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The transcriptional repressors Snail and Slug are situated at the core of several signaling pathways proposed to mediate epithelial to mesenchymal transition or EMT, which has been implicated in tumor metastasis. EMT involves an alteration from an organized, epithelial cell structure to a mesenchymal, invasive and migratory phenotype. In order to obtain a global view of the impact of Snail and Slug expression, we performed a microarray experiment using the MCF-7 breast cancer cell line, which does not express detectable levels of Snail or Slug. MCF-7 cells were infected with Snail, Slug or control adenovirus, and RNA samples isolated at various time points were analyzed across all transcripts. Our analyses indicated that Snail and Slug regulate many genes in common, but also have distinct sets of gene targets. Gene set enrichment analyses indicated that Snail and Slug directed the transcriptome of MCF-7 cells from a luminal towards a more complex pattern that includes many features of the claudin-low breast cancer signature. Of particular interest, genes involved in the TGF-beta signaling pathway are upregulated, while genes responsible for a differentiated morphology are downregulated following Snail or Slug expression. Further we noticed increased histone acetylation at the promoter region of the transforming growth factor beta-receptor II (TGFBR2) gene following Snail or Slug expression. Inhibition of the TGF-beta signaling pathway using selective small-molecule inhibitors following Snail or Slug addition resulted in decreased cell migration with no impact on the repression of cell junction molecules by Snail and Slug. We propose that there are two regulatory modules embedded within EMT: one that involves repression of cell junction molecules, and the other involving cell migration via TGF-beta and/or other pathways.

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.

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. In mammals, DNA methylation patterns are established by two de novo DNA methyltransferases, DNMT3A and DNMT3B. Here the authors report the crystal structures of DNMT3B in complex with both CpG and CpA DNA, providing insight into the substrate-recognition mechanism underpinning the divergent genomic methylation activities of DNMT3A and DNMT3B.

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. GATA 3 functions as a pioneer factor during cellular reprogramming. Here the authors delineate nucleosome positioning relative to GATA3 binding motifs and describe the structure of a GATA3–nucleosome complex; providing insight into how a pioneer factor interacts with nucleosomes and catalyze their local remodelling to produce an accessible enhancer.

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. Metabolic adaptation to different diets results in changes to gene expression. Here, the authors characterise the chromatin landscape and transcriptional network in mice on a diet of high saturated fat, compared to a diet high in carbohydrate, finding a dramatic reprogramming of the liver 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. In breast cancer GATA3 is known to be frequently mutated, but the function of these mutations is unclear. Here, the authors utilise CRISPR-Cas9 to model frame-shift mutations in zinc finger 2 of GATA3, highlighting that GATA3 mutation can have gain- or loss-of function effects in breast cancer.

Background Transcription factor-dependent cellular reprogramming is integral to normal development and is central to production of induced pluripotent stem cells. This process typically requires pioneer transcription factors (TFs) to induce de novo formation of enhancers at previously closed chromatin. Mechanistic information on this process is currently sparse. Results Here we explore the mechanistic basis by which GATA3 functions as a pioneer TF in a cellular reprogramming event relevant to breast cancer, the mesenchymal to epithelial transition (MET). In some instances, GATA3 binds previously inaccessible chromatin, characterized by stable, positioned nucleosomes where it induces nucleosome eviction, alters local histone modifications, and remodels local chromatin architecture. At other loci, GATA3 binding induces nucleosome sliding without concomitant generation of accessible chromatin. Deletion of the transactivation domain retains the chromatin binding ability of GATA3 but cripples chromatin reprogramming ability, resulting in failure to induce MET. Conclusions These data provide mechanistic insights into GATA3-mediated chromatin reprogramming during MET, and suggest unexpected complexity to TF pioneering. Successful reprogramming requires stable binding to a nucleosomal site; activation domain-dependent recruitment of co-factors including BRG1, the ATPase subunit of the SWI/SNF chromatin remodeling complex; and appropriate genomic context. The resulting model provides a new conceptual framework for de novo enhancer establishment by a pioneer TF.

The Mi-2/nucleosome remodeling and histone deacetylase (NuRD) complex is a multiprotein machine proposed to regulate chromatin structure by nucleosome remodeling and histone deacetylation activities. Recent reports describing localization of NuRD provide new insights that question previous models on NuRD action, but are not in complete agreement. Here, we provide location analysis of endogenous MBD3, a component of NuRD complex, in two human breast cancer cell lines (MCF-7 and MDA-MB-231) using two independent genomic techniques: DNA adenine methyltransferase identification (DamID) and ChIP-seq. We observed concordance of the resulting genomic localization, suggesting that these studies are converging on a robust map for NuRD in the cancer cell genome. MBD3 preferentially associated with CpG rich promoters marked by H3K4me3 and showed cell-type specific localization across gene bodies, peaking around the transcription start site. A subset of sites bound by MBD3 was enriched in H3K27ac and was in physical proximity to promoters in three-dimensional space, suggesting function as enhancers. MBD3 enrichment was also noted at promoters modified by H3K27me3. Functional analysis of chromatin indicated that MBD3 regulates nucleosome occupancy near promoters and in gene bodies. These data suggest that MBD3, and by extension the NuRD complex, may have multiple roles in fine tuning expression for both active and silent genes, representing an important step in defining regulatory mechanisms by which NuRD complex controls chromatin structure and modification status.

Maternal smoking during pregnancy is associated with significant infant morbidity and mortality, and may influence later disease risk. One mechanism by which smoking (and other environmental factors) might have long-lasting effects is through epigenetic modifications such as DNA methylation. We conducted an epigenome-wide association study (EWAS) investigating alterations in DNA methylation in infants exposed in utero to maternal tobacco smoke, using the Norway Facial Clefts Study. The Illumina HumanMethylation450 BeadChip was used to assess DNA methylation in whole blood from 889 infants shortly after delivery. Of 889 mothers, 287 reported smoking-twice as many smokers as in any previous EWAS of maternal smoking. CpG sites related to maternal smoking during the first trimester were identified using robust linear regression. We identified 185 CpGs with altered methylation in infants of smokers at genome-wide significance (q-value < 0.05; mean Δβ = ± 2%). These correspond to 110 gene regions, of which 7 have been previously reported and 10 are newly confirmed using publicly available results. Among these 10, the most noteworthy are FRMD4A, ATP9A, GALNT2, and MEG3, implicated in processes related to nicotine dependence, smoking cessation, and placental and embryonic development. Our study identified 10 genes with newly established links to maternal smoking. Further, we note differences between smoking-related methylation changes in newborns and adults, suggesting possible distinct effects of direct versus indirect tobacco smoke exposure as well as potential differences due to age. Further work would be needed to determine whether these small changes in DNA methylation are biologically or clinically relevant. The methylation changes identified in newborns may mediate the association between in utero maternal smoking exposure and later health outcomes.

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.