Persistent gene expression and DNA methylation alterations linked to carcinogenic effects of dichloroacetic acid.

Background: Mechanistic understanding of transient exposures that lead to adverse health outcomes will enhance our ability to recognize biological signatures of disease. Here, we measured the transcriptomic and epigenomic alterations due to exposure to the metabolic reprogramming agent, dichloroacetic acid (DCA). Previously, we showed that exposure to DCA increased liver tumor incidence in B6C3F1 mice after continuous or early life exposures significantly over background level. Methods: Using archived formalin-fixed liver samples, we utilized modern methodologies to measure gene expression and DNA methylation levels to link to previously generated phenotypic measures. Gene expression was measured by targeted RNA sequencing (TempO-seq 1500+ toxicity panel: 2754 total genes) in liver samples collected from 10-, 32-, 57-, and 78-week old mice exposed to deionized water (controls), 3.5 g/L DCA continuously in drinking water ("Direct" group), or DCA for 10-, 32-, or 57-weeks followed by deionized water until sample collection ("Stop" groups). Genome-scaled alterations in DNA methylation were measured by Reduced Representation Bisulfite Sequencing (RRBS) in 78-week liver samples for control, Direct, 10-week Stop DCA exposed mice. Results: Transcriptomic changes were most robust with concurrent or adjacent timepoints after exposure was withdrawn. We observed a similar pattern with DNA methylation alterations where we noted attenuated differentially methylated regions (DMRs) in the 10-week Stop DCA exposure groups compared to the Direct group at 78-weeks. Gene pathway analysis indicated cellular effects linked to increased oxidative metabolism, a primary mechanism of action for DCA, closer to exposure windows especially early in life. Conversely, many gene signatures and pathways reversed patterns later in life and reflected more pro-tumorigenic patterns for both current and prior DCA exposures. DNA methylation patterns correlated to early gene pathway perturbations, such as cellular signaling, regulation and metabolism, suggesting persistence in the epigenome and possible regulatory effects. Conclusion: Liver metabolic reprogramming effects of DCA interacted with normal age mechanisms, increasing tumor burden with both continuous and prior DCA exposure in the male B6C3F1 rodent model.

Proteasome Inhibition Reprograms Chromatin Landscape in Breast Cancer.

The 26S proteasome is the major protein degradation machinery in cells. Cancer cells use the proteasome to modulate gene expression networks that promote tumor growth. Proteasome inhibitors have emerged as effective cancer therapeutics, but how they work mechanistically remains unclear. Here, using integrative genomic analysis, we discovered unexpected reprogramming of the chromatin landscape and RNA polymerase II (RNAPII) transcription initiation in breast cancer cells treated with the proteasome inhibitor MG132. The cells acquired dynamic changes in chromatin accessibility at specific genomic loci termed differentially open chromatin regions (DOCR). DOCRs with decreased accessibility were promoter proximal and exhibited unique chromatin architecture associated with divergent RNAPII transcription. Conversely, DOCRs with increased accessibility were primarily distal to transcription start sites and enriched in oncogenic superenhancers predominantly accessible in non-basal breast tumor subtypes. These findings describe the mechanisms by which the proteasome modulates the expression of gene networks intrinsic to breast cancer biology. SIGNIFICANCE: Our study provides a strong basis for understanding the mechanisms by which proteasome inhibitors exert anticancer effects. We find open chromatin regions that change during proteasome inhibition, are typically accessible in non-basal breast cancers.

Comprehensive evaluation of smoking exposures and their interactions on DNA methylation.

BACKGROUND: Smoking impacts DNA methylation, but data are lacking on smoking-related differential methylation by sex or dietary intake, recent smoking cessation (<1 year), persistence of differential methylation from in utero smoking exposure, and effects of environmental tobacco smoke (ETS). METHODS: We meta-analysed data from up to 15,014 adults across 5 cohorts with DNA methylation measured in blood using Illumina's EPIC array for current smoking (2560 exposed), quit < 1 year (500 exposed), in utero (286 exposed), and ETS exposure (676 exposed). We also evaluated the interaction of current smoking with sex or diet (fibre, folate, and vitamin C). FINDINGS: Using false discovery rate (FDR < 0.05), 65,857 CpGs were differentially methylated in relation to current smoking, 4025 with recent quitting, 594 with in utero exposure, and 6 with ETS. Most current smoking CpGs attenuated within a year of quitting. CpGs related to in utero exposure in adults were enriched for those previously observed in newborns. Differential methylation by current smoking at 4-71 CpGs may be modified by sex or dietary intake. Nearly half (35-50%) of differentially methylated CpGs on the 450 K array were associated with blood gene expression. Current smoking and in utero smoking CpGs implicated 3049 and 1067 druggable targets, including chemotherapy drugs. INTERPRETATION: Many smoking-related methylation sites were identified with Illumina's EPIC array. Most signals revert to levels observed in never smokers within a year of cessation. Many in utero smoking CpGs persist into adulthood. Smoking-related druggable targets may provide insights into cancer treatment response and shared mechanisms across smoking-related diseases. FUNDING: Intramural Research Program of the National Institutes of Health, Norwegian Ministry of Health and Care Services and the Ministry of Education and Research, Chief Scientist Office of the Scottish Government Health Directorates and the Scottish Funding Council, Medical Research Council UK and the Wellcome Trust.

Proteasome inhibition reprograms chromatin landscape in breast cancer.

The 26S proteasome is the major protein degradation machinery in cells. Cancer cells use the proteasome to modulate gene expression networks that promote tumor growth. Proteasome inhibitors have emerged as effective cancer therapeutics, but how they work mechanistically remains unclear. Here, using integrative genomic analysis, we discovered unexpected reprogramming of the chromatin landscape and RNAPII transcription initiation in breast cancer cells treated with the proteasome inhibitor MG132. The cells acquired dynamic changes in chromatin accessibility at specific genomic loci termed Differentially Open Chromatin Regions (DOCRs). DOCRs with decreased accessibility were promoter proximal and exhibited unique chromatin architecture associated with divergent RNAPII transcription. Conversely, DOCRs with increased accessibility were primarily distal to transcription start sites and enriched in oncogenic super enhancers predominantly accessible in non-basal breast tumor subtypes. These findings describe the mechanisms by which the proteasome modulates the expression of gene networks intrinsic to breast cancer biology. Highlights: Proteasome inhibition uncovers de novo Differential Open Chromatin Regions (DOCRs) in breast cancer cells. Proteasome inhibitor sensitive promoters exhibit a distinctive chromatin architecture with discrete transcription initiation patterns.Proteasome inhibition reprograms accessibility of super enhancers.Proteasome inhibitor sensitive super enhancers distinguish basal from non-basal breast cancer subtypes.

Prospective epigenome and transcriptome analyses of cord and peripheral blood from preterm infants at risk of bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a prevalent chronic lung disease of prematurity with limited treatment options. To uncover biomarkers of BPD risk, this study investigated epigenetic and transcriptomic signatures of prematurity at birth and during the neonatal period at day 14 and 28. Peripheral blood DNAs from preterm infants were applied to methylation arrays and cell-type composition was estimated by deconvolution. Covariate-adjusted robust linear regression elucidated BPD- and prolonged oxygen (≥ 14 days) exposure-associated CpGs. RNAs from cord and peripheral blood were sequenced, and differentially expressed genes (DEGs) for BPD or oxygen exposure were determined. Estimated neutrophil-lymphocyte ratios in peripheral blood at day 14 in BPD infants were significantly higher than nonBPD infants, suggesting an heightened inflammatory response in developing BPD. BPD-DEGs in cord blood indicated lymphopoiesis inhibition, altered Th1/Th2 responses, DNA damage, and organ degeneration. On day 14, BPD-associated CpGs were highly enriched in neutrophil activation, infection, and CD4 + T cell quantity, and BPD-DEGs were involved in DNA damage, cellular senescence, T cell homeostasis, and hyper-cytokinesis. On day 28, BPD-associated CpGs along with BPD-DEGs were enriched for phagocytosis, neurological disorder, and nucleotide metabolism. Oxygen supplementation markedly downregulated mitochondrial biogenesis genes and altered CpGs annotated to developmental genes. Prematurity-altered DNA methylation could cause abnormal lymphopoiesis, cellular assembly and cell cycle progression to increase BPD risk. Similar pathways between epigenome and transcriptome networks suggest coordination of the two in dysregulating leukopoiesis, adaptive immunity, and innate immunity. The results provide molecular insights into biomarkers for early detection and prevention of BPD.

Single-cell analyses identify dysfunctional CD16+ CD8 T cells in smokers.

Tobacco smoke exposure contributes to the global burden of communicable and chronic diseases. To identify immune cells affected by smoking, we use single-cell RNA sequencing on peripheral blood from smokers and nonsmokers. Transcriptomes reveal a subpopulation of FCGR3A (CD16)-expressing Natural Killer (NK)-like CD8 T lymphocytes that increase in smokers. Mass cytometry confirms elevated CD16+ CD8 T cells in smokers. Inferred as highly differentiated by pseudotime analysis, NK-like CD8 T cells express markers characteristic of effector memory re-expressing CD45RA T (TEMRA) cells. Indicative of immune aging, smokers' CD8 T cells are biased toward differentiated cells and smokers have fewer naïve cells than nonsmokers. DNA methylation-based models show that smoking dose is associated with accelerated aging and decreased telomere length, a biomarker of T cell senescence. Immune aging accompanies T cell senescence, which can ultimately lead to impaired immune function. This suggests a role for smoking-induced, senescence-associated immune dysregulation in smoking-mediated pathologies.

Ancestry-dependent gene expression correlates with reprogramming to pluripotency and multiple dynamic biological processes.

Induced pluripotent stem cells (iPSCs) can be derived from differentiated cells, enabling the generation of personalized disease models by differentiating patient-derived iPSCs into disease-relevant cell lines. While genetic variability between different iPSC lines affects differentiation potential, how this variability in somatic cells affects pluripotent potential is less understood. We generated and compared transcriptomic data from 72 dermal fibroblast-iPSC pairs with consistent variation in reprogramming efficiency. By considering equal numbers of samples from self-reported African Americans and White Americans, we identified both ancestry-dependent and ancestry-independent transcripts associated with reprogramming efficiency, suggesting that transcriptomic heterogeneity can substantially affect reprogramming. Moreover, reprogramming efficiency-associated genes are involved in diverse dynamic biological processes, including cancer and wound healing, and are predictive of 5-year breast cancer survival in an independent cohort. Candidate genes may provide insight into mechanisms of ancestry-dependent regulation of cell fate transitions and motivate additional studies for improvement of reprogramming.

Epigenome-wide association study of DNA methylation and adult asthma in the Agricultural Lung Health Study.

Epigenome-wide studies of methylation in children support a role for epigenetic mechanisms in asthma; however, studies in adults are rare and few have examined non-atopic asthma. We conducted the largest epigenome-wide association study (EWAS) of blood DNA methylation in adults in relation to non-atopic and atopic asthma.We measured DNA methylation in blood using the Illumina MethylationEPIC array among 2286 participants in a case-control study of current adult asthma nested within a United States agricultural cohort. Atopy was defined by serum specific immunoglobulin E (IgE). Participants were categorised as atopy without asthma (n=185), non-atopic asthma (n=673), atopic asthma (n=271), or a reference group of neither atopy nor asthma (n=1157). Analyses were conducted using logistic regression.No associations were observed with atopy without asthma. Numerous cytosine-phosphate-guanine (CpG) sites were differentially methylated in non-atopic asthma (eight at family-wise error rate (FWER) p<9×10-8, 524 at false discovery rate (FDR) less than 0.05) and implicated 382 novel genes. More CpG sites were identified in atopic asthma (181 at FWER, 1086 at FDR) and implicated 569 novel genes. 104 FDR CpG sites overlapped. 35% of CpG sites in non-atopic asthma and 91% in atopic asthma replicated in studies of whole blood, eosinophils, airway epithelium, or nasal epithelium. Implicated genes were enriched in pathways related to the nervous system or inflammation.We identified numerous, distinct differentially methylated CpG sites in non-atopic and atopic asthma. Many CpG sites from blood replicated in asthma-relevant tissues. These circulating biomarkers reflect risk and sequelae of disease, as well as implicate novel genes associated with non-atopic and atopic asthma.

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

Estrogen receptors (ER) are activated by the steroid hormone 17ß-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.

Proteasome inhibition creates a chromatin landscape favorable to RNA Pol II processivity.

Proteasome activity is required for diverse cellular processes, including transcriptional and epigenetic regulation. However, inhibiting proteasome activity can lead to an increase in transcriptional output that is correlated with enriched levels of trimethyl H3K4 and phosphorylated forms of RNA polymerase (Pol) II at the promoter and gene body. Here, we perform gene expression analysis and ChIP followed by sequencing (ChIP-seq) in MCF-7 breast cancer cells treated with the proteasome inhibitor MG132, and we further explore genome-wide effects of proteasome inhibition on the chromatin state and RNA Pol II transcription. Analysis of gene expression programs and chromatin architecture reveals that chemically inhibiting proteasome activity creates a distinct chromatin state, defined by spreading of the H3K4me3 mark into the gene bodies of differentially-expressed genes. The distinct H3K4me3 chromatin profile and hyperacetylated nucleosomes at transcription start sites establish a chromatin landscape that facilitates recruitment of Ser-5- and Ser-2-phosphorylated RNA Pol II. Subsequent transcriptional events result in diverse gene expression changes. Alterations of H3K36me3 levels in the gene body reflect productive RNA Pol II elongation of transcripts of genes that are induced, underscoring the requirement for proteasome activity at multiple phases of the transcriptional cycle. Finally, by integrating genomics data and pathway analysis, we find that the differential effects of proteasome inhibition on the chromatin state modulate genes that are fundamental for cancer cell survival. Together, our results uncover underappreciated downstream effects of proteasome inhibitors that may underlie targeting of distinct chromatin states and key steps of RNA Pol II-mediated transcription in cancer cells.

Comparison of smoking-related DNA methylation between newborns from prenatal exposure and adults from personal smoking.

Aim: Cigarette smoking influences DNA methylation genome wide, in newborns from pregnancy exposure and in adults from personal smoking. Whether a unique methylation signature exists for in utero exposure in newborns is unknown. Materials & methods: We separately meta-analyzed newborn blood DNA methylation (assessed using Illumina450k Beadchip), in relation to sustained maternal smoking during pregnancy (9 cohorts, 5648 newborns, 897 exposed) and adult blood methylation and personal smoking (16 cohorts, 15907 participants, 2433 current smokers). Results & conclusion: Comparing meta-analyses, we identified numerous signatures specific to newborns along with many shared between newborns and adults. Unique smoking-associated genes in newborns were enriched in xenobiotic metabolism pathways. Our findings may provide insights into specific health impacts of prenatal exposure on offspring.

NF-Y controls fidelity of transcription initiation at gene promoters through maintenance of the nucleosome-depleted region.

Faithful transcription initiation is critical for accurate gene expression, yet the mechanisms underlying specific transcription start site (TSS) selection in mammals remain unclear. Here, we show that the histone-fold domain protein NF-Y, a ubiquitously expressed transcription factor, controls the fidelity of transcription initiation at gene promoters in mouse embryonic stem cells. We report that NF-Y maintains the region upstream of TSSs in a nucleosome-depleted state while simultaneously protecting this accessible region against aberrant and/or ectopic transcription initiation. We find that loss of NF-Y binding in mammalian cells disrupts the promoter chromatin landscape, leading to nucleosomal encroachment over the canonical TSS. Importantly, this chromatin rearrangement is accompanied by upstream relocation of the transcription pre-initiation complex and ectopic transcription initiation. Further, this phenomenon generates aberrant extended transcripts that undergo translation, disrupting gene expression profiles. These results suggest NF-Y is a central player in TSS selection in metazoans and highlight the deleterious consequences of inaccurate transcription initiation.

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

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.

Dioxin and AHR impairs mesoderm gene expression and cardiac differentiation in human embryonic stem cells.

Dioxin and dioxin-related polychlorinated biphenyls are potent toxicants with association with developmental heart defects and congenital heart diseases. However, the underlying mechanism of their developmental toxicity is not fully understood. Further, different animals show distinct susceptibility and phenotypes after exposure, suggesting possible species-specific effects. Using a human embryonic stem cell (ESC) cardiomyocyte differentiation model, we examined the impact, susceptible window, and dosage of 2,3,7,8­tetrachlorodibenzo­p­dioxin (TCDD) on human cardiac development. We showed that treatment of human ESCs with TCDD at the ESC stage inhibits cardiomyocyte differentiation, and the effect is largely mediated by the aryl hydrocarbon receptor (AHR). We further identified genes that are differentially expressed after TCDD treatment by RNA-sequencing, and genomic regions that are occupied by AHR by chromatin immunoprecipitation and high-throughput sequencing. Our results support the model that TCDD impairs human ESC cardiac differentiation by promoting AHR binding and repression of key mesoderm genes. More importantly, our study demonstrates the toxicity of dioxin in human embryonic development and uncovered a novel mechanism by which dioxin and AHR regulates lineage commitment. It also illustrates the power of ESC-based models in the systematic study of developmental toxicology.

Epigenome-wide meta-analysis of DNA methylation and childhood asthma.

BACKGROUND: Epigenetic mechanisms, including methylation, can contribute to childhood asthma. Identifying DNA methylation profiles in asthmatic patients can inform disease pathogenesis. OBJECTIVE: We sought to identify differential DNA methylation in newborns and children related to childhood asthma. METHODS: Within the Pregnancy And Childhood Epigenetics consortium, we performed epigenome-wide meta-analyses of school-age asthma in relation to CpG methylation (Illumina450K) in blood measured either in newborns, in prospective analyses, or cross-sectionally in school-aged children. We also identified differentially methylated regions. RESULTS: In newborns (8 cohorts, 668 cases), 9 CpGs (and 35 regions) were differentially methylated (epigenome-wide significance, false discovery rate < 0.05) in relation to asthma development. In a cross-sectional meta-analysis of asthma and methylation in children (9 cohorts, 631 cases), we identified 179 CpGs (false discovery rate < 0.05) and 36 differentially methylated regions. In replication studies of methylation in other tissues, most of the 179 CpGs discovered in blood replicated, despite smaller sample sizes, in studies of nasal respiratory epithelium or eosinophils. Pathway analyses highlighted enrichment for asthma-relevant immune processes and overlap in pathways enriched both in newborns and children. Gene expression correlated with methylation at most loci. Functional annotation supports a regulatory effect on gene expression at many asthma-associated CpGs. Several implicated genes are targets for approved or experimental drugs, including IL5RA and KCNH2. CONCLUSION: Novel loci differentially methylated in newborns represent potential biomarkers of risk of asthma by school age. Cross-sectional associations in children can reflect both risk for and effects of disease. Asthma-related differential methylation in blood in children was substantially replicated in eosinophils and respiratory epithelium.

Revealing a human p53 universe.

p53 transcriptional networks are well-characterized in many organisms. However, a global understanding of requirements for in vivo p53 interactions with DNA and relationships with transcription across human biological systems in response to various p53 activating situations remains limited. Using a common analysis pipeline, we analyzed 41 data sets from genome-wide ChIP-seq studies of which 16 have associated gene expression data, including our recent primary data with normal human lymphocytes. The resulting extensive analysis, accessible at p53 BAER hub via the UCSC browser, provides a robust platform to characterize p53 binding throughout the human genome including direct influence on gene expression and underlying mechanisms. We establish the impact of spacers and mismatches from consensus on p53 binding in vivo and propose that once bound, neither significantly influences the likelihood of expression. Our rigorous approach revealed a large p53 genome-wide cistrome composed of >900 genes directly targeted by p53. Importantly, we identify a core cistrome signature composed of genes appearing in over half the data sets, and we identify signatures that are treatment- or cell-specific, demonstrating new functions for p53 in cell biology. Our analysis reveals a broad homeostatic role for human p53 that is relevant to both basic and translational studies.

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

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.

Identification of Smoking-Associated Differentially Methylated Regions Using Reduced Representation Bisulfite Sequencing and Cell type-Specific Enhancer Activation and Gene Expression.

BACKGROUND: Cigarette smoke is a causal factor in cancers and cardiovascular disease. Smoking-associated differentially methylated regions (SM-DMRs) have been observed in disease studies, but the causal link between altered DNA methylation and transcriptional change is obscure. OBJECTIVE: Our objectives were to finely resolve SM-DMRs and to interrogate the mechanistic link between SM-DMRs and altered transcription of enhancer noncoding RNA (eRNA) and mRNA in human circulating monocytes. METHOD: We integrated SM-DMRs identified by reduced representation bisulfite sequencing (RRBS) of circulating CD14+ monocyte DNA collected from two independent human studies [n=38 from Clinical Research Unit (CRU) and n=55 from the Multi-Ethnic Study of Atherosclerosis (MESA), about half of whom were active smokers] with gene expression for protein-coding genes and noncoding RNAs measured by RT-PCR or RNA sequencing. Candidate SM-DMRs were compared with RRBS of purified CD4+ T cells, CD8+ T cells, CD15+ granulocytes, CD19+ B cells, and CD56+ NK cells (n=19 females, CRU). DMRs were validated using pyrosequencing or bisulfite amplicon sequencing in up to 85 CRU volunteers, who also provided saliva DNA. RESULTS: RRBS identified monocyte SM-DMRs frequently located in putative gene regulatory regions. The most significant monocyte DMR occurred at a poised enhancer in the aryl-hydrocarbon receptor repressor gene (AHRR) and it was also detected in both granulocytes and saliva DNA. To our knowledge, we identify for the first time that SM-DMRs in or near AHRR, C5orf55-EXOC-AS, and SASH1 were associated with increased noncoding eRNA as well as mRNA in monocytes. Functionally, the AHRR SM-DMR appeared to up-regulate AHRR mRNA through activating the AHRR enhancer, as suggested by increased eRNA in the monocytes, but not granulocytes, from smokers compared with nonsmokers. CONCLUSIONS: Our findings suggest that AHRR SM-DMR up-regulates AHRR mRNA in a monocyte-specific manner by activating the AHRR enhancer. Cell type-specific activation of enhancers at SM-DMRs may represent a mechanism driving smoking-related disease. https://doi.org/10.1289/EHP2395.

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

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.

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

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.