Publications by authors named "Jason D Lieb"

110 Publications

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

Dev Cell 2020 03;52(6):699-713.e11

Department of Pediatrics, The University of Chicago, Chicago, IL 60637, USA; Department of Human Genetics, The University of Chicago, Chicago, IL 60637, USA; Department of Pathology, The University of Chicago, Chicago, IL 60637, USA.

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.
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http://dx.doi.org/10.1016/j.devcel.2020.02.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7201903PMC
March 2020

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

Mamm Genome 2018 02 10;29(1-2):153-167. Epub 2018 Feb 10.

Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.

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.
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http://dx.doi.org/10.1007/s00335-018-9739-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6095468PMC
February 2018

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.

Environ Health Perspect 2017 10 16;125(10):107006. Epub 2017 Oct 16.

Department of Genetics, University of North Carolina , Chapel Hill, North Carolina, USA.

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 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.
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http://dx.doi.org/10.1289/EHP1937DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5944832PMC
October 2017

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

Genome Res 2017 01 14;27(1):75-86. Epub 2016 Nov 14.

Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey 08544, USA.

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.
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http://dx.doi.org/10.1101/gr.208173.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5204346PMC
January 2017

Chromatin profiling of Drosophila CNS subpopulations identifies active transcriptional enhancers.

Development 2016 10;143(20):3723-3732

Department of Biochemistry and Biophysics, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280, USA

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.
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http://dx.doi.org/10.1242/dev.136895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5087646PMC
October 2016

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

Proc Natl Acad Sci U S A 2016 10 29;113(41):11507-11512. Epub 2016 Sep 29.

Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7260;

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.
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http://dx.doi.org/10.1073/pnas.1614430113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5068337PMC
October 2016

A Transcriptional Lineage of the Early C. elegans Embryo.

Dev Cell 2016 08;38(4):430-44

Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.devcel.2016.07.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4999266PMC
August 2016

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

Eur J Immunol 2016 08 1;46(8):1912-25. Epub 2016 Jun 1.

Department of Genetics, University of North Carolina, Chapel Hill, NC, USA.

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.
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http://dx.doi.org/10.1002/eji.201546237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5486994PMC
August 2016

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

Proc Natl Acad Sci U S A 2016 Apr 28;113(15):E2124-33. Epub 2016 Mar 28.

Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina, Chapel Hill, NC 27599;

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.
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http://dx.doi.org/10.1073/pnas.1603388113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4839430PMC
April 2016

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

Development 2016 Feb 23;143(3):483-91. Epub 2015 Dec 23.

Department of Biochemistry and Molecular Biology, Alberta Children's Hospital Research Institute, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4N1

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.
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http://dx.doi.org/10.1242/dev.130914DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4760314PMC
February 2016

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

Genome Res 2015 Nov 2;25(11):1715-26. Epub 2015 Sep 2.

Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin Madison, Madison, Wisconsin 53706, USA;

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.
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http://dx.doi.org/10.1101/gr.192682.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4617967PMC
November 2015

Single-cell ATAC-seq: strength in numbers.

Genome Biol 2015 Aug 21;16:172. Epub 2015 Aug 21.

Department of Human Genetics, The University of Chicago, 920 E. 58th Street, Chicago, IL, 60637, USA.

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.
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http://dx.doi.org/10.1186/s13059-015-0737-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4546161PMC
August 2015

Histone modifications predispose genome regions to breakage and translocation.

Genes Dev 2015 Jul 23;29(13):1393-402. Epub 2015 Jun 23.

National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA;

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.
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http://dx.doi.org/10.1101/gad.262170.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4511214PMC
July 2015

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

Genes Dev 2015 May;29(9):948-60

Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA;

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.
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http://dx.doi.org/10.1101/gad.261271.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4421983PMC
May 2015

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

PLoS Genet 2015 Apr 13;11(4):e1005117. Epub 2015 Apr 13.

Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, Chapel Hill, North Carolina, United States of America; Department of Biology, University of North Carolina, Chapel Hill, Chapel Hill, North Carolina, United States of America; Department of Human Genetics, University of Chicago, Chicago, Illinois, United States of America.

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.
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http://dx.doi.org/10.1371/journal.pgen.1005117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4395330PMC
April 2015

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

Epigenetics Chromatin 2015 14;8. Epub 2015 Feb 14.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115 USA.

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.
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http://dx.doi.org/10.1186/1756-8935-8-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4362815PMC
March 2015

Interrogating the function of metazoan histones using engineered gene clusters.

Dev Cell 2015 Feb;32(3):373-86

Department of Biology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Curriculum in Genetics and Molecular Biology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Integrative Program for Biological and Genome Sciences, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Genetics, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.devcel.2014.12.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4385256PMC
February 2015

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

Cell Rep 2015 Feb 29;10(4):551-61. Epub 2015 Jan 29.

Yale Systems Biology Institute and Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT 06511, USA.

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.
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http://dx.doi.org/10.1016/j.celrep.2014.12.052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4447085PMC
February 2015

What are super-enhancers?

Nat Genet 2015 Jan;47(1):8-12

Department of Human Genetics, University of Chicago, Chicago, Illinois, USA.

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.
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http://dx.doi.org/10.1038/ng.3167DOI Listing
January 2015

A split personality for nucleosomes.

Cell 2014 Dec;159(6):1249-51

Department of Human Genetics, The University of Chicago, 920 East 58(th) Street, Chicago, IL, 60637, USA. Electronic address:

A high-resolution look at where histones touch DNA reveals a surprisingly intricate, dynamic, and modular nucleosome. Three advances in the study by Rhee et al. include unexpected interactions between the H3 tail and linker DNA, new evidence for existence of subnucleosomal particles, and asymmetric patterns of histone modification within a single nucleosome that correspond to the direction of transcription.
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http://dx.doi.org/10.1016/j.cell.2014.11.030DOI Listing
December 2014

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

PLoS Genet 2014 Nov 20;10(11):e1004798. Epub 2014 Nov 20.

The European Molecular Biology Laboratory, The European Bioinformatics Institute (EMBL-EBI), Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, United Kingdom.

Associating genetic variation with quantitative measures of gene regulation offers a way to bridge the gap between genotype and complex phenotypes. In order to identify quantitative trait loci (QTLs) that influence the binding of a transcription factor in humans, we measured binding of the multifunctional transcription and chromatin factor CTCF in 51 HapMap cell lines. We identified thousands of QTLs in which genotype differences were associated with differences in CTCF binding strength, hundreds of them confirmed by directly observable allele-specific binding bias. The majority of QTLs were either within 1 kb of the CTCF binding motif, or in linkage disequilibrium with a variant within 1 kb of the motif. On the X chromosome we observed three classes of binding sites: a minority class bound only to the active copy of the X chromosome, the majority class bound to both the active and inactive X, and a small set of female-specific CTCF sites associated with two non-coding RNA genes. In sum, our data reveal extensive genetic effects on CTCF binding, both direct and indirect, and identify a diversity of patterns of CTCF binding on the X chromosome.
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http://dx.doi.org/10.1371/journal.pgen.1004798DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4238955PMC
November 2014

Comparative analysis of metazoan chromatin organization.

Nature 2014 Aug;512(7515):449-52

1] Department of Biology and Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA [2] Department of Biology, Center for Genomics and Systems Biology, New York University, New York, New York 10003, USA.

Genome function is dynamically regulated in part by chromatin, which consists of the histones, non-histone proteins and RNA molecules that package DNA. Studies in Caenorhabditis elegans and Drosophila melanogaster have contributed substantially to our understanding of molecular mechanisms of genome function in humans, and have revealed conservation of chromatin components and mechanisms. Nevertheless, the three organisms have markedly different genome sizes, chromosome architecture and gene organization. On human and fly chromosomes, for example, pericentric heterochromatin flanks single centromeres, whereas worm chromosomes have dispersed heterochromatin-like regions enriched in the distal chromosomal 'arms', and centromeres distributed along their lengths. To systematically investigate chromatin organization and associated gene regulation across species, we generated and analysed a large collection of genome-wide chromatin data sets from cell lines and developmental stages in worm, fly and human. Here we present over 800 new data sets from our ENCODE and modENCODE consortia, bringing the total to over 1,400. Comparison of combinatorial patterns of histone modifications, nuclear lamina-associated domains, organization of large-scale topological domains, chromatin environment at promoters and enhancers, nucleosome positioning, and DNA replication patterns reveals many conserved features of chromatin organization among the three organisms. We also find notable differences in the composition and locations of repressive chromatin. These data sets and analyses provide a rich resource for comparative and species-specific investigations of chromatin composition, organization and function.
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http://dx.doi.org/10.1038/nature13415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4227084PMC
August 2014

Defining functional DNA elements in the human genome.

Proc Natl Acad Sci U S A 2014 Apr 21;111(17):6131-8. Epub 2014 Apr 21.

Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge, MA 02139.

With the completion of the human genome sequence, attention turned to identifying and annotating its functional DNA elements. As a complement to genetic and comparative genomics approaches, the Encyclopedia of DNA Elements Project was launched to contribute maps of RNA transcripts, transcriptional regulator binding sites, and chromatin states in many cell types. The resulting genome-wide data reveal sites of biochemical activity with high positional resolution and cell type specificity that facilitate studies of gene regulation and interpretation of noncoding variants associated with human disease. However, the biochemically active regions cover a much larger fraction of the genome than do evolutionarily conserved regions, raising the question of whether nonconserved but biochemically active regions are truly functional. Here, we review the strengths and limitations of biochemical, evolutionary, and genetic approaches for defining functional DNA segments, potential sources for the observed differences in estimated genomic coverage, and the biological implications of these discrepancies. We also analyze the relationship between signal intensity, genomic coverage, and evolutionary conservation. Our results reinforce the principle that each approach provides complementary information and that we need to use combinations of all three to elucidate genome function in human biology and disease.
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http://dx.doi.org/10.1073/pnas.1318948111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4035993PMC
April 2014

Regulation of the X chromosomes in Caenorhabditis elegans.

Cold Spring Harb Perspect Biol 2014 Mar 1;6(3). Epub 2014 Mar 1.

Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, Santa Cruz, California 95064.

Dosage compensation, which regulates the expression of genes residing on the sex chromosomes, has provided valuable insights into chromatin-based mechanisms of gene regulation. The nematode Caenorhabditis elegans has adopted various strategies to down-regulate and even nearly silence the X chromosomes. This article discusses the different chromatin-based strategies used in somatic tissues and in the germline to modulate gene expression from the C. elegans X chromosomes and compares these strategies to those used by other organisms to cope with similar X-chromosome dosage differences.
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http://dx.doi.org/10.1101/cshperspect.a018366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3942922PMC
March 2014

Genome-wide analysis links emerin to neuromuscular junction activity in Caenorhabditis elegans.

Genome Biol 2014 Feb 3;15(2):R21. Epub 2014 Feb 3.

Background: Laminopathies are diseases characterized by defects in nuclear envelope structure. A well-known example is Emery-Dreifuss muscular dystrophy, which is caused by mutations in the human lamin A/C and emerin genes. While most nuclear envelope proteins are ubiquitously expressed, laminopathies often affect only a subset of tissues. The molecular mechanisms underlying these tissue-specific manifestations remain elusive. We hypothesize that different functional subclasses of genes might be differentially affected by defects in specific nuclear envelope components.

Results: Here we determine genome-wide DNA association profiles of two nuclear envelope components, lamin/LMN-1 and emerin/EMR-1 in adult Caenorhabditis elegans. Although both proteins bind to transcriptionally inactive regions of the genome, EMR-1 is enriched at genes involved in muscle and neuronal function. Deletion of either EMR-1 or LEM-2, another integral envelope protein, causes local changes in nuclear architecture as evidenced by altered association between DNA and LMN-1. Transcriptome analyses reveal that EMR-1 and LEM-2 are associated with gene repression, particularly of genes implicated in muscle and nervous system function. We demonstrate that emr-1, but not lem-2, mutants are sensitive to the cholinesterase inhibitor aldicarb, indicating altered activity at neuromuscular junctions.

Conclusions: We identify a class of elements that bind EMR-1 but do not associate with LMN-1, and these are enriched for muscle and neuronal genes. Our data support a redundant function of EMR-1 and LEM-2 in chromatin anchoring to the nuclear envelope and gene repression. We demonstrate a specific role of EMR-1 in neuromuscular junction activity that may contribute to Emery-Dreifuss muscular dystrophy in humans.
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http://dx.doi.org/10.1186/gb-2014-15-2-r21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4053756PMC
February 2014

Gene model 129 (Gm129) encodes a novel transcriptional repressor that modulates circadian gene expression.

J Biol Chem 2014 Feb 2;289(8):5013-24. Epub 2014 Jan 2.

From the Departments of Biochemistry and Biophysics and.

The mammalian circadian clock is a molecular oscillator composed of a feedback loop that involves transcriptional activators CLOCK and BMAL1, and repressors Cryptochrome (CRY) and Period (PER). Here we show that a direct CLOCK·BMAL1 target gene, Gm129, is a novel regulator of the feedback loop. ChIP analysis revealed that the CLOCK·BMAL1·CRY1 complex strongly occupies the promoter region of Gm129. Both mRNA and protein levels of GM129 exhibit high amplitude circadian oscillations in mouse liver, and Gm129 gene encodes a nuclear-localized protein that directly interacts with BMAL1 and represses CLOCK·BMAL1 activity. In vitro and in vivo protein-DNA interaction results demonstrate that, like CRY1, GM129 functions as a repressor by binding to the CLOCK·BMAL1 complex on DNA. Although Gm129(-/-) or Cry1(-/-) Gm129(-/-) mice retain a robust circadian rhythm, the peaks of Nr1d1 and Dbp mRNAs in liver exhibit a significant phase delay compared with control. Our results suggest that, in addition to CRYs and PERs, the GM129 protein contributes to the transcriptional feedback loop by modulating CLOCK·BMAL1 activity as a transcriptional repressor.
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http://dx.doi.org/10.1074/jbc.M113.534651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3931061PMC
February 2014

A common set of DNA regulatory elements shapes Drosophila appendages.

Dev Cell 2013 Nov;27(3):306-18

Department of Biology, Carolina Center for Genome Sciences, and Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280, USA. Electronic address:

Animals have body parts made of similar cell types located at different axial positions, such as limbs. The identity and distinct morphology of each structure is often specified by the activity of different "master regulator" transcription factors. Although similarities in gene expression have been observed between body parts made of similar cell types, how regulatory information in the genome is differentially utilized to create morphologically diverse structures in development is not known. Here, we use genome-wide open chromatin profiling to show that among the Drosophila appendages, the same DNA regulatory modules are accessible throughout the genome at a given stage of development, except at the loci encoding the master regulators themselves. In addition, open chromatin profiles change over developmental time, and these changes are coordinated between different appendages. We propose that master regulators create morphologically distinct structures by differentially influencing the function of the same set of DNA regulatory modules.
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http://dx.doi.org/10.1016/j.devcel.2013.10.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3866527PMC
November 2013

Variation in chromatin accessibility in human kidney cancer links H3K36 methyltransferase loss with widespread RNA processing defects.

Genome Res 2014 Feb 24;24(2):241-50. Epub 2013 Oct 24.

Department of Genetics, University of North Carolina, Chapel Hill, North Carolina 27514, USA;

Comprehensive sequencing of human cancers has identified recurrent mutations in genes encoding chromatin regulatory proteins. For clear cell renal cell carcinoma (ccRCC), three of the five commonly mutated genes encode the chromatin regulators PBRM1, SETD2, and BAP1. How these mutations alter the chromatin landscape and transcriptional program in ccRCC or other cancers is not understood. Here, we identified alterations in chromatin organization and transcript profiles associated with mutations in chromatin regulators in a large cohort of primary human kidney tumors. By associating variation in chromatin organization with mutations in SETD2, which encodes the enzyme responsible for H3K36 trimethylation, we found that changes in chromatin accessibility occurred primarily within actively transcribed genes. This increase in chromatin accessibility was linked with widespread alterations in RNA processing, including intron retention and aberrant splicing, affecting ∼25% of all expressed genes. Furthermore, decreased nucleosome occupancy proximal to misspliced exons was observed in tumors lacking H3K36me3. These results directly link mutations in SETD2 to chromatin accessibility changes and RNA processing defects in cancer. Detecting the functional consequences of specific mutations in chromatin regulatory proteins in primary human samples could ultimately inform the therapeutic application of an emerging class of chromatin-targeted compounds.
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http://dx.doi.org/10.1101/gr.158253.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3912414PMC
February 2014
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