Phosphorylated Lamin A/C in the Nuclear Interior Binds Active Enhancers Associated with Abnormal Transcription in Progeria.

LMNA encodes nuclear Lamin A/C that tethers lamina-associated domains (LADs) to the nuclear periphery. Mutations in LMNA cause degenerative disorders including the premature aging disorder Hutchinson-Gilford progeria, but the mechanisms are unknown. We report that Ser22-phosphorylated (pS22) Lamin A/C was localized to the nuclear interior in human fibroblasts throughout the cell cycle. pS22-Lamin A/C interacted with a subset of putative active enhancers, not LADs, at locations co-bound by the transcriptional activator c-Jun. In progeria-patient fibroblasts, a subset of pS22-Lamin A/C-binding sites were lost, whereas new pS22-Lamin A/C-binding sites emerged in normally quiescent loci. New pS22-Lamin A/C binding was accompanied by increased histone acetylation, increased c-Jun binding, and upregulation of nearby genes implicated in progeria pathophysiology. These results suggest that Lamin A/C regulates gene expression by enhancer binding. Disruption of the gene regulatory rather than LAD tethering function of Lamin A/C may underlie the pathogenesis of disorders caused by LMNA mutations.

Tissue- and strain-specific effects of a genotoxic carcinogen 1,3-butadiene on chromatin and transcription.

Epigenetic effects of environmental chemicals are under intense investigation to fill existing knowledge gaps between environmental/occupational exposures and adverse health outcomes. Chromatin accessibility is one prominent mechanism of epigenetic control of transcription, and understanding of the chemical effects on both could inform the causal role of epigenetic alterations in disease mechanisms. In this study, we hypothesized that baseline variability in chromatin organization and transcription profiles among various tissues and mouse strains influence the outcome of exposure to the DNA damaging chemical 1,3-butadiene. To test this hypothesis, we evaluated DNA damage along with comprehensive quantification of RNA transcripts (RNA-seq), identification of accessible chromatin (ATAC-seq), and characterization of regions with histone modifications associated with active transcription (ChIP-seq for acetylation at histone 3 lysine 27, H3K27ac). We collected these data in the lung, liver, and kidney of mice from two genetically divergent strains, C57BL/6J and CAST/EiJ, that were exposed to clean air or to 1,3-butadiene (~600 ppm) for 2 weeks. We found that tissue effects dominate differences in both gene expression and chromatin states, followed by strain effects. At baseline, xenobiotic metabolism was consistently more active in CAST/EiJ, while immune system pathways were more active in C57BL/6J across tissues. Surprisingly, even though all three tissues in both strains harbored butadiene-induced DNA damage, little transcriptional effect of butadiene was observed in liver and kidney. Toxicologically relevant effects of butadiene in the lung were on the pathways of xenobiotic metabolism and inflammation. We also found that variability in chromatin accessibility across individuals (i.e., strains) only partially explains the variability in transcription. This study showed that variation in the basal states of epigenome and transcriptome may be useful indicators for individuals or tissues susceptible to genotoxic environmental chemicals.

Variation in DNA-Damage Responses to an Inhalational Carcinogen (1,3-Butadiene) in Relation to Strain-Specific Differences in Chromatin Accessibility and Gene Transcription Profiles in C57BL/6J and CAST/EiJ Mice.

BACKGROUND: The damaging effects of exposure to environmental toxicants differentially affect genetically distinct individuals, but the mechanisms contributing to these differences are poorly understood. Genetic variation affects the establishment of the gene regulatory landscape and thus gene expression, and we hypothesized that this contributes to the observed heterogeneity in individual responses to exogenous cellular insults. OBJECTIVES: We performed an in vivo study of how genetic variation and chromatin organization may dictate susceptibility to DNA damage, and influence the cellular response to such damage, caused by an environmental toxicant. MATERIALS AND METHODS: We measured DNA damage, messenger RNA (mRNA) and microRNA (miRNA) expression, and genome-wide chromatin accessibility in lung tissue from two genetically divergent inbred mouse strains, C57BL/6J and CAST/EiJ, both in unexposed mice and in mice exposed to a model DNA-damaging chemical, 1,3-butadiene. RESULTS: Our results showed that unexposed CAST/EiJ and C57BL/6J mice have very different chromatin organization and transcription profiles in the lung. Importantly, in unexposed CAST/EiJ mice, which acquired relatively less 1,3-butadiene-induced DNA damage, we observed increased transcription and a more accessible chromatin landscape around genes involved in detoxification pathways. Upon chemical exposure, chromatin was significantly remodeled in the lung of C57BL/6J mice, a strain that acquired higher levels of 1,3-butadiene-induced DNA damage, around the same genes, ultimately resembling the molecular profile of CAST/EiJ. CONCLUSIONS: These results suggest that strain-specific changes in chromatin and transcription in response to chemical exposure lead to a "compensation" for underlying genetic-driven interindividual differences in the baseline chromatin and transcriptional state. This work represents an example of how chemical and environmental exposures can be evaluated to better understand gene-by-environment interactions, and it demonstrates the important role of chromatin response in transcriptomic changes and, potentially, in deleterious effects of exposure. https://doi.org/10.1289/EHP1937.

Nucleosome fragility is associated with future transcriptional response to developmental cues and stress in C. elegans.

Nucleosomes have structural and regulatory functions in all eukaryotic DNA-templated processes. The position of nucleosomes on DNA and the stability of the underlying histone-DNA interactions affect the access of regulatory proteins to DNA. Both stability and position are regulated through DNA sequence, histone post-translational modifications, histone variants, chromatin remodelers, and transcription factors. Here, we explored the functional implications of nucleosome properties on gene expression and development in Caenorhabditis elegans embryos. We performed a time-course of micrococcal nuclease (MNase) digestion and measured the relative sensitivity or resistance of nucleosomes throughout the genome. Fragile nucleosomes were defined by nucleosomal DNA fragments that were recovered preferentially in early MNase-digestion time points. Nucleosome fragility was strongly and positively correlated with the AT content of the underlying DNA sequence. There was no correlation between promoter nucleosome fragility and the levels of histone modifications or histone variants. Genes with fragile nucleosomes in their promoters tended to be lowly expressed and expressed in a context-specific way, operating in neuronal response, the immune system, and stress response. In addition to DNA-encoded nucleosome fragility, we also found fragile nucleosomes at locations where we expected to find destabilized nucleosomes, for example, at transcription factor binding sites where nucleosomes compete with DNA-binding factors. Our data suggest that in C. elegans promoters, nucleosome fragility is in large part DNA-encoded and that it poises genes for future context-specific activation in response to environmental stress and developmental cues.

Chromatin profiling of Drosophila CNS subpopulations identifies active transcriptional enhancers.

One of the key issues in studying transcriptional regulation during development is how to employ genome-wide assays that reveals sites of open chromatin and transcription factor binding to efficiently identify biologically relevant genes and enhancers. Analysis of Drosophila CNS midline cell development provides a useful system for studying transcriptional regulation at the genomic level due to a large, well-characterized set of midline-expressed genes and in vivo validated enhancers. In this study, FAIRE-seq on FACS-purified midline cells was performed and the midline FAIRE data were compared with whole-embryo FAIRE data. We find that regions of the genome with a strong midline FAIRE peak and weak whole-embryo FAIRE peak overlap with known midline enhancers and provide a useful predictive tool for enhancer identification. In a complementary analysis, we compared a large dataset of fragments that drive midline expression in vivo with the FAIRE data. Midline enhancer fragments with a midline FAIRE peak tend to be near midline-expressed genes, whereas midline enhancers without a midline FAIRE peak were often distant from midline-expressed genes and unlikely to drive midline transcription in vivo.

Cisplatin DNA damage and repair maps of the human genome at single-nucleotide resolution.

Cisplatin is a major anticancer drug that kills cancer cells by damaging their DNA. Cancer cells cope with the drug by removal of the damages with nucleotide excision repair. We have developed methods to measure cisplatin adduct formation and its repair at single-nucleotide resolution. "Damage-seq" relies on the replication-blocking properties of the bulky base lesions to precisely map their location. "XR-seq" independently maps the removal of these damages by capturing and sequencing the excised oligomer released during repair. The damage and repair maps we generated reveal that damage distribution is essentially uniform and is dictated mostly by the underlying sequence. In contrast, cisplatin repair is heterogeneous in the genome and is affected by multiple factors including transcription and chromatin states. Thus, the overall effect of damages in the genome is primarily driven not by damage formation but by the repair efficiency. The combination of the Damage-seq and XR-seq methods has the potential for developing novel cancer therapeutic strategies.

A Transcriptional Lineage of the Early C. elegans Embryo.

During embryonic development, cells must establish fates, morphologies, and behaviors in coordination with one another to form a functional body. A prevalent hypothesis for how this coordination is achieved is that each cell's fate and behavior is determined by a defined mixture of RNAs. Only recently has it become possible to measure the full suite of transcripts in a single cell. Here we quantify genome-wide mRNA abundance in each cell of the Caenorhabditis elegans embryo up to the 16-cell stage. We describe spatially dynamic expression, quantify cell-specific differential activation of the zygotic genome, and identify genes that were previously unappreciated as being critical for development. We present an interactive data visualization tool that allows broad access to our dataset. This genome-wide single-cell map of mRNA abundance, alongside the well-studied life history and fate of each cell, describes at a cellular resolution the mRNA landscape that guides development.

Alterations to chromatin in intestinal macrophages link IL-10 deficiency to inappropriate inflammatory responses.

Intestinal macrophages (IMs) are uniquely programmed to tolerate exposure to bacteria without mounting potent inflammatory responses. The cytokine IL-10 maintains the macrophage anti-inflammatory response such that loss of IL-10 results in chronic intestinal inflammation. To investigate how IL-10-deficiency alters IM programming and bacterial tolerance, we studied changes in chromatin accessibility in response to bacteria in macrophages from two distinct niches, the intestine and bone-marrow, from both wild-type and IL-10-deficient (Il10(-/-) ) mice. We identified chromatin accessibility changes associated with bacterial exposure and IL-10 deficiency in both bone marrow derived macrophages and IMs. Surprisingly, Il10(-/-) IMs adopted chromatin and gene expression patterns characteristic of an inflammatory response, even in the absence of bacteria. Further, when recombinant IL-10 was added to Il10(-/-) cells, it could not revert the chromatin landscape to a normal state. Our results demonstrate that IL-10 deficiency results in stable chromatin alterations in macrophages, even in the absence of bacteria. This supports a model in which IL-10-deficiency leads to chromatin alterations that contribute to a loss of IM tolerance to bacteria, which is a primary initiating event in chronic intestinal inflammation.

Genome-wide kinetics of DNA excision repair in relation to chromatin state and mutagenesis.

We recently developed a high-resolution genome-wide assay for mapping DNA excision repair named eXcision Repair-sequencing (XR-seq) and have now used XR-seq to determine which regions of the genome are subject to repair very soon after UV exposure and which regions are repaired later. Over a time course, we measured repair of the UV-induced damage of cyclobutane pyrimidine dimers (CPDs) (at 1, 4, 8, 16, 24, and 48 h) and (6-4)pyrimidine-pyrimidone photoproducts [(6-4)PPs] (at 5 and 20 min and 1, 2, and 4 h) in normal human skin fibroblasts. Each type of damage has distinct repair kinetics. The (6-4)PPs are detected as early as 5 min after UV treatment, with the bulk of repair completed by 4 h. Repair of CPDs, which we previously showed is intimately coupled to transcription, is slower and in certain regions persists even 2 d after UV irradiation. We compared our results to the Encyclopedia of DNA Elements data regarding histone modifications, chromatin state, and transcription. For both damage types, and for both transcription-coupled and general excision repair, the earliest repair occurred preferentially in active and open chromatin states. Conversely, repair in regions classified as "heterochromatic" and "repressed" was relatively low at early time points, with repair persisting into the late time points. Damage that remains during DNA replication increases the risk for mutagenesis. Indeed, late-repaired regions are associated with a higher level of cancer-linked mutations. In summary, we show that XR-seq is a powerful approach for studying relationships among chromatin state, DNA repair, genome stability, mutagenesis, and carcinogenesis.

The function and regulation of the GATA factor ELT-2 in the C. elegans endoderm.

ELT-2 is the major regulator of genes involved in differentiation, maintenance and function of C. elegans intestine from the early embryo to mature adult. elt-2 responds to overexpression of the GATA transcription factors END-1 and END-3, which specify the intestine, as well as to overexpression of the two GATA factors that are normally involved in intestinal differentiation, ELT-7 and ELT-2 itself. Little is known about the molecular mechanisms underlying these interactions, how ELT-2 levels are maintained throughout development or how such systems respond to developmental perturbations. Here, we analyse elt-2 gene regulation through transgenic reporter assays, ELT-2 ChIP and characterisation of in vitro DNA-protein interactions. Our results indicate that elt-2 is controlled by three discrete regulatory regions conserved between C. elegans and C. briggsae that span >4 kb of 5' flanking sequence. These regions are superficially interchangeable but have quantitatively different enhancer properties, and their combined activities indicate inter-region synergies. Their regulatory activity is mediated by a small number of conserved TGATAA sites that are largely interchangeable and interact with different endodermal GATA factors with only modest differences in affinity. The redundant molecular mechanism that forms the elt-2 regulatory network is robust and flexible, as loss of end-3 halves ELT-2 levels in the early embryo but levels fully recover by the time of hatching. When ELT-2 is expressed under the control of end-1 regulatory elements, in addition to its own endogenous promoter, it can replace the complete set of endoderm-specific GATA factors: END-1, END-3, ELT-7 and (the probably non-functional) ELT-4. Thus, in addition to controlling gene expression during differentiation, ELT-2 is capable of specifying the entire C. elegans endoderm.

Zelda is differentially required for chromatin accessibility, transcription factor binding, and gene expression in the early Drosophila embryo.

The transition from a specified germ cell to a population of pluripotent cells occurs rapidly following fertilization. During this developmental transition, the zygotic genome is largely transcriptionally quiescent and undergoes significant chromatin remodeling. In Drosophila, the DNA-binding protein Zelda (also known as Vielfaltig) is required for this transition and for transcriptional activation of the zygotic genome. Open chromatin is associated with Zelda-bound loci, as well as more generally with regions of active transcription. Nonetheless, the extent to which Zelda influences chromatin accessibility across the genome is largely unknown. Here we used formaldehyde-assisted isolation of regulatory elements to determine the role of Zelda in regulating regions of open chromatin in the early embryo. We demonstrate that Zelda is essential for hundreds of regions of open chromatin. This Zelda-mediated chromatin accessibility facilitates transcription-factor recruitment and early gene expression. Thus, Zelda possesses some key characteristics of a pioneer factor. Unexpectedly, chromatin at a large subset of Zelda-bound regions remains open even in the absence of Zelda. The GAGA factor-binding motif and embryonic GAGA factor binding are specifically enriched in these regions. We propose that both Zelda and GAGA factor function to specify sites of open chromatin and together facilitate the remodeling of the early embryonic genome.

Single-cell ATAC-seq: strength in numbers.

Single-cell ATAC-seq detects open chromatin in individual cells. Currently data are sparse, but combining information from many single cells can identify determinants of cell-to-cell chromatin variation.

Histone modifications predispose genome regions to breakage and translocation.

Chromosome translocations are well-established hallmarks of cancer cells and often occur at nonrandom sites in the genome. The molecular features that define recurrent chromosome breakpoints are largely unknown. Using a combination of bioinformatics, biochemical analysis, and cell-based assays, we identify here specific histone modifications as facilitators of chromosome breakage and translocations. We show enrichment of several histone modifications over clinically relevant translocation-prone genome regions. Experimental modulation of histone marks sensitizes genome regions to breakage by endonuclease challenge or irradiation and promotes formation of chromosome translocations of endogenous gene loci. Our results demonstrate that histone modifications predispose genome regions to chromosome breakage and translocations.

Genome-wide analysis of human global and transcription-coupled excision repair of UV damage at single-nucleotide resolution.

We developed a method for genome-wide mapping of DNA excision repair named XR-seq (excision repair sequencing). Human nucleotide excision repair generates two incisions surrounding the site of damage, creating an ∼30-mer. In XR-seq, this fragment is isolated and subjected to high-throughput sequencing. We used XR-seq to produce stranded, nucleotide-resolution maps of repair of two UV-induced DNA damages in human cells: cyclobutane pyrimidine dimers (CPDs) and (6-4) pyrimidine-pyrimidone photoproducts [(6-4)PPs]. In wild-type cells, CPD repair was highly associated with transcription, specifically with the template strand. Experiments in cells defective in either transcription-coupled excision repair or general excision repair isolated the contribution of each pathway to the overall repair pattern and showed that transcription-coupled repair of both photoproducts occurs exclusively on the template strand. XR-seq maps capture transcription-coupled repair at sites of divergent gene promoters and bidirectional enhancer RNA (eRNA) production at enhancers. XR-seq data also uncovered the repair characteristics and novel sequence preferences of CPDs and (6-4)PPs. XR-seq and the resulting repair maps will facilitate studies of the effects of genomic location, chromatin context, transcription, and replication on DNA repair in human cells.

Correction: Quantitative genetics of CTCF binding reveal local sequence effects and different modes of X-chromosome association.

[This corrects the article DOI: 10.1371/journal.pgen.1004798.].

Asymmetric transcript discovery by RNA-seq in C. elegans blastomeres identifies neg-1, a gene important for anterior morphogenesis.

After fertilization but prior to the onset of zygotic transcription, the C. elegans zygote cleaves asymmetrically to create the anterior AB and posterior P1 blastomeres, each of which goes on to generate distinct cell lineages. To understand how patterns of RNA inheritance and abundance arise after this first asymmetric cell division, we pooled hand-dissected AB and P1 blastomeres and performed RNA-seq. Our approach identified over 200 asymmetrically abundant mRNA transcripts. We confirmed symmetric or asymmetric abundance patterns for a subset of these transcripts using smFISH. smFISH also revealed heterogeneous subcellular patterning of the P1-enriched transcripts chs-1 and bpl-1. We screened transcripts enriched in a given blastomere for embryonic defects using RNAi. The gene neg-1 (F32D1.6) encoded an AB-enriched (anterior) transcript and was required for proper morphology of anterior tissues. In addition, analysis of the asymmetric transcripts yielded clues regarding the post-transcriptional mechanisms that control cellular mRNA abundance during asymmetric cell divisions, which are common in developing organisms.

STAT3 acts through pre-existing nucleosome-depleted regions bound by FOS during an epigenetic switch linking inflammation to cancer.

BACKGROUND: Transient induction of the Src oncoprotein in a non-transformed breast cell line can initiate an epigenetic switch to a cancer cell via a positive feedback loop that involves activation of the signal transducer and activator of transcription 3 protein (STAT3) and NF-κB transcription factors. RESULTS: We show that during the transformation process, nucleosome-depleted regions (defined by formaldehyde-assisted isolation of regulatory elements (FAIRE)) are largely unchanged and that both before and during transformation, STAT3 binds almost exclusively to previously open chromatin regions. Roughly, a third of the transformation-inducible genes require STAT3 for the induction. STAT3 and NF-κB appear to drive the regulation of different gene sets during the transformation process. Interestingly, STAT3 directly regulates the expression of NFKB1, which encodes a subunit of NF-κB, and IL6, a cytokine that stimulates STAT3 activity. Lastly, many STAT3 binding sites are also bound by FOS and the expression of several AP-1 factors is altered during transformation in a STAT3-dependent manner, suggesting that STAT3 may cooperate with AP-1 proteins. CONCLUSIONS: These observations uncover additional complexities to the inflammatory feedback loop that are likely to contribute to the epigenetic switch. In addition, gene expression changes during transformation, whether driven by pre-existing or induced transcription factors, occur largely through pre-existing nucleosome-depleted regions.

Ancient transposable elements transformed the uterine regulatory landscape and transcriptome during the evolution of mammalian pregnancy.

A major challenge in biology is determining how evolutionarily novel characters originate; however, mechanistic explanations for the origin of new characters are almost completely unknown. The evolution of pregnancy is an excellent system in which to study the origin of novelties because mammals preserve stages in the transition from egg laying to live birth. To determine the molecular bases of this transition, we characterized the pregnant/gravid uterine transcriptome from tetrapods to trace the evolutionary history of uterine gene expression. We show that thousands of genes evolved endometrial expression during the origins of mammalian pregnancy, including genes that mediate maternal-fetal communication and immunotolerance. Furthermore, thousands of cis-regulatory elements that mediate decidualization and cell-type identity in decidualized stromal cells are derived from ancient mammalian transposable elements (TEs). Our results indicate that one of the defining mammalian novelties evolved from DNA sequences derived from ancient mammalian TEs co-opted into hormone-responsive regulatory elements distributed throughout the genome.

Interrogating the function of metazoan histones using engineered gene clusters.

Histones and their posttranslational modifications influence the regulation of many DNA-dependent processes. Although an essential role for histone-modifying enzymes in these processes is well established, defining the specific contribution of individual histone residues remains a challenge because many histone-modifying enzymes have nonhistone targets. This challenge is exacerbated by the paucity of suitable approaches to genetically engineer histone genes in metazoans. Here, we describe a platform in Drosophila for generating and analyzing any desired histone genotype, and we use it to test the in vivo function of three histone residues. We demonstrate that H4K20 is neither essential for DNA replication nor for completion of development, unlike inferences drawn from analyses of H4K20 methyltransferases. We also show that H3K36 is required for viability and H3K27 is essential for maintenance of cellular identity but not for gene activation. These findings highlight the power of engineering histones to interrogate genome structure and function in animals.

What are super-enhancers?

The term 'super-enhancer' has been used to describe groups of putative enhancers in close genomic proximity with unusually high levels of Mediator binding, as measured by chromatin immunoprecipitation and sequencing (ChIP-seq). Here we review the identification and composition of super-enhancers, describe links between super-enhancers, gene regulation and disease, and discuss the functional significance of enhancer clustering. We also provide our perspective regarding the proposition that super-enhancers are a regulatory entity conceptually distinct from what was known before the introduction of the term. Our opinion is that there is not yet strong evidence that super-enhancers are a novel paradigm in gene regulation and that use of the term in this context is not currently justified. However, the term likely identifies strong enhancers that exhibit behaviors consistent with previous models and concepts of transcriptional regulation. In this respect, the super-enhancer definition is useful in identifying regulatory elements likely to control genes important for cell type specification.