Publications by authors named "Alexias Safi"

32 Publications

Integrative QTL analysis of gene expression and chromatin accessibility identifies multi-tissue patterns of genetic regulation.

PLoS Genet 2020 01 21;16(1):e1008537. Epub 2020 Jan 21.

Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.

Gene transcription profiles across tissues are largely defined by the activity of regulatory elements, most of which correspond to regions of accessible chromatin. Regulatory element activity is in turn modulated by genetic variation, resulting in variable transcription rates across individuals. The interplay of these factors, however, is poorly understood. Here we characterize expression and chromatin state dynamics across three tissues-liver, lung, and kidney-in 47 strains of the Collaborative Cross (CC) mouse population, examining the regulation of these dynamics by expression quantitative trait loci (eQTL) and chromatin QTL (cQTL). QTL whose allelic effects were consistent across tissues were detected for 1,101 genes and 133 chromatin regions. Also detected were eQTL and cQTL whose allelic effects differed across tissues, including local-eQTL for Pik3c2g detected in all three tissues but with distinct allelic effects. Leveraging overlapping measurements of gene expression and chromatin accessibility on the same mice from multiple tissues, we used mediation analysis to identify chromatin and gene expression intermediates of eQTL effects. Based on QTL and mediation analyses over multiple tissues, we propose a causal model for the distal genetic regulation of Akr1e1, a gene involved in glycogen metabolism, through the zinc finger transcription factor Zfp985 and chromatin intermediates. This analysis demonstrates the complexity of transcriptional and chromatin dynamics and their regulation over multiple tissues, as well as the value of the CC and related genetic resource populations for identifying specific regulatory mechanisms within cells and tissues.
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http://dx.doi.org/10.1371/journal.pgen.1008537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7010298PMC
January 2020

Integrated chromatin and transcriptomic profiling of patient-derived colon cancer organoids identifies personalized drug targets to overcome oxaliplatin resistance.

Genes Dis 2021 Mar 29;8(2):203-214. Epub 2019 Oct 29.

Department of Biomedical Engineering, Pratt School of Engineering, Duke University, Durham, NC, 27708, USA.

Colorectal cancer is a leading cause of cancer deaths. Most colorectal cancer patients eventually develop chemoresistance to the current standard-of-care therapies. Here, we used patient-derived colorectal cancer organoids to demonstrate that resistant tumor cells undergo significant chromatin changes in response to oxaliplatin treatment. Integrated transcriptomic and chromatin accessibility analyses using ATAC-Seq and RNA-Seq identified a group of genes associated with significantly increased chromatin accessibility and upregulated gene expression. CRISPR/Cas9 silencing of fibroblast growth factor receptor 1 (FGFR1) and oxytocin receptor (OXTR) helped overcome oxaliplatin resistance. Similarly, treatment with oxaliplatin in combination with an FGFR1 inhibitor (PD166866) or an antagonist of OXTR (L-368,899) suppressed chemoresistant organoids. However, oxaliplatin treatment did not activate either FGFR1 or OXTR expression in another resistant organoid, suggesting that chromatin accessibility changes are patient-specific. The use of patient-derived cancer organoids in combination with transcriptomic and chromatin profiling may lead to precision treatments to overcome chemoresistance in colorectal cancer.
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http://dx.doi.org/10.1016/j.gendis.2019.10.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8099686PMC
March 2021

Evaluating Chromatin Accessibility Differences Across Multiple Primate Species Using a Joint Modeling Approach.

Genome Biol Evol 2019 10;11(10):3035-3053

Department of Biostatistics and Bioinformatics, Duke University.

Changes in transcriptional regulation are thought to be a major contributor to the evolution of phenotypic traits, but the contribution of changes in chromatin accessibility to the evolution of gene expression remains almost entirely unknown. To address this important gap in knowledge, we developed a new method to identify DNase I Hypersensitive (DHS) sites with differential chromatin accessibility between species using a joint modeling approach. Our method overcomes several limitations inherent to conventional threshold-based pairwise comparisons that become increasingly apparent as the number of species analyzed rises. Our approach employs a single quantitative test which is more sensitive than existing pairwise methods. To illustrate, we applied our joint approach to DHS sites in fibroblast cells from five primates (human, chimpanzee, gorilla, orangutan, and rhesus macaque). We identified 89,744 DHS sites, of which 41% are identified as differential between species using the joint model compared with 33% using the conventional pairwise approach. The joint model provides a principled approach to distinguishing single from multiple chromatin accessibility changes among species. We found that nondifferential DHS sites are enriched for nucleotide conservation. Differential DHS sites with decreased chromatin accessibility relative to rhesus macaque occur more commonly near transcription start sites (TSS), while those with increased chromatin accessibility occur more commonly distal to TSS. Further, differential DHS sites near TSS are less cell type-specific than more distal regulatory elements. Taken together, these results point to distinct classes of DHS sites, each with distinct characteristics of selection, genomic location, and cell type specificity.
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http://dx.doi.org/10.1093/gbe/evz218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821351PMC
October 2019

Comparative Analyses of Chromatin Landscape in White Adipose Tissue Suggest Humans May Have Less Beigeing Potential than Other Primates.

Genome Biol Evol 2019 07;11(7):1997-2008

Biology Department, Duke University.

Humans carry a much larger percentage of body fat than other primates. Despite the central role of adipose tissue in metabolism, little is known about the evolution of white adipose tissue in primates. Phenotypic divergence is often caused by genetic divergence in cis-regulatory regions. We examined the cis-regulatory landscape of fat during human origins by performing comparative analyses of chromatin accessibility in human and chimpanzee adipose tissue using rhesus macaque as an outgroup. We find that many regions that have decreased accessibility in humans are enriched for promoter and enhancer sequences, are depleted for signatures of negative selection, are located near genes involved with lipid metabolism, and contain a short sequence motif involved in the beigeing of fat, the process in which lipid-storing white adipocytes are transdifferentiated into thermogenic beige adipocytes. The collective closing of many putative regulatory regions associated with beigeing of fat suggests a mechanism that increases body fat in humans.
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http://dx.doi.org/10.1093/gbe/evz134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6648876PMC
July 2019

Open Chromatin Profiling in Adipose Tissue Marks Genomic Regions with Functional Roles in Cardiometabolic Traits.

G3 (Bethesda) 2019 08 8;9(8):2521-2533. Epub 2019 Aug 8.

Department of Genetics,

Identifying the regulatory mechanisms of genome-wide association study (GWAS) loci affecting adipose tissue has been restricted due to limited characterization of adipose transcriptional regulatory elements. We profiled chromatin accessibility in three frozen human subcutaneous adipose tissue needle biopsies and preadipocytes and adipocytes from the Simpson Golabi-Behmel Syndrome (SGBS) cell strain using an assay for transposase-accessible chromatin (ATAC-seq). We identified 68,571 representative accessible chromatin regions (peaks) across adipose tissue samples (FDR < 5%). GWAS loci for eight cardiometabolic traits were enriched in these peaks ( < 0.005), with the strongest enrichment for waist-hip ratio. Of 110 recently described cardiometabolic GWAS loci colocalized with adipose tissue eQTLs, 59 loci had one or more variants overlapping an adipose tissue peak. Annotated variants at the waist-hip ratio locus and the body mass index locus showed allelic differences in regulatory assays. These adipose tissue accessible chromatin regions elucidate genetic variants that may alter adipose tissue function to impact cardiometabolic traits.
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http://dx.doi.org/10.1534/g3.119.400294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6686932PMC
August 2019

Transcriptome and epigenome landscape of human cortical development modeled in organoids.

Science 2018 12;362(6420)

Genes implicated in neuropsychiatric disorders are active in human fetal brain, yet difficult to study in a longitudinal fashion. We demonstrate that organoids from human pluripotent cells model cerebral cortical development on the molecular level before 16 weeks postconception. A multiomics analysis revealed differentially active genes and enhancers, with the greatest changes occurring at the transition from stem cells to progenitors. Networks of converging gene and enhancer modules were assembled into six and four global patterns of expression and activity across time. A pattern with progressive down-regulation was enriched with human-gained enhancers, suggesting their importance in early human brain development. A few convergent gene and enhancer modules were enriched in autism-associated genes and genomic variants in autistic children. The organoid model helps identify functional elements that may drive disease onset.
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http://dx.doi.org/10.1126/science.aat6720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6426303PMC
December 2018

Human cardiac -regulatory elements, their cognate transcription factors, and regulatory DNA sequence variants.

Genome Res 2018 10 23;28(10):1577-1588. Epub 2018 Aug 23.

McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

-regulatory elements (CRE), short DNA sequences through which transcription factors (TFs) exert regulatory control on gene expression, are postulated to be the major sites of causal sequence variation underlying the genetics of complex traits and diseases. We present integrative analyses, combining high-throughput genomic and epigenomic data with sequence-based computations, to identify the causal transcriptional components in a given tissue. We use data on adult human hearts to demonstrate that (1) sequence-based predictions detect numerous, active, tissue-specific CREs missed by experimental observations, (2) learned sequence features identify the cognate TFs, (3) CRE variants are specifically associated with cardiac gene expression, and (4) a significant fraction of the heritability of exemplar cardiac traits (QT interval, blood pressure, pulse rate) is attributable to these variants. This general systems approach can thus identify candidate causal variants and the components of gene regulatory networks (GRN) to enable understanding of the mechanisms of complex disorders on a tissue- or cell-type basis.
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http://dx.doi.org/10.1101/gr.234633.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6169896PMC
October 2018

Glucocorticoid receptor recruits to enhancers and drives activation by motif-directed binding.

Genome Res 2018 09 10;28(9):1272-1284. Epub 2018 Aug 10.

Graduate Program in Computational Biology and Bioinformatics, Duke University, Durham, North Carolina 27708, USA.

Glucocorticoids are potent steroid hormones that regulate immunity and metabolism by activating the transcription factor (TF) activity of glucocorticoid receptor (GR). Previous models have proposed that DNA binding motifs and sites of chromatin accessibility predetermine GR binding and activity. However, there are vast excesses of both features relative to the number of GR binding sites. Thus, these features alone are unlikely to account for the specificity of GR binding and activity. To identify genomic and epigenetic contributions to GR binding specificity and the downstream changes resultant from GR binding, we performed hundreds of genome-wide measurements of TF binding, epigenetic state, and gene expression across a 12-h time course of glucocorticoid exposure. We found that glucocorticoid treatment induces GR to bind to nearly all pre-established enhancers within minutes. However, GR binds to only a small fraction of the set of accessible sites that lack enhancer marks. Once GR is bound to enhancers, a combination of enhancer motif composition and interactions between enhancers then determines the strength and persistence of GR binding, which consequently correlates with dramatic shifts in enhancer activation. Over the course of several hours, highly coordinated changes in TF binding and histone modification occupancy occur specifically within enhancers, and these changes correlate with changes in the expression of nearby genes. Following GR binding, changes in the binding of other TFs precede changes in chromatin accessibility, suggesting that other TFs are also sensitive to genomic features beyond that of accessibility.
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http://dx.doi.org/10.1101/gr.233346.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120625PMC
September 2018

Evaluation of chromatin accessibility in prefrontal cortex of individuals with schizophrenia.

Nat Commun 2018 08 7;9(1):3121. Epub 2018 Aug 7.

Center for Genomic and Computational Biology, Duke University, Durham, NC, 27708, USA.

Schizophrenia genome-wide association studies have identified >150 regions of the genome associated with disease risk, yet there is little evidence that coding mutations contribute to this disorder. To explore the mechanism of non-coding regulatory elements in schizophrenia, we performed ATAC-seq on adult prefrontal cortex brain samples from 135 individuals with schizophrenia and 137 controls, and identified 118,152 ATAC-seq peaks. These accessible chromatin regions in the brain are highly enriched for schizophrenia SNP heritability. Accessible chromatin regions that overlap evolutionarily conserved regions exhibit an even higher heritability enrichment, indicating that sequence conservation can further refine functional risk variants. We identify few differences in chromatin accessibility between cases and controls, in contrast to thousands of age-related differential accessible chromatin regions. Altogether, we characterize chromatin accessibility in the human prefrontal cortex, the effect of schizophrenia and age on chromatin accessibility, and provide evidence that our dataset will allow for fine mapping of risk variants.
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http://dx.doi.org/10.1038/s41467-018-05379-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6081462PMC
August 2018

Pre-established Chromatin Interactions Mediate the Genomic Response to Glucocorticoids.

Cell Syst 2018 08 18;7(2):146-160.e7. Epub 2018 Jul 18.

University Program in Genetics and Genomics, Duke University, Durham, NC 27708, USA; Center for Genomic and Computational Biology, Duke University, Durham, NC 27708, USA; Computational Biology and Bioinformatics Graduate Program, Duke University, Durham, NC 27708, USA; Department of Biostatistics and Bioinformatics, Duke University Medical Center, Durham, NC 27710, USA; Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA; Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA. Electronic address:

The glucocorticoid receptor (GR) is a hormone-inducible transcription factor involved in metabolic and anti-inflammatory gene expression responses. To investigate what controls interactions between GR binding sites and their target genes, we used in situ Hi-C to generate high-resolution, genome-wide maps of chromatin interactions before and after glucocorticoid treatment. We found that GR binding to the genome typically does not cause new chromatin interactions to target genes but instead acts through chromatin interactions that already exist prior to hormone treatment. Both glucocorticoid-induced and glucocorticoid-repressed genes increased interactions with distal GR binding sites. In addition, while glucocorticoid-induced genes increased interactions with transcriptionally active chromosome compartments, glucocorticoid-repressed genes increased interactions with transcriptionally silent compartments. Lastly, while the architectural DNA-binding proteins CTCF and RAD21 were bound to most chromatin interactions, we found that glucocorticoid-responsive chromatin interactions were depleted for CTCF binding but enriched for RAD21. Together, these findings offer new insights into the mechanisms underlying GC-mediated gene activation and repression.
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http://dx.doi.org/10.1016/j.cels.2018.06.007DOI Listing
August 2018

Transcriptome-wide association study of schizophrenia and chromatin activity yields mechanistic disease insights.

Nat Genet 2018 04 9;50(4):538-548. Epub 2018 Apr 9.

Department of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, MA, USA.

Genome-wide association studies (GWAS) have identified over 100 risk loci for schizophrenia, but the causal mechanisms remain largely unknown. We performed a transcriptome-wide association study (TWAS) integrating a schizophrenia GWAS of 79,845 individuals from the Psychiatric Genomics Consortium with expression data from brain, blood, and adipose tissues across 3,693 primarily control individuals. We identified 157 TWAS-significant genes, of which 35 did not overlap a known GWAS locus. Of these 157 genes, 42 were associated with specific chromatin features measured in independent samples, thus highlighting potential regulatory targets for follow-up. Suppression of one identified susceptibility gene, mapk3, in zebrafish showed a significant effect on neurodevelopmental phenotypes. Expression and splicing from the brain captured most of the TWAS effect across all genes. This large-scale connection of associations to target genes, tissues, and regulatory features is an essential step in moving toward a mechanistic understanding of GWAS.
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http://dx.doi.org/10.1038/s41588-018-0092-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5942893PMC
April 2018

Comparative Serum Challenges Show Divergent Patterns of Gene Expression and Open Chromatin in Human and Chimpanzee.

Genome Biol Evol 2018 03;10(3):826-839

Department of Biology, University of Massachusetts Amherst.

Humans experience higher rates of age-associated diseases than our closest living evolutionary relatives, chimpanzees. Environmental factors can explain many of these increases in disease risk, but species-specific genetic changes can also play a role. Alleles that confer increased disease susceptibility later in life can persist in a population in the absence of selective pressure if those changes confer positive adaptation early in life. One age-associated disease that disproportionately affects humans compared with chimpanzees is epithelial cancer. Here, we explored genetic differences between humans and chimpanzees in a well-defined experimental assay that mimics gene expression changes that happen during cancer progression: A fibroblast serum challenge. We used this assay with fibroblasts isolated from humans and chimpanzees to explore species-specific differences in gene expression and chromatin state with RNA-Seq and DNase-Seq. Our data reveal that human fibroblasts increase expression of genes associated with wound healing and cancer pathways; in contrast, chimpanzee gene expression changes are not concentrated around particular functional categories. Chromatin accessibility dramatically increases in human fibroblasts, yet decreases in chimpanzee cells during the serum response. Many regions of opening and closing chromatin are in close proximity to genes encoding transcription factors or genes involved in wound healing processes, further supporting the link between changes in activity of regulatory elements and changes in gene expression. Together, these expression and open chromatin data show that humans and chimpanzees have dramatically different responses to the same physiological stressor, and how a core physiological process can evolve quickly over relatively short evolutionary time scales.
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http://dx.doi.org/10.1093/gbe/evy041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5848805PMC
March 2018

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

CRISPR-Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome.

Nat Biotechnol 2017 06 3;35(6):561-568. Epub 2017 Apr 3.

Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA.

Large genome-mapping consortia and thousands of genome-wide association studies have identified non-protein-coding elements in the genome as having a central role in various biological processes. However, decoding the functions of the millions of putative regulatory elements discovered in these studies remains challenging. CRISPR-Cas9-based epigenome editing technologies have enabled precise perturbation of the activity of specific regulatory elements. Here we describe CRISPR-Cas9-based epigenomic regulatory element screening (CERES) for improved high-throughput screening of regulatory element activity in the native genomic context. Using dCas9 repressor and dCas9 activator constructs and lentiviral single guide RNA libraries to target DNase I hypersensitive sites surrounding a gene of interest, we carried out both loss- and gain-of-function screens to identify regulatory elements for the β-globin and HER2 loci in human cells. CERES readily identified known and previously unidentified regulatory elements, some of which were dependent on cell type or direction of perturbation. This technology allows the high-throughput functional annotation of putative regulatory elements in their native chromosomal context.
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http://dx.doi.org/10.1038/nbt.3853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5462860PMC
June 2017

Direct GR Binding Sites Potentiate Clusters of TF Binding across the Human Genome.

Cell 2016 Aug;166(5):1269-1281.e19

Center for Genomic & Computational Biology, Duke University, Durham, NC 27708, USA; Department of Biostatistics & Bioinformatics, Duke University, Durham, NC 27708, USA. Electronic address:

The glucocorticoid receptor (GR) binds the human genome at >10,000 sites but only regulates the expression of hundreds of genes. To determine the functional effect of each site, we measured the glucocorticoid (GC) responsive activity of nearly all GR binding sites (GBSs) captured using chromatin immunoprecipitation (ChIP) in A549 cells. 13% of GBSs assayed had GC-induced activity. The responsive sites were defined by direct GR binding via a GC response element (GRE) and exclusively increased reporter-gene expression. Meanwhile, most GBSs lacked GC-induced reporter activity. The non-responsive sites had epigenetic features of steady-state enhancers and clustered around direct GBSs. Together, our data support a model in which clusters of GBSs observed with ChIP-seq reflect interactions between direct and tethered GBSs over tens of kilobases. We further show that those interactions can synergistically modulate the activity of direct GBSs and may therefore play a major role in driving gene activation in response to GCs.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5046229PMC
http://dx.doi.org/10.1016/j.cell.2016.07.049DOI Listing
August 2016

Differential contribution of cis-regulatory elements to higher order chromatin structure and expression of the CFTR locus.

Nucleic Acids Res 2016 Apr 15;44(7):3082-94. Epub 2015 Dec 15.

Human Molecular Genetics Program, Lurie Children's Research Center, Chicago, IL 60614, USA Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA

Higher order chromatin structure establishes domains that organize the genome and coordinate gene expression. However, the molecular mechanisms controlling transcription of individual loci within a topological domain (TAD) are not fully understood. The cystic fibrosis transmembrane conductance regulator (CFTR) gene provides a paradigm for investigating these mechanisms.CFTR occupies a TAD bordered by CTCF/cohesin binding sites within which are cell-type-selective cis-regulatory elements for the locus. We showed previously that intronic and extragenic enhancers, when occupied by specific transcription factors, are recruited to the CFTR promoter by a looping mechanism to drive gene expression. Here we use a combination of CRISPR/Cas9 editing of cis-regulatory elements and siRNA-mediated depletion of architectural proteins to determine the relative contribution of structural elements and enhancers to the higher order structure and expression of the CFTR locus. We found the boundaries of the CFTRTAD are conserved among diverse cell types and are dependent on CTCF and cohesin complex. Removal of an upstream CTCF-binding insulator alters the interaction profile, but has little effect on CFTR expression. Within the TAD, intronic enhancers recruit cell-type selective transcription factors and deletion of a pivotal enhancer element dramatically decreases CFTR expression, but has minor effect on its 3D structure.
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http://dx.doi.org/10.1093/nar/gkv1358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4838340PMC
April 2016

Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements.

Nat Methods 2015 Dec 26;12(12):1143-9. Epub 2015 Oct 26.

Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA.

Epigenome editing with the CRISPR (clustered, regularly interspaced, short palindromic repeats)-Cas9 platform is a promising technology for modulating gene expression to direct cell phenotype and to dissect the causal epigenetic mechanisms of gene regulation. Fusions of nuclease-inactive dCas9 to the Krüppel-associated box (KRAB) repressor (dCas9-KRAB) can silence target gene expression, but the genome-wide specificity and the extent of heterochromatin formation catalyzed by dCas9-KRAB are not known. We targeted dCas9-KRAB to the HS2 enhancer, a distal regulatory element that orchestrates the expression of multiple globin genes, and observed highly specific induction of H3K9 trimethylation (H3K9me3) at the enhancer and decreased chromatin accessibility of both the enhancer and its promoter targets. Targeted epigenetic modification of HS2 silenced the expression of multiple globin genes, with minimal off-target changes in global gene expression. These results demonstrate that repression mediated by dCas9-KRAB is sufficiently specific to disrupt the activity of individual enhancers via local modification of the epigenome.
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http://dx.doi.org/10.1038/nmeth.3630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4666778PMC
December 2015

Genetic variants and cellular stressors associated with exfoliation syndrome modulate promoter activity of a lncRNA within the LOXL1 locus.

Hum Mol Genet 2015 Nov 25;24(22):6552-63. Epub 2015 Aug 25.

Division of Ophthalmology, Department of Neurosciences, University of the Witwatersrand, Johannesburg, South Africa and.

Exfoliation syndrome (XFS) is a common, age-related, systemic fibrillinopathy. It greatly increases risk of exfoliation glaucoma (XFG), a major worldwide cause of irreversible blindness. Coding variants in the lysyl oxidase-like 1 (LOXL1) gene are strongly associated with XFS in all studied populations, but a functional role for these variants has not been established. To identify additional candidate functional variants, we sequenced the entire LOXL1 genomic locus (∼40 kb) in 50 indigenous, black South African XFS cases and 50 matched controls. The variants with the strongest evidence of association were located in a well-defined 7-kb region bounded by the 3'-end of exon 1 and the adjacent region of intron 1 of LOXL1. We replicated this finding in US Caucasian (91 cases/1031 controls), German (771 cases/1365 controls) and Japanese (1484 cases/1188 controls) populations. The region of peak association lies upstream of LOXL1-AS1, a long non-coding RNA (lncRNA) encoded on the opposite strand of LOXL1. We show that this region contains a promoter and, importantly, that the strongly associated XFS risk alleles in the South African population are functional variants that significantly modulate the activity of this promoter. LOXL1-AS1 expression is also significantly altered in response to oxidative stress in human lens epithelial cells and in response to cyclic mechanical stress in human Schlemm's canal endothelial cells. Taken together, these findings support a functional role for the LOXL1-AS1 lncRNA in cellular stress response and suggest that dysregulation of its expression by genetic risk variants plays a key role in XFS pathogenesis.
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http://dx.doi.org/10.1093/hmg/ddv347DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4614704PMC
November 2015

Genomic analysis reveals distinct mechanisms and functional classes of SOX10-regulated genes in melanocytes.

Hum Mol Genet 2015 Oct 23;24(19):5433-50. Epub 2015 Jul 23.

Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA,

SOX10 is required for melanocyte development and maintenance, and has been linked to melanoma initiation and progression. However, the molecular mechanisms by which SOX10 guides the appropriate gene expression programs necessary to promote the melanocyte lineage are not fully understood. Here we employ genetic and epigenomic analysis approaches to uncover novel genomic targets and previously unappreciated molecular roles of SOX10 in melanocytes. Through global analysis of SOX10-binding sites and epigenetic characteristics of chromatin states, we uncover an extensive catalog of SOX10 targets genome-wide. Our findings reveal that SOX10 predominantly engages 'open' chromatin regions and binds to distal regulatory elements, including novel and previously known melanocyte enhancers. Integrated chromatin occupancy and transcriptome analysis suggest a role for SOX10 in both transcriptional activation and repression to regulate functionally distinct classes of genes. We demonstrate that distinct epigenetic signatures and cis-regulatory sequence motifs predicted to bind putative co-regulatory transcription factors define SOX10-activated and SOX10-repressed target genes. Collectively, these findings uncover a central role of SOX10 as a global regulator of gene expression in the melanocyte lineage by targeting diverse regulatory pathways.
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http://dx.doi.org/10.1093/hmg/ddv267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4572067PMC
October 2015

Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators.

Genome Res 2015 Aug 29;25(8):1158-69. Epub 2015 May 29.

Department of Biomedical Engineering, Duke University, Durham, North Carolina 27708, USA; Center for Genomic and Computational Biology, Duke University, Durham, North Carolina 27708, USA; Department of Orthopaedic Surgery, Duke University Medical Center, Durham, North Carolina 27710, USA.

Genome engineering technologies based on the CRISPR/Cas9 and TALE systems are enabling new approaches in science and biotechnology. However, the specificity of these tools in complex genomes and the role of chromatin structure in determining DNA binding are not well understood. We analyzed the genome-wide effects of TALE- and CRISPR-based transcriptional activators in human cells using ChIP-seq to assess DNA-binding specificity and RNA-seq to measure the specificity of perturbing the transcriptome. Additionally, DNase-seq was used to assess genome-wide chromatin remodeling that occurs as a result of their action. Our results show that these transcription factors are highly specific in both DNA binding and gene regulation and are able to open targeted regions of closed chromatin independent of gene activation. Collectively, these results underscore the potential for these technologies to make precise changes to gene expression for gene and cell therapies or fundamental studies of gene function.
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http://dx.doi.org/10.1101/gr.179044.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4510000PMC
August 2015

Regulation of chromatin accessibility and Zic binding at enhancers in the developing cerebellum.

Nat Neurosci 2015 May 6;18(5):647-56. Epub 2015 Apr 6.

Department of Neurobiology, Duke University Medical Center, Durham, North Carolina, USA.

To identify chromatin mechanisms of neuronal differentiation, we characterized chromatin accessibility and gene expression in cerebellar granule neurons (CGNs) of the developing mouse. We used DNase-seq to map accessibility of cis-regulatory elements and RNA-seq to profile transcript abundance across postnatal stages of neuronal differentiation in vivo and in culture. We observed thousands of chromatin accessibility changes as CGNs differentiated, and verified, using H3K27ac ChIP-seq, reporter gene assays and CRISPR-mediated activation, that many of these regions function as neuronal enhancers. Motif discovery in differentially accessible chromatin regions suggested a previously unknown role for the Zic family of transcription factors in CGN maturation. We confirmed the association of Zic with these elements by ChIP-seq and found, using knockdown, that Zic1 and Zic2 are required for coordinating mature neuronal gene expression patterns. Together, our data reveal chromatin dynamics at thousands of gene regulatory elements that facilitate the gene expression patterns necessary for neuronal differentiation and function.
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http://dx.doi.org/10.1038/nn.3995DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4414887PMC
May 2015

Interactions of chromatin context, binding site sequence content, and sequence evolution in stress-induced p53 occupancy and transactivation.

PLoS Genet 2015 Jan 8;11(1):e1004885. Epub 2015 Jan 8.

Environmental Genomics Group, Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, United States of America.

Cellular stresses activate the tumor suppressor p53 protein leading to selective binding to DNA response elements (REs) and gene transactivation from a large pool of potential p53 REs (p53REs). To elucidate how p53RE sequences and local chromatin context interact to affect p53 binding and gene transactivation, we mapped genome-wide binding localizations of p53 and H3K4me3 in untreated and doxorubicin (DXR)-treated human lymphoblastoid cells. We examined the relationships among p53 occupancy, gene expression, H3K4me3, chromatin accessibility (DNase 1 hypersensitivity, DHS), ENCODE chromatin states, p53RE sequence, and evolutionary conservation. We observed that the inducible expression of p53-regulated genes was associated with the steady-state chromatin status of the cell. Most highly inducible p53-regulated genes were suppressed at baseline and marked by repressive histone modifications or displayed CTCF binding. Comparison of p53RE sequences residing in different chromatin contexts demonstrated that weaker p53REs resided in open promoters, while stronger p53REs were located within enhancers and repressed chromatin. p53 occupancy was strongly correlated with similarity of the target DNA sequences to the p53RE consensus, but surprisingly, inversely correlated with pre-existing nucleosome accessibility (DHS) and evolutionary conservation at the p53RE. Occupancy by p53 of REs that overlapped transposable element (TE) repeats was significantly higher (p<10-7) and correlated with stronger p53RE sequences (p<10-110) relative to nonTE-associated p53REs, particularly for MLT1H, LTR10B, and Mer61 TEs. However, binding at these elements was generally not associated with transactivation of adjacent genes. Occupied p53REs located in L2-like TEs were unique in displaying highly negative PhyloP scores (predicted fast-evolving) and being associated with altered H3K4me3 and DHS levels. These results underscore the systematic interaction between chromatin status and p53RE context in the induced transactivation response. This p53 regulated response appears to have been tuned via evolutionary processes that may have led to repression and/or utilization of p53REs originating from primate-specific transposon elements.
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http://dx.doi.org/10.1371/journal.pgen.1004885DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4287438PMC
January 2015

Distinct properties of cell-type-specific and shared transcription factor binding sites.

Mol Cell 2013 Oct 26;52(1):25-36. Epub 2013 Sep 26.

HudsonAlpha Institute for Biotechnology, 601 Genome Way, Huntsville, AL 35806, USA.

Most human transcription factors bind a small subset of potential genomic sites and often use different subsets in different cell types. To identify mechanisms that govern cell-type-specific transcription factor binding, we used an integrative approach to study estrogen receptor α (ER). We found that ER exhibits two distinct modes of binding. Shared sites, bound in multiple cell types, are characterized by high-affinity estrogen response elements (EREs), inaccessible chromatin, and a lack of DNA methylation, while cell-specific sites are characterized by a lack of EREs, co-occurrence with other transcription factors, and cell-type-specific chromatin accessibility and DNA methylation. These observations enabled accurate quantitative models of ER binding that suggest tethering of ER to one-third of cell-specific sites. The distinct properties of cell-specific binding were also observed with glucocorticoid receptor and for ER in primary mouse tissues, representing an elegant genomic encoding scheme for generating cell-type-specific gene regulation.
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http://dx.doi.org/10.1016/j.molcel.2013.08.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3811135PMC
October 2013

Patterns of regulatory activity across diverse human cell types predict tissue identity, transcription factor binding, and long-range interactions.

Genome Res 2013 May 12;23(5):777-88. Epub 2013 Mar 12.

Program in Computational Biology and Bioinformatics, Duke University, Durham, North Carolina 27710, USA.

Regulatory elements recruit transcription factors that modulate gene expression distinctly across cell types, but the relationships among these remains elusive. To address this, we analyzed matched DNase-seq and gene expression data for 112 human samples representing 72 cell types. We first defined more than 1800 clusters of DNase I hypersensitive sites (DHSs) with similar tissue specificity of DNase-seq signal patterns. We then used these to uncover distinct associations between DHSs and promoters, CpG islands, conserved elements, and transcription factor motif enrichment. Motif analysis within clusters identified known and novel motifs in cell-type-specific and ubiquitous regulatory elements and supports a role for AP-1 regulating open chromatin. We developed a classifier that accurately predicts cell-type lineage based on only 43 DHSs and evaluated the tissue of origin for cancer cell types. A similar classifier identified three sex-specific loci on the X chromosome, including the XIST lincRNA locus. By correlating DNase I signal and gene expression, we predicted regulated genes for more than 500K DHSs. Finally, we introduce a web resource to enable researchers to use these results to explore these regulatory patterns and better understand how expression is modulated within and across human cell types.
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http://dx.doi.org/10.1101/gr.152140.112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3638134PMC
May 2013

The accessible chromatin landscape of the human genome.

Nature 2012 Sep;489(7414):75-82

Department of Genome Sciences, University of Washington, Seattle, Washington 98195, USA.

DNase I hypersensitive sites (DHSs) are markers of regulatory DNA and have underpinned the discovery of all classes of cis-regulatory elements including enhancers, promoters, insulators, silencers and locus control regions. Here we present the first extensive map of human DHSs identified through genome-wide profiling in 125 diverse cell and tissue types. We identify ∼2.9 million DHSs that encompass virtually all known experimentally validated cis-regulatory sequences and expose a vast trove of novel elements, most with highly cell-selective regulation. Annotating these elements using ENCODE data reveals novel relationships between chromatin accessibility, transcription, DNA methylation and regulatory factor occupancy patterns. We connect ∼580,000 distal DHSs with their target promoters, revealing systematic pairing of different classes of distal DHSs and specific promoter types. Patterning of chromatin accessibility at many regulatory regions is organized with dozens to hundreds of co-activated elements, and the transcellular DNase I sensitivity pattern at a given region can predict cell-type-specific functional behaviours. The DHS landscape shows signatures of recent functional evolutionary constraint. However, the DHS compartment in pluripotent and immortalized cells exhibits higher mutation rates than that in highly differentiated cells, exposing an unexpected link between chromatin accessibility, proliferative potential and patterns of human variation.
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http://dx.doi.org/10.1038/nature11232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3721348PMC
September 2012

A region of the nucleosome required for multiple types of transcriptional silencing in Saccharomyces cerevisiae.

Genetics 2011 Jul 5;188(3):535-48. Epub 2011 May 5.

Institute for Genome Sciences and Policy and Biochemistry Department, Duke University, Durham, North Carolina 27710, USA.

Extended heterochromatin domains, which are repressive to transcription and help define centromeres and telomeres, are formed through specific interactions between silencing proteins and nucleosomes. This study reveals that in Saccharomyces cerevisiae, the same nucleosomal surface is critical for the formation of multiple types of heterochromatin, but not for local repression mediated by a related transcriptional repressor. Thus, this region of the nucleosome may be generally important to long-range silencing. In S. cerevisiae, the Sir proteins perform long-range silencing, whereas the Sum1 complex acts locally to repress specific genes. A mutant form of Sum1p, Sum1-1p, achieves silencing in the absence of Sir proteins. A genetic screen identified mutations in histones H3 and H4 that disrupt Sum1-1 silencing and fall in regions of the nucleosome previously known to disrupt Sir silencing and rDNA silencing. In contrast, no mutations were identified that disrupt wild-type Sum1 repression. Mutations that disrupt silencing fall in two regions of the nucleosome, the tip of the H3 tail and a surface of the nucleosomal core (LRS domain) and the adjacent base of the H4 tail. The LRS/H4 tail region interacts with the Sir3p bromo-adjacent homology (BAH) domain to facilitate Sir silencing. By analogy, this study is consistent with the LRS/H4 tail region interacting with Orc1p, a paralog of Sir3p, to facilitate Sum1-1 silencing. Thus, the LRS/H4 tail region of the nucleosome may be relatively accessible and facilitate interactions between silencing proteins and nucleosomes to stabilize long-range silencing.
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http://dx.doi.org/10.1534/genetics.111.129197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3176532PMC
July 2011

Evolution of new function through a single amino acid change in the yeast repressor Sum1p.

Mol Cell Biol 2008 Apr 11;28(8):2567-78. Epub 2008 Feb 11.

Institute for Genome Sciences and Policy, Box 3382, Duke University, Durham, NC 27710, USA.

The SUM1-1 mutation is an example of a single amino acid change that results in new function. Wild-type Sum1p in Saccharomyces cerevisiae is a DNA-binding repressor that acts locally, whereas mutant Sum1-1p forms an extended repressive chromatin structure. By characterizing a panel of mutations in which various amino acids replaced the critical residue, threonine 988, we found that threonine was required for wild-type function and that in the absence of threonine the association of Sum1p with DNA was reduced. Isoleucine, the amino acid in mutant Sum1-1p, was required for the novel spreading property. Thus, the SUM1-1 mutation results in both a loss and a gain of function. The presence of isoleucine caused Sum1-1p to self-associate, a property that may promote spreading. In addition, isoleucine enabled Sum1-1p to associate with the origin recognition complex (ORC) and accumulate near ORC binding sites. Thus, both threonine and isoleucine at position 988 enable Sum1p to form intermolecular interactions. We propose that interaction domains may be hotspots for gain-of-function mutations because alterations in such domains have the potential to redirect a protein to new sets of binding partners. In addition, self-association of chromatin proteins may promote the formation of extended chromatin structures.
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http://dx.doi.org/10.1128/MCB.01785-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2293110PMC
April 2008

Autophosphorylation of Ser66 on Xenopus Myt1 is a prerequisite for meiotic inactivation of Myt1.

Cell Cycle 2006 Feb 15;5(4):421-7. Epub 2006 Feb 15.

Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.

Myt1 is a dual-specificity kinase that contributes to the regulation of the cell cycle by adding inhibitory phosphates to the cyclin-dependent kinases (Cdk/cyclins). Myt1 is found to be phosphorylated and less active in M-phase compared to interphase. Although Myt1 can be phosphorylated by several different kinases in vitro, it is not well understood how Myt1 is regulated in vivo. Additionally, the interplay between phosphorylation by other kinases and autophosphorylation has not been investigated. Since phosphorylation is an important mode of regulation for Myt1, we have investigated the properties and physiological significance of the autophosphorylation of Myt1 from Xenopus laevis (XMyt1). Using MALDI mass spectrometry we have identified Ser66 and Ser76 as autophosphorylation sites. Autophosphorylation is important for the activity of XMyt1 in intact cells, as found by comparing the timing of the cell cycle in Xenopus oocytes expressing either exogenous wild type XMyt1 or its autophosphorylation site mutants. Specifically, S66A is significantly more potent than wild type XMyt1 at delaying entry into meiosis and concomitantly is hypophosphorylated as evident by a loss of mobility shift. However, this cannot be accounted for by a simple increase in kinase activity towards Cdk/cyclins in vitro. We therefore propose that Myt1 catalyzed autophosphorylation of residue S66 is a prerequisite and/or trigger for the further phosphorylation and inactivation of Myt1. Thus autophosphorylation of Myt1 is a novel inhibitory mechanism that adds another layer of complexity to the phosphorylation-dependent mechanism of Myt1 regulation.
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http://dx.doi.org/10.4161/cc.5.4.2492DOI Listing
February 2006

Experimental validation of the docking orientation of Cdc25 with its Cdk2-CycA protein substrate.

Biochemistry 2005 Dec;44(50):16563-73

Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.

Cdc25 phosphatases are key activators of the eukaryotic cell cycle and compelling anticancer targets because their overexpression has been associated with numerous cancers. However, drug discovery targeting these phosphatases has been hampered by the lack of structural information about how Cdc25s interact with their native protein substrates, the cyclin-dependent kinases. Herein, we predict a docked orientation for Cdc25B with its Cdk2-pTpY-CycA protein substrate by a rigid-body docking method and refine the docked models with full-scale molecular dynamics simulations and minimization. We validate the stable ensemble structure experimentally by a variety of in vitro and in vivo techniques. Specifically, we compare our model with a crystal structure of the substrate-trapping mutant of Cdc25B. We identify and validate in vivo a novel hot-spot residue on Cdc25B (Arg492) that plays a central role in protein substrate recognition. We identify a hot-spot residue on the substrate Cdk2 (Asp206) and confirm its interaction with hot-spot residues on Cdc25 using hot-spot swapping and double mutant cycles to derive interaction energies. Our experimentally validated model is consistent with previous studies of Cdk2 and its interaction partners and initiates the opportunity for drug discovery of inhibitors that target the remote binding sites of this protein-protein interaction.
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http://dx.doi.org/10.1021/bi0516879DOI Listing
December 2005