 in GATA3-mutant cells is associated with altered chromatin architecture, which leads to differential gene expression. These results suggest an active role for GATA3 zinc finger 2 mutants in ER-α positive breast tumors.
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http://dx.doi.org/10.1093/nar/gkaa179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229857PMC
May 2020

Alterations in promoter interaction landscape and transcriptional network underlying metabolic adaptation to diet.

Nat Commun 2020 02 19;11(1):962. Epub 2020 Feb 19.

Eukaryotic Transcriptional Regulation Group, Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, 27709, USA.

Metabolic adaptation to nutritional state requires alterations in gene expression in key tissues. Here, we investigated chromatin interaction dynamics, as well as alterations in cis-regulatory loci and transcriptional network in a mouse model system. Chronic consumption of a diet high in saturated fat, when compared to a diet high in carbohydrate, led to dramatic reprogramming of the liver transcriptional network. Long-range interaction of promoters with distal regulatory loci, monitored by promoter capture Hi-C, was regulated by metabolic status in distinct fashion depending on diet. Adaptation to a lipid-rich diet, mediated largely by nuclear receptors including Hnf4α, relied on activation of preformed enhancer/promoter loops. Adaptation to carbohydrate-rich diet led to activation of preformed loops and to de novo formation of new promoter/enhancer interactions. These results suggest that adaptation to nutritional changes and metabolic stress occurs through both de novo and pre-existing chromatin interactions which respond differently to metabolic signals.
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http://dx.doi.org/10.1038/s41467-020-14796-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031266PMC
February 2020

The CHD4-related syndrome: a comprehensive investigation of the clinical spectrum, genotype-phenotype correlations, and molecular basis.

Genet Med 2020 02 7;22(2):389-397. Epub 2019 Aug 7.

Division of Genomic Diagnostics, Children's Hospital of Philadelphia, Philadelphia, PA, USA.

Purpose: Sifrim-Hitz-Weiss syndrome (SIHIWES) is a recently described multisystemic neurodevelopmental disorder caused by de novo variants inCHD4. In this study, we investigated the clinical spectrum of the disorder, genotype-phenotype correlations, and the effect of different missense variants on CHD4 function.

Methods: We collected clinical and molecular data from 32 individuals with mostly de novo variants in CHD4, identified through next-generation sequencing. We performed adenosine triphosphate (ATP) hydrolysis and nucleosome remodeling assays on variants from five different CHD4 domains.

Results: The majority of participants had global developmental delay, mild to moderate intellectual disability, brain anomalies, congenital heart defects, and dysmorphic features. Macrocephaly was a frequent but not universal finding. Additional common abnormalities included hypogonadism in males, skeletal and limb anomalies, hearing impairment, and ophthalmic abnormalities. The majority of variants were nontruncating and affected the SNF2-like region of the protein. We did not identify genotype-phenotype correlations based on the type or location of variants. Alterations in ATP hydrolysis and chromatin remodeling activities were observed in variants from different domains.

Conclusion: The CHD4-related syndrome is a multisystemic neurodevelopmental disorder. Missense substitutions in different protein domains alter CHD4 function in a variant-specific manner, but result in a similar phenotype in humans.
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http://dx.doi.org/10.1038/s41436-019-0612-0DOI Listing
February 2020

Maternal Influence and Murine Housing Confound Impact of NLRP1 Inflammasome on Microbiome Composition.

J Innate Immun 2019 13;11(5):416-431. Epub 2019 Feb 13.

Graduate Program in Translational Biology, Medicine, and Health, Virginia Tech, Blacksburg, Virginia, USA,

The NLRP1 inflammasome attenuates inflammatory bowel disease (IBD) progression and colitis-associated tumorigenesis. A possible mechanism postulates that the lack of the NLRP1 inflammasome creates permissive niches in the gut for pathogenic bacteria to flourish, causing dysbiosis and increased IBD susceptibility. To evaluate this hypothesis, we characterized the gut microbiome of wild-type, Nlrp1b-/-, and Asc-/- mice under naïve conditions by sequencing the V3 region of the 16s rRNA gene. For both genetically modified mouse lines, the microbiome composition reflected overrepresentation of bacteria associated with dysbiosis relative to wild-type animals. Measurement of short- and medium-chain fatty acids by mass spectrometry further revealed significant differences between genotypes. However, prior to concluding that the NLRP1 inflammasome plays a role in regulating the composition of the microbiome, we evaluated two additional strategies for cohousing wild-type and Nlrp1b-/- mice: breeding homozygous parents and cohousing at weaning, and breeding from heterozygous parents and cohousing littermates. We found that maternal influence was the greater predictor of microbiome composition rather than genotype. With the rise in microbiome research across disciplines, our study should be viewed as a cautionary example that illustrates the importance of careful breeding and housing strategies when evaluating host-microbiome interactions.
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http://dx.doi.org/10.1159/000495850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6738257PMC
June 2020

DNA methylation in mice is influenced by genetics as well as sex and life experience.

Nat Commun 2019 01 18;10(1):305. Epub 2019 Jan 18.

Division of Intramural Research, NIEHS, 111 TW Alexander Drive, Research Triangle Park, NC, 27709, USA.

DNA methylation is an essential epigenetic process in mammals, intimately involved in gene regulation. Here we address the extent to which genetics, sex, and pregnancy influence genomic DNA methylation by intercrossing 2 inbred mouse strains, C57BL/6N and C3H/HeN, and analyzing DNA methylation in parents and offspring using whole-genome bisulfite sequencing. Differential methylation across genotype is detected at thousands of loci and is preserved on parental alleles in offspring. In comparison of autosomal DNA methylation patterns across sex, hundreds of differentially methylated regions are detected. Comparison of animals with different histories of pregnancy within our study reveals a CpG methylation pattern that is restricted to female animals that had borne offspring. Collectively, our results demonstrate the stability of CpG methylation across generations, clarify the interplay of epigenetics with genetics and sex, and suggest that CpG methylation may serve as an epigenetic record of life events in somatic tissues at loci whose expression is linked to the relevant biology.
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http://dx.doi.org/10.1038/s41467-018-08067-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6338756PMC
January 2019

CHD3 helicase domain mutations cause a neurodevelopmental syndrome with macrocephaly and impaired speech and language.

Nat Commun 2018 11 5;9(1):4619. Epub 2018 Nov 5.

AP-HP, Hôpital de la Pitié-Salpêtrière, Département de Génétique, Paris, 75013, France.

Chromatin remodeling is of crucial importance during brain development. Pathogenic alterations of several chromatin remodeling ATPases have been implicated in neurodevelopmental disorders. We describe an index case with a de novo missense mutation in CHD3, identified during whole genome sequencing of a cohort of children with rare speech disorders. To gain a comprehensive view of features associated with disruption of this gene, we use a genotype-driven approach, collecting and characterizing 35 individuals with de novo CHD3 mutations and overlapping phenotypes. Most mutations cluster within the ATPase/helicase domain of the encoded protein. Modeling their impact on the three-dimensional structure demonstrates disturbance of critical binding and interaction motifs. Experimental assays with six of the identified mutations show that a subset directly affects ATPase activity, and all but one yield alterations in chromatin remodeling. We implicate de novo CHD3 mutations in a syndrome characterized by intellectual disability, macrocephaly, and impaired speech and language.
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http://dx.doi.org/10.1038/s41467-018-06014-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218476PMC
November 2018

Dosage compensation and DNA methylation landscape of the X chromosome in mouse liver.

Sci Rep 2018 07 4;8(1):10138. Epub 2018 Jul 4.

Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA.

DNA methylation plays a key role in X-chromosome inactivation (XCI), a process that achieves dosage compensation for X-encoded gene products between mammalian female and male cells. However, differential sex chromosome dosage complicates genome-wide epigenomic assessments, and the X chromosome is frequently excluded from female-to-male comparative analyses. Using the X chromosome in the sexually dimorphic mouse liver as a model, we provide a general framework for comparing base-resolution DNA methylation patterns across samples that have different chromosome numbers and ask at a systematic level if predictions by historical analyses of X-linked DNA methylation hold true at a base-resolution chromosome-wide level. We demonstrate that sex-specific methylation patterns on the X chromosome largely reflect the effects of XCI. While our observations concur with longstanding observations of XCI at promoter-proximal CpG islands, we provide evidence that sex-specific DNA methylation differences are not limited to CpG island boundaries. Moreover, these data support a model in which maintenance of CpG islands in the inactive state does not require complete regional methylation. Further, we validate an intragenic non-CpG methylation signature in genes escaping XCI in mouse liver. Our analyses provide insight into underlying methylation patterns that should be considered when assessing sex differences in genome-wide methylation analyses.
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http://dx.doi.org/10.1038/s41598-018-28356-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6031675PMC
July 2018

CHD4 and the NuRD complex directly control cardiac sarcomere formation.

Proc Natl Acad Sci U S A 2018 06 11;115(26):6727-6732. Epub 2018 Jun 11.

University of North Carolina McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599;

Cardiac development relies on proper cardiomyocyte differentiation, including expression and assembly of cell-type-specific actomyosin subunits into a functional cardiac sarcomere. Control of this process involves not only promoting expression of cardiac sarcomere subunits but also repressing expression of noncardiac myofibril paralogs. This level of transcriptional control requires broadly expressed multiprotein machines that modify and remodel the chromatin landscape to restrict transcription machinery access. Prominent among these is the nucleosome remodeling and deacetylase (NuRD) complex, which includes the catalytic core subunit CHD4. Here, we demonstrate that direct CHD4-mediated repression of skeletal and smooth muscle myofibril isoforms is required for normal cardiac sarcomere formation, function, and embryonic survival early in gestation. Through transcriptomic and genome-wide analyses of CHD4 localization, we identified unique CHD4 binding sites in smooth muscle myosin heavy chain, fast skeletal α-actin, and the fast skeletal troponin complex genes. We further demonstrate that in the absence of CHD4, cardiomyocytes in the developing heart form a hybrid muscle cell that contains cardiac, skeletal, and smooth muscle myofibril components. These misexpressed paralogs intercalate into the nascent cardiac sarcomere to disrupt sarcomere formation and cause impaired cardiac function in utero. These results demonstrate the genomic and physiological requirements for CHD4 in mammalian cardiac development.
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http://dx.doi.org/10.1073/pnas.1722219115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6042121PMC
June 2018

DNA methylation and transcriptome aberrations mediated by ERα in mouse seminal vesicles following developmental DES exposure.

Proc Natl Acad Sci U S A 2018 05 16;115(18):E4189-E4198. Epub 2018 Apr 16.

Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709;

Early transient developmental exposure to an endocrine active compound, diethylstilbestrol (DES), a synthetic estrogen, causes late-stage effects in the reproductive tract of adult mice. Estrogen receptor alpha (ERα) plays a role in mediating these developmental effects. However, the developmental mechanism is not well known in male tissues. Here, we present genome-wide transcriptome and DNA methylation profiling of the seminal vesicles (SVs) during normal development and after DES exposure. ERα mediates aberrations of the mRNA transcriptome in SVs of adult mice following neonatal DES exposure. This developmental exposure impacts differential diseases between male (SVs) and female (uterus) tissues when mice reach adulthood due to most DES-altered genes that appear to be tissue specific during mouse development. Certain estrogen-responsive gene changes in SVs are cell-type specific. DNA methylation dynamically changes during development in the SVs of wild-type (WT) and ERα-knockout (αERKO) mice, which increases both the loss and gain of differentially methylated regions (DMRs). There are more gains of DMRs in αERKO compared with WT. Interestingly, the methylation changes between the two genotypes are in different genomic loci. Additionally, the expression levels of a subset of DES-altered genes are associated with their DNA methylation status following developmental DES exposure. Taken together, these findings provide an important basis for understanding the molecular and cellular mechanism of endocrine-disrupting chemicals (EDCs), such as DES, during development in the male mouse tissues. This unique evidence contributes to our understanding of developmental actions of EDCs in human health.
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http://dx.doi.org/10.1073/pnas.1719010115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5939078PMC
May 2018

Cryo-EM structure of the nucleosome containing the enhancer DNA sequence.

Open Biol 2018 03;8(3)

Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan

Pioneer transcription factors specifically target their recognition DNA sequences within nucleosomes. FoxA is the pioneer transcription factor that binds to the gene enhancer in liver precursor cells, and is required for liver differentiation in embryos. The enhancer DNA sequence is reportedly incorporated into nucleosomes in cells, although the nucleosome structure containing the targeting sites for FoxA has not been clarified yet. In this study, we determined the nucleosome structure containing the enhancer (N1) sequence, by cryogenic electron microscopy at 4.0 Å resolution. The nucleosome structure with the enhancer DNA is not significantly different from the previously reported nucleosome structure with the Widom 601 DNA. Interestingly, in the nucleosomes, the enhancer DNA contains local flexible regions, as compared to the Widom 601 DNA. Consistently, DNaseI treatments revealed that, in the nucleosome, the enhancer (N1) DNA is more accessible than the Widom 601 sequence. The histones also associated less strongly with the enhancer (N1) DNA than the Widom 601 DNA in the nucleosome. Therefore, the local histone-DNA contacts may be responsible for the enhanced DNA accessibility in the nucleosome with the enhancer DNA.
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http://dx.doi.org/10.1098/rsob.170255DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5881032PMC
March 2018

GATA3 zinc finger 2 mutations reprogram the breast cancer transcriptional network.

Nat Commun 2018 03 13;9(1):1059. Epub 2018 Mar 13.

Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, Durham, NC, 27709, USA.

GATA3 is frequently mutated in breast cancer; these mutations are widely presumed to be loss-of function despite a dearth of information regarding their effect on disease course or their mechanistic impact on the breast cancer transcriptional network. Here, we address molecular and clinical features associated with GATA3 mutations. A novel classification scheme defines distinct clinical features for patients bearing breast tumors with mutations in the second GATA3 zinc-finger (ZnFn2). An engineered ZnFn2 mutant cell line by CRISPR-Cas9 reveals that mutation of one allele of the GATA3 second zinc finger (ZnFn2) leads to loss of binding and decreased expression at a subset of genes, including Progesterone Receptor. At other loci, associated with epithelial to mesenchymal transition, gain of binding correlates with increased gene expression. These results demonstrate that not all GATA3 mutations are equivalent and that ZnFn2 mutations impact breast cancer through gain and loss-of function.
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http://dx.doi.org/10.1038/s41467-018-03478-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5849768PMC
March 2018

An obesity-associated gut microbiome reprograms the intestinal epigenome and leads to altered colonic gene expression.

Genome Biol 2018 01 23;19(1). Epub 2018 Jan 23.

Eukaryotic Transcriptional Regulation Group, Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, 27709, USA.

Background: The gut microbiome, a key constituent of the colonic environment, has been implicated as an important modulator of human health. The eukaryotic epigenome is postulated to respond to environmental stimuli through alterations in chromatin features and, ultimately, gene expression. How the host mediates epigenomic responses to gut microbiota is an emerging area of interest. Here, we profile the gut microbiome and chromatin characteristics in colon epithelium from mice fed either an obesogenic or control diet, followed by an analysis of the resultant changes in gene expression.

Results: The obesogenic diet shapes the microbiome prior to the development of obesity, leading to altered bacterial metabolite production which predisposes the host to obesity. This microbiota-diet interaction leads to changes in histone modification at active enhancers that are enriched for binding sites for signal responsive transcription factors. These alterations of histone methylation and acetylation are associated with signaling pathways integral to the development of colon cancer. The transplantation of obesogenic diet-conditioned microbiota into germ free mice, combined with an obesogenic diet, recapitulates the features of the long-term diet regimen. The diet/microbiome-dependent changes are reflected in both the composition of the recipient animals' microbiome as well as in the set of transcription factor motifs identified at diet-influenced enhancers.

Conclusions: These findings suggest that the gut microbiome, under specific dietary exposures, stimulates a reprogramming of the enhancer landscape in the colon, with downstream effects on transcription factors. These chromatin changes may be associated with those seen during colon cancer development.
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http://dx.doi.org/10.1186/s13059-018-1389-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5782396PMC
January 2018

Transcriptome and DNA Methylome Analysis in a Mouse Model of Diet-Induced Obesity Predicts Increased Risk of Colorectal Cancer.

Cell Rep 2018 01;22(3):624-637

Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA. Electronic address:

Colorectal cancer (CRC) tends to occur at older age; however, CRC incidence rates have been rising sharply among young age groups. The increasing prevalence of obesity is recognized as a major risk, yet the mechanistic underpinnings remain poorly understood. Using a diet-induced obesity mouse model, we identified obesity-associated molecular changes in the colonic epithelium of young and aged mice, and we further investigated whether the changes were reversed after weight loss. Transcriptome analysis indicated that obesity-related colonic cellular metabolic switch favoring long-chain fatty acid oxidation happened in young mice, while obesity-associated downregulation of negative feedback regulators of pro-proliferative signaling pathways occurred in older mice. Strikingly, colonic DNA methylome was pre-programmed by obesity at young age, priming for a tumor-prone gene signature after aging. Furthermore, obesity-related changes were substantially preserved after short-term weight loss, but they were largely reversed after long-term weight loss. We provided mechanistic insights into increased CRC risk in obesity.
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http://dx.doi.org/10.1016/j.celrep.2017.12.071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5793878PMC
January 2018

Base-Resolution Analysis of DNA Methylation Patterns Downstream of in Mouse Naïve B Cells.

G3 (Bethesda) 2018 03 2;8(3):805-813. Epub 2018 Mar 2.

Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

The DNA methyltransferase, , is dynamically regulated throughout mammalian B cell development and upon activation by antigenic stimulation. inactivation in hematopoietic stem cells has been shown to drive B cell-related malignancies, including chronic lymphocytic leukemia, and associates with specific DNA methylation patterns in transformed cells. However, while it is clear that inactivation of in hematopoietic stem cells has profound functional effects, the consequences of inactivation in cells of the B lineage are unclear. To assess whether loss of at the earliest stages of B cell development lead to DNA methylation defects that might impair function, we selectively inactivated early in mouse B cell development and then utilized whole genome bisulfite sequencing to generate base-resolution profiles of and naïve splenic B cells. Overall, we find that global methylation patterns are largely consistent between and naïve B cells, indicating a minimal functional effect of DNMT3A in mature B cells. However, loss of induced 449 focal DNA methylation changes, dominated by loss-of-methylation events. Regions found to be hypomethylated in naïve splenic B cells were enriched in gene bodies of transcripts expressed in B cells, a fraction of which are implicated in B cell-related disease. Overall, the results from this study suggest that factors other than are the major drivers for methylome maintenance in B cell development.
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http://dx.doi.org/10.1534/g3.117.300446DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5844302PMC
March 2018

Crosstalk between the microbiome and epigenome: messages from bugs.

J Biochem 2018 Feb;163(2):105-112

Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.

Mammals exist in a complicated symbiotic relationship with their gut microbiome, which is postulated to have broad impacts on host health and disease. As omics-based technologies have matured, the potential mechanisms by which the microbiome affects host physiology are being addressed. The gut microbiome, which provides environmental cues, can modify host cell responses to stimuli through alterations in the host epigenome and, ultimately, gene expression. Increasing evidence highlights microbial generation of bioactive compounds that impact the transcriptional machinery in host cells. Here, we review current understanding of the crosstalk between gut microbiota and the host epigenome, including DNA methylation, histone modification and non-coding RNAs. These studies are providing insights into how the host responds to microbial signalling and are predicted to provide information for the application of precision medicine.
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http://dx.doi.org/10.1093/jb/mvx080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5892391PMC
February 2018

Reversing SKI-SMAD4-mediated suppression is essential for T17 cell differentiation.

Nature 2017 11 25;551(7678):105-109. Epub 2017 Oct 25.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, North Carolina 27599, USA.

T helper 17 (T17) cells are critically involved in host defence, inflammation, and autoimmunity. Transforming growth factor β (TGFβ) is instrumental in T17 cell differentiation by cooperating with interleukin-6 (refs 6, 7). Yet, the mechanism by which TGFβ enables T17 cell differentiation remains elusive. Here we reveal that TGFβ enables T17 cell differentiation by reversing SKI-SMAD4-mediated suppression of the expression of the retinoic acid receptor (RAR)-related orphan receptor γt (RORγt). We found that, unlike wild-type T cells, SMAD4-deficient T cells differentiate into T17 cells in the absence of TGFβ signalling in a RORγt-dependent manner. Ectopic SMAD4 expression suppresses RORγt expression and T17 cell differentiation of SMAD4-deficient T cells. However, TGFβ neutralizes SMAD4-mediated suppression without affecting SMAD4 binding to the Rorc locus. Proteomic analysis revealed that SMAD4 interacts with SKI, a transcriptional repressor that is degraded upon TGFβ stimulation. SKI controls histone acetylation and deacetylation of the Rorc locus and T17 cell differentiation via SMAD4: ectopic SKI expression inhibits H3K9 acetylation of the Rorc locus, Rorc expression, and T17 cell differentiation in a SMAD4-dependent manner. Therefore, TGFβ-induced disruption of SKI reverses SKI-SMAD4-mediated suppression of RORγt to enable T17 cell differentiation. This study reveals a critical mechanism by which TGFβ controls T17 cell differentiation and uncovers the SKI-SMAD4 axis as a potential therapeutic target for treating T17-related diseases.
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http://dx.doi.org/10.1038/nature24283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5743442PMC
November 2017

Rif1 promotes a repressive chromatin state to safeguard against endogenous retrovirus activation.

Nucleic Acids Res 2017 Dec;45(22):12723-12738

Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, RTP, NC 27709, USA.

Transposable elements, including endogenous retroviruses (ERVs), constitute a large fraction of the mammalian genome. They are transcriptionally silenced during early development to protect genome integrity and aberrant transcription. However, the mechanisms that control their repression are not fully understood. To systematically study ERV repression, we carried out an RNAi screen in mouse embryonic stem cells (ESCs) and identified a list of novel regulators. Among them, Rif1 displays the strongest effect. Rif1 depletion by RNAi or gene deletion led to increased transcription and increased chromatin accessibility at ERV regions and their neighboring genes. This transcriptional de-repression becomes more severe when DNA methylation is lost. On the mechanistic level, Rif1 directly occupies ERVs and is required for repressive histone mark H3K9me3 and H3K27me3 assembly and DNA methylation. It interacts with histone methyltransferases and facilitates their recruitment to ERV regions. Importantly, Rif1 represses ERVs in human ESCs as well, and the evolutionally-conserved HEAT-like domain is essential for its function. Finally, Rif1 acts as a barrier during somatic cell reprogramming, and its depletion significantly enhances reprogramming efficiency. Together, our study uncovered many previously uncharacterized repressors of ERVs, and defined an essential role of Rif1 in the epigenetic defense against ERV activation.
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http://dx.doi.org/10.1093/nar/gkx884DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5727408PMC
December 2017

Covalent Modifications of Histone H3K9 Promote Binding of CHD3.

Cell Rep 2017 Oct;21(2):455-466

Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO 80045, USA. Electronic address:

Chromatin remodeling is required for genome function and is facilitated by ATP-dependent complexes, such as nucleosome remodeling and deacetylase (NuRD). Among its core components is the chromodomain helicase DNA binding protein 3 (CHD3) whose functional significance is not well established. Here, we show that CHD3 co-localizes with the other NuRD subunits, including HDAC1, near the H3K9ac-enriched promoters of the NuRD target genes. The tandem PHD fingers of CHD3 bind histone H3 tails and posttranslational modifications that increase hydrophobicity of H3K9-methylation or acetylation (H3K9me3 or H3K9ac)-enhance this interaction. Binding of CHD3 PHDs promotes H3K9me3-nucleosome unwrapping in vitro and perturbs the pericentric heterochromatin structure in vivo. Methylation or acetylation of H3K9 uniquely alleviates the intra-nucleosomal interaction of histone H3 tails, increasing H3K9 accessibility. Collectively, our data suggest that the targeting of covalently modified H3K9 by CHD3 might be essential in diverse functions of NuRD.
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http://dx.doi.org/10.1016/j.celrep.2017.09.054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5653232PMC
October 2017

DNA Methylation Changes in Tbx3 in a Mouse Model Exposed to Polybrominated Diphenyl Ethers.

Int J Toxicol 2017 May/Jun;36(3):229-238. Epub 2017 May 3.

1 Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA.

DE-71, a commercial mixture of polybrominated diphenyl ethers widely used in flame retardants, is a pervasive environmental contaminant due to its continuing release from waste material and its long half-life in humans. Although the genotoxic potential of DE-71 appears to be low based on bacterial mutagenicity, it remains a public health concern due to its reported involvement in tumor development. Molecular mechanisms by which DE-71 influences tumor incidence or progression remain understudied. We used liver carcinoma tissue from mice exposed to DE-71 to test the hypothesis that epigenetic alterations consistent with tumor development, specifically DNA methylation, result from long-term DE-71 exposure. We profiled DNA methylation status using the methylated-CpG island recovery assay coupled with microarray analysis of hepatocellular carcinoma DNA from animals exposed to DE-71. DE-71 exposure had little impact on global DNA methylation. However, we detected gene body-specific hypomethylation within the Tbx3 locus, a transcription factor important in liver tumorigenesis and in embryonic and cancer stem cell proliferation. This nonpromoter hypomethylation was accompanied by upregulation of Tbx3 mRNA and protein and by alterations in downstream cell cycle-associated marker expression. Thus, exposure to DE-71 may facilitate tumor development by inducing epigenetic programs that favor expansion of progenitor cell populations.
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http://dx.doi.org/10.1177/1091581817706676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5477986PMC
March 2018

Proteins That Read DNA Methylation.

Adv Exp Med Biol 2016 ;945:303-320

Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, Durham, NC, USA.

Covalent modification of DNA via deposition of a methyl group at the 5' position on cytosine residues alters the chemical groups available for interaction in the major groove of DNA. The information content inherent in this modification alters the affinity and the specificity of DNA binding; some proteins favor interaction with methylated DNA, and others disfavor it. Molecular recognition of cytosine methylation by proteins often initiates sequential regulatory events which impact gene expression and chromatin structure. The known methyl-DNA-binding proteins have unique domains responsible for DNA methylation recognition: (1) the methyl-CpG-binding domain (MBD), (2) the C2H2 zinc finger domain, and (3) the SET- and RING finger-associated (SRA) domain. Structural analyses have revealed that each domain has a characteristic methylated DNA-binding pattern, and this difference in the recognition mechanism renders the DNA methylation mark able to transmit complicated biological information. Recent genetic and genomic studies have revealed novel functions of methyl-DNA-binding proteins. These emerging data have also provided glimpses into how methyl-DNA-binding proteins possess unique features and, presumably, functions. In this review, we summarize structural and biochemical analyses elucidating the mechanism for recognition of DNA methylation and correlate this information with emerging genomic and functional data.
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http://dx.doi.org/10.1007/978-3-319-43624-1_13DOI Listing
June 2017

Maternal Age at Delivery Is Associated with an Epigenetic Signature in Both Newborns and Adults.

PLoS One 2016 6;11(7):e0156361. Epub 2016 Jul 6.

Epidemiology Branch, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina, United States of America.

Offspring of older mothers are at increased risk of adverse birth outcomes, childhood cancers, type 1 diabetes, and neurodevelopmental disorders. The underlying biologic mechanisms for most of these associations remain obscure. One possibility is that maternal aging may produce lasting changes in the epigenetic features of a child's DNA. To test this, we explored the association of mothers' age at pregnancy with methylation in her offspring, using blood samples from 890 Norwegian newborns and measuring DNA methylation at more than 450,000 CpG sites across the genome. We examined replication of a maternal-age finding in an independent group of 1062 Norwegian newborns, and then in 200 US middle-aged women. Older maternal age was significantly associated with reduced methylation at four adjacent CpGs near the 2nd exon of KLHL35 in newborns (p-values ranging from 3x10-6 to 8x10-7). These associations were replicated in the independent set of newborns, and replicated again in women 40 to 60 years after their birth. This study provides the first example of parental age permanently affecting the epigenetic profile of offspring. While the specific functions of the affected gene are unknown, this finding opens the possibility that a mother's age at pregnancy could affect her child's health through epigenetic mechanisms.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0156361PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4934688PMC
August 2017

Epigenetic Perturbations by Arg882-Mutated DNMT3A Potentiate Aberrant Stem Cell Gene-Expression Program and Acute Leukemia Development.

Cancer Cell 2016 07 23;30(1):92-107. Epub 2016 Jun 23.

Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA; Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA; Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address:

DNA methyltransferase 3A (DNMT3A) is frequently mutated in hematological cancers; however, the underlying oncogenic mechanism remains elusive. Here, we report that the DNMT3A mutational hotspot at Arg882 (DNMT3A(R882H)) cooperates with NRAS mutation to transform hematopoietic stem/progenitor cells and induce acute leukemia development. Mechanistically, DNMT3A(R882H) directly binds to and potentiates transactivation of stemness genes critical for leukemogenicity including Meis1, Mn1, and Hoxa gene cluster. DNMT3A(R882H) induces focal epigenetic alterations, including CpG hypomethylation and concurrent gain of active histone modifications, at cis-regulatory elements such as enhancers to facilitate gene transcription. CRISPR/Cas9-mediated ablation of a putative Meis1 enhancer carrying DNMT3A(R882H)-induced DNA hypomethylation impairs Meis1 expression. Importantly, DNMT3A(R882H)-induced gene-expression programs can be repressed through Dot1l inhibition, providing an attractive therapeutic strategy for DNMT3A-mutated leukemias.
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http://dx.doi.org/10.1016/j.ccell.2016.05.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4945461PMC
July 2016

High-quality ChIP-seq analysis of MBD3 in human breast cancer cells.

Genom Data 2016 Mar 6;7:173-4. Epub 2016 Jan 6.

Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, USA.

Chromatin accessibility is tightly regulated by multiple factors/mechanisms to establish different cell type-specific gene expression programs from a single genome. Dysregulation of this process can lead to diseases including cancer. The Mi-2/nucleosome remodeling and deacetylase (NuRD) complex is thought to orchestrate chromatin structure using its intrinsic nucleosome remodeling and histone deacetylase activities. However, the detailed mechanisms by which the NuRD complex regulates chromatin structure in vivo are not yet known. To explore the regulatory mechanisms of the NuRD complex, we mapped genome-wide localization of MBD3, a structural component of NuRD, in a human breast cancer cell line (MDA-MB-231) using a modified ChIP-seq protocol. Our data showed high quality localization information (i.e., high mapping efficiency and low PCR duplication rate) and excellent consistency between biological replicates. The data are deposited in the Gene Expression Omnibus (GSE76116).
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http://dx.doi.org/10.1016/j.gdata.2015.12.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4778645PMC
March 2016