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1.
Cell Stem Cell ; 30(1): 96-111.e6, 2023 01 05.
Article in English | MEDLINE | ID: mdl-36516837

ABSTRACT

The efficacy and safety of gene-therapy strategies for indications like tissue damage hinge on precision; yet, current methods afford little spatial or temporal control of payload delivery. Here, we find that tissue-regeneration enhancer elements (TREEs) isolated from zebrafish can direct targeted, injury-associated gene expression from viral DNA vectors delivered systemically in small and large adult mammalian species. When employed in combination with CRISPR-based epigenome editing tools in mice, zebrafish TREEs stimulated or repressed the expression of endogenous genes after ischemic myocardial infarction. Intravenously delivered recombinant AAV vectors designed with a TREE to direct a constitutively active YAP factor boosted indicators of cardiac regeneration in mice and improved the function of the injured heart. Our findings establish the application of contextual enhancer elements as a potential therapeutic platform for spatiotemporally controlled tissue regeneration in mammals.


Subject(s)
Enhancer Elements, Genetic , Genetic Therapy , Heart , Myocardial Infarction , Myocytes, Cardiac , Regeneration , Animals , Mice , Cell Proliferation , Heart/physiology , Myocardial Infarction/genetics , Myocardial Infarction/therapy , Myocytes, Cardiac/metabolism , Zebrafish/genetics , Genetic Therapy/methods , Regeneration/genetics
2.
Nat Methods ; 18(8): 965-974, 2021 08.
Article in English | MEDLINE | ID: mdl-34341582

ABSTRACT

CRISPR-Cas9 technologies have dramatically increased the ease of targeting DNA sequences in the genomes of living systems. The fusion of chromatin-modifying domains to nuclease-deactivated Cas9 (dCas9) has enabled targeted epigenome editing in both cultured cells and animal models. However, delivering large dCas9 fusion proteins to target cells and tissues is an obstacle to the widespread adoption of these tools for in vivo studies. Here, we describe the generation and characterization of two conditional transgenic mouse lines for epigenome editing, Rosa26:LSL-dCas9-p300 for gene activation and Rosa26:LSL-dCas9-KRAB for gene repression. By targeting the guide RNAs to transcriptional start sites or distal enhancer elements, we demonstrate regulation of target genes and corresponding changes to epigenetic states and downstream phenotypes in the brain and liver in vivo, and in T cells and fibroblasts ex vivo. These mouse lines are convenient and valuable tools for facile, temporally controlled, and tissue-restricted epigenome editing and manipulation of gene expression in vivo.


Subject(s)
CRISPR-Cas Systems , Epigenesis, Genetic , Epigenome , Gene Editing/methods , Gene Expression Regulation , Animals , Brain/metabolism , Female , Fibroblasts/metabolism , Humans , Liver/metabolism , Male , Mice , Mice, Transgenic , T-Lymphocytes/metabolism
3.
Nat Med ; 25(3): 427-432, 2019 03.
Article in English | MEDLINE | ID: mdl-30778238

ABSTRACT

Duchenne muscular dystrophy (DMD) is a monogenic disorder and a candidate for therapeutic genome editing. There have been several recent reports of genome editing in preclinical models of Duchenne muscular dystrophy1-6, however, the long-term persistence and safety of these genome editing approaches have not been addressed. Here we show that genome editing and dystrophin protein restoration is sustained in the mdx mouse model of Duchenne muscular dystrophy for 1 year after a single intravenous administration of an adeno-associated virus that encodes CRISPR (AAV-CRISPR). We also show that AAV-CRISPR is immunogenic when administered to adult mice7; however, humoral and cellular immune responses can be avoided by treating neonatal mice. Additionally, we describe unintended genome and transcript alterations induced by AAV-CRISPR that should be considered for the development of AAV-CRISPR as a therapeutic approach. This study shows the potential of AAV-CRISPR for permanent genome corrections and highlights aspects of host response and alternative genome editing outcomes that require further study.


Subject(s)
CRISPR-Cas Systems/genetics , Gene Editing/methods , Muscular Dystrophy, Duchenne/therapy , Animals , Animals, Newborn , CRISPR-Cas Systems/immunology , Dependovirus , Disease Models, Animal , Dystrophin/genetics , Genetic Therapy/methods , Genetic Vectors , Immunity, Cellular/immunology , Immunity, Humoral/immunology , Mice , Mice, Inbred mdx , Muscular Dystrophy, Duchenne/genetics
4.
Nat Commun ; 9(1): 1674, 2018 04 26.
Article in English | MEDLINE | ID: mdl-29700298

ABSTRACT

CRISPR-Cas9 transcriptional repressors have emerged as robust tools for disrupting gene regulation in vitro but have not yet been adapted for systemic delivery in adult animal models. Here we describe a Staphylococcus aureus Cas9-based repressor (dSaCas9KRAB) compatible with adeno-associated viral (AAV) delivery. To evaluate dSaCas9KRAB efficacy for gene silencing in vivo, we silenced transcription of Pcsk9, a regulator of cholesterol levels, in the liver of adult mice. Systemic administration of a dual-vector AAV8 system expressing dSaCas9KRAB and a Pcsk9-targeting guide RNA (gRNA) results in significant reductions of serum Pcsk9 and cholesterol levels. Despite a moderate host response to dSaCas9KRAB expression, Pcsk9 repression is maintained for 24 weeks after a single treatment, demonstrating the potential for long-term gene silencing in post-mitotic tissues with dSaCas9KRAB. In vivo programmable gene silencing enables studies that link gene regulation to complex phenotypes and expands the CRISPR-Cas9 perturbation toolbox for basic research and gene therapy applications.


Subject(s)
Bacterial Proteins/metabolism , Endonucleases/metabolism , Gene Silencing , Proprotein Convertase 9/genetics , RNA, Guide, Kinetoplastida/genetics , Staphylococcus aureus/enzymology , Animals , Bacterial Proteins/genetics , CRISPR-Cas Systems , Cholesterol/blood , Endonucleases/genetics , Genetic Therapy , Liver/enzymology , Male , Mice , Mice, Inbred C57BL , Proprotein Convertase 9/blood , RNA, Guide, Kinetoplastida/metabolism , Staphylococcus aureus/genetics , Transcription, Genetic
6.
Dev Cell ; 40(4): 392-404.e5, 2017 02 27.
Article in English | MEDLINE | ID: mdl-28245924

ABSTRACT

Chromatin regulation is a principal mechanism governing animal development, yet it is unclear to what extent structural changes in chromatin underlie tissue regeneration. Non-mammalian vertebrates such as zebrafish activate cardiomyocyte (CM) division after tissue damage to regenerate lost heart muscle. Here, we generated transgenic zebrafish expressing a biotinylatable H3.3 histone variant in CMs and derived cell-type-specific profiles of histone replacement. We identified an emerging program of putative enhancers that revise H3.3 occupancy during regeneration, overlaid upon a genome-wide reduction of H3.3 from promoters. In transgenic reporter lines, H3.3-enriched elements directed gene expression in subpopulations of CMs. Other elements increased H3.3 enrichment and displayed enhancer activity in settings of injury- and/or Neuregulin1-elicited CM proliferation. Dozens of consensus sequence motifs containing predicted transcription factor binding sites were enriched in genomic regions with regeneration-responsive H3.3 occupancy. Thus, cell-type-specific regulatory programs of tissue regeneration can be revealed by genome-wide H3.3 profiling.


Subject(s)
Heart/physiology , Histones/metabolism , Regeneration/physiology , Zebrafish/physiology , Animals , Animals, Genetically Modified , Base Sequence , Binding Sites , Enhancer Elements, Genetic/genetics , Gene Expression Regulation, Developmental , Histones/genetics , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Nucleotide Motifs/genetics , Regeneration/genetics , Transcription Factors/metabolism , Zebrafish/genetics , Zebrafish/metabolism
7.
Nat Rev Genet ; 18(1): 24-40, 2017 01.
Article in English | MEDLINE | ID: mdl-27795562

ABSTRACT

Our understanding of the genetic mechanisms that underlie biological processes has relied extensively on loss-of-function (LOF) analyses. LOF methods target DNA, RNA or protein to reduce or to ablate gene function. By analysing the phenotypes that are caused by these perturbations the wild-type function of genes can be elucidated. Although all LOF methods reduce gene activity, the choice of approach (for example, mutagenesis, CRISPR-based gene editing, RNA interference, morpholinos or pharmacological inhibition) can have a major effect on phenotypic outcomes. Interpretation of the LOF phenotype must take into account the biological process that is targeted by each method. The practicality and efficiency of LOF methods also vary considerably between model systems. We describe parameters for choosing the optimal combination of method and system, and for interpreting phenotypes within the constraints of each method.


Subject(s)
CRISPR-Cas Systems , Gene Silencing , Models, Animal , Morpholinos/pharmacology , Mutagenesis , Mutation/genetics , RNA Interference , Animals , Genotype , Humans , Phenotype , Species Specificity
8.
Nature ; 532(7598): 201-6, 2016 Apr 14.
Article in English | MEDLINE | ID: mdl-27049946

ABSTRACT

How tissue regeneration programs are triggered by injury has received limited research attention. Here we investigate the existence of enhancer regulatory elements that are activated in regenerating tissue. Transcriptomic analyses reveal that leptin b (lepb) is highly induced in regenerating hearts and fins of zebrafish. Epigenetic profiling identified a short DNA sequence element upstream and distal to lepb that acquires open chromatin marks during regeneration and enables injury-dependent expression from minimal promoters. This element could activate expression in injured neonatal mouse tissues and was divisible into tissue-specific modules sufficient for expression in regenerating zebrafish fins or hearts. Simple enhancer-effector transgenes employing lepb-linked sequences upstream of pro- or anti-regenerative factors controlled the efficacy of regeneration in zebrafish. Our findings provide evidence for 'tissue regeneration enhancer elements' (TREEs) that trigger gene expression in injury sites and can be engineered to modulate the regenerative potential of vertebrate organs.


Subject(s)
Enhancer Elements, Genetic/genetics , Organ Specificity/genetics , Regeneration/genetics , Regeneration/physiology , Wound Healing/genetics , Zebrafish/genetics , Zebrafish/physiology , Acetylation , Animal Fins/injuries , Animal Fins/metabolism , Animals , Animals, Newborn , Cell Proliferation , Chromatin Assembly and Disassembly/genetics , Epigenesis, Genetic/genetics , Female , Gene Expression Profiling , Gene Expression Regulation/genetics , Heart , Histones/chemistry , Histones/metabolism , Leptin/biosynthesis , Leptin/genetics , Lysine/metabolism , Male , Mice , Myocytes, Cardiac/cytology , Promoter Regions, Genetic/genetics , Transgenes/genetics , Zebrafish Proteins/genetics
9.
Development ; 143(2): 232-43, 2016 Jan 15.
Article in English | MEDLINE | ID: mdl-26657776

ABSTRACT

In contrast to mammals, adult zebrafish have a high capacity to regenerate damaged or lost myocardium through proliferation of cardiomyocytes spared from damage. The epicardial sheet covering the heart is activated by injury and aids muscle regeneration through paracrine effects and as a multipotent cell source, and has received recent attention as a target in cardiac repair strategies. Although it is recognized that epicardium is required for muscle regeneration and itself has high regenerative potential, the extent of cellular heterogeneity within epicardial tissue is largely unexplored. Here, we performed transcriptome analysis on dozens of epicardial lineage cells purified from zebrafish harboring a transgenic reporter for the pan-epicardial gene tcf21. Hierarchical clustering analysis suggested the presence of at least three epicardial cell subsets defined by expression signatures. We validated many new pan-epicardial and epicardial markers by alternative expression assays. Additionally, we explored the function of the scaffolding protein and main component of caveolae, caveolin 1 (cav1), which was present in each epicardial subset. In BAC transgenic zebrafish, cav1 regulatory sequences drove strong expression in ostensibly all epicardial cells and in coronary vascular endothelial cells. Moreover, cav1 mutant zebrafish generated by genome editing showed grossly normal heart development and adult cardiac anatomy, but displayed profound defects in injury-induced cardiomyocyte proliferation and heart regeneration. Our study defines a new platform for the discovery of epicardial lineage markers, genetic tools, and mechanisms of heart regeneration.


Subject(s)
Caveolin 1/metabolism , Heart/physiology , Pericardium/cytology , Regeneration/physiology , Animals , Caveolin 1/genetics , Myocytes, Cardiac/cytology , Zebrafish , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism
10.
Dev Cell ; 34(4): 387-99, 2015 Aug 24.
Article in English | MEDLINE | ID: mdl-26256209

ABSTRACT

Some organisms, such as adult zebrafish and newborn mice, have the capacity to regenerate heart tissue following injury. Unraveling the mechanisms of heart regeneration is fundamental to understanding why regeneration fails in adult humans. Numerous studies have revealed that nerves are crucial for organ regeneration, thus we aimed to determine whether nerves guide heart regeneration. Here, we show using transgenic zebrafish that inhibition of cardiac innervation leads to reduction of myocyte proliferation following injury. Specifically, pharmacological inhibition of cholinergic nerve function reduces cardiomyocyte proliferation in the injured hearts of both zebrafish and neonatal mice. Direct mechanical denervation impairs heart regeneration in neonatal mice, which was rescued by the administration of neuregulin 1 (NRG1) and nerve growth factor (NGF) recombinant proteins. Transcriptional analysis of mechanically denervated hearts revealed a blunted inflammatory and immune response following injury. These findings demonstrate that nerve function is required for both zebrafish and mouse heart regeneration.


Subject(s)
Cholinergic Neurons/physiology , Heart/innervation , Heart/physiology , Myocytes, Cardiac/cytology , Regeneration , Animals , Animals, Genetically Modified , Animals, Newborn , Cell Proliferation/drug effects , Denervation , Gene Expression Regulation/drug effects , Immunity/drug effects , Immunity/genetics , Inflammation/genetics , Mice , Models, Biological , Molecular Sequence Data , Nerve Growth Factor/pharmacology , Neuregulin-1/pharmacology , Regeneration/drug effects , Synaptic Transmission/drug effects , Vagotomy , Zebrafish
11.
Elife ; 42015 Apr 01.
Article in English | MEDLINE | ID: mdl-25830562

ABSTRACT

Heart regeneration is limited in adult mammals but occurs naturally in adult zebrafish through the activation of cardiomyocyte division. Several components of the cardiac injury microenvironment have been identified, yet no factor on its own is known to stimulate overt myocardial hyperplasia in a mature, uninjured animal. In this study, we find evidence that Neuregulin1 (Nrg1), previously shown to have mitogenic effects on mammalian cardiomyocytes, is sharply induced in perivascular cells after injury to the adult zebrafish heart. Inhibition of Erbb2, an Nrg1 co-receptor, disrupts cardiomyocyte proliferation in response to injury, whereas myocardial Nrg1 overexpression enhances this proliferation. In uninjured zebrafish, the reactivation of Nrg1 expression induces cardiomyocyte dedifferentiation, overt muscle hyperplasia, epicardial activation, increased vascularization, and causes cardiomegaly through persistent addition of wall myocardium. Our findings identify Nrg1 as a potent, induced mitogen for the endogenous adult heart regeneration program.


Subject(s)
Heart/physiology , Mitogens/metabolism , Neuregulin-1/metabolism , Regeneration , Zebrafish Proteins/metabolism , Zebrafish/physiology , Animals , Cardiomegaly/pathology , Cell Proliferation , Echocardiography , Heart Ventricles/pathology , Hyperplasia , Myocardium/metabolism , Myocardium/pathology , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Signal Transduction
12.
Trends Genet ; 29(11): 611-20, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23927865

ABSTRACT

For centuries, philosophers and scientists have been fascinated by the principles and implications of regeneration in lower vertebrate species. Two features have made zebrafish an informative model system for determining mechanisms of regenerative events. First, they are highly regenerative, able to regrow amputated fins, as well as a lesioned brain, retina, spinal cord, heart, and other tissues. Second, they are amenable to both forward and reverse genetic approaches, with a research toolset regularly updated by an expanding community of zebrafish researchers. Zebrafish studies have helped identify new mechanistic underpinnings of regeneration in multiple tissues and, in some cases, have served as a guide for contemplating regenerative strategies in mammals. Here, we review the recent history of zebrafish as a genetic model system for understanding how and why tissue regeneration occurs.


Subject(s)
Animal Fins/physiology , Brain/physiology , Heart/physiology , Regeneration , Retina/physiology , Spinal Cord/physiology , Animals , Models, Animal , Zebrafish/genetics
13.
Curr Biol ; 23(13): 1221-7, 2013 Jul 08.
Article in English | MEDLINE | ID: mdl-23791730

ABSTRACT

A common principle of tissue regeneration is the reactivation of previously employed developmental programs. During zebrafish heart regeneration, cardiomyocytes in the cortical layer of the ventricle induce the transcription factor gene gata4 and proliferate to restore lost muscle. A dynamic cellular mechanism initially creates this cortical muscle in juvenile zebrafish, where a small number of internal cardiomyocytes breach the ventricular wall and expand upon its surface. Here, we find that emergent juvenile cortical cardiomyocytes induce expression of gata4 in a manner similar to during regeneration. Clonal analysis indicates that these cardiomyocytes make biased contributions to build the ventricular wall, whereas gata4(+) cardiomyocytes have little or no proliferation hierarchy during regeneration. Experimental microinjuries or conditions of rapid organismal growth stimulate production of ectopic gata4(+) cortical muscle, implicating biomechanical stress in morphogenesis of this tissue and revealing clonal plasticity. Induced transgenic inhibition defined an essential role for Gata4 activity in morphogenesis of the cortical layer and the preservation of normal cardiac function in growing juveniles, and again in adults during heart regeneration. Our experiments uncover an injury-responsive program that prevents heart failure in juveniles by fortifying the ventricular wall, one that is reiterated in adults to promote regeneration after cardiac damage.


Subject(s)
GATA Transcription Factors/metabolism , Heart Ventricles/metabolism , Myocytes, Cardiac/metabolism , Zebrafish Proteins/metabolism , Zebrafish/growth & development , Zebrafish/genetics , Animals , Animals, Genetically Modified/genetics , Animals, Genetically Modified/growth & development , Animals, Genetically Modified/metabolism , GATA Transcription Factors/genetics , Heart Ventricles/cytology , Heart Ventricles/growth & development , Morphogenesis , Myocytes, Cardiac/cytology , Zebrafish/metabolism , Zebrafish Proteins/genetics
14.
Development ; 140(3): 660-6, 2013 Feb 01.
Article in English | MEDLINE | ID: mdl-23293297

ABSTRACT

Adult mammalian cardiomyocytes have little capacity to proliferate in response to injury, a deficiency that underlies the poor regenerative ability of human hearts after myocardial infarction. By contrast, zebrafish regenerate heart muscle after trauma by inducing proliferation of spared cardiomyocytes, providing a model for identifying manipulations that block or enhance these events. Although direct genetic or chemical screens of heart regeneration in adult zebrafish present several challenges, zebrafish embryos are ideal for high-throughput screening. Here, to visualize cardiomyocyte proliferation events in live zebrafish embryos, we generated transgenic zebrafish lines that employ fluorescent ubiquitylation-based cell cycle indicator (FUCCI) technology. We then performed a chemical screen and identified several small molecules that increase or reduce cardiomyocyte proliferation during heart development. These compounds act via Hedgehog, Insulin-like growth factor or Transforming growth factor ß signaling pathways. Direct examination of heart regeneration after mechanical or genetic ablation injuries indicated that these pathways are activated in regenerating cardiomyocytes and that they can be pharmacologically manipulated to inhibit or enhance cardiomyocyte proliferation during adult heart regeneration. Our findings describe a new screening system that identifies molecules and pathways with the potential to modify heart regeneration.


Subject(s)
Cell Proliferation , Heart/physiology , High-Throughput Screening Assays/methods , Myocytes, Cardiac/cytology , Regeneration , Animals , Animals, Genetically Modified/embryology , Animals, Genetically Modified/metabolism , Animals, Genetically Modified/physiology , Biomarkers/metabolism , Catechols/pharmacology , Cell Count , Cyclohexylamines/pharmacology , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/drug effects , Embryo, Nonmammalian/physiology , Female , Heart/embryology , Hedgehog Proteins/agonists , Hedgehog Proteins/genetics , Hedgehog Proteins/metabolism , Isoquinolines/pharmacology , Male , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction , Thiophenes/pharmacology , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolism , Transgenes , Ubiquitination , Zebrafish/genetics , Zebrafish/injuries , Zebrafish/physiology
15.
Development ; 138(16): 3421-30, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21752928

ABSTRACT

Natural models of heart regeneration in lower vertebrates such as zebrafish are based on invasive surgeries causing mechanical injuries that are limited in size. Here, we created a genetic cell ablation model in zebrafish that facilitates inducible destruction of a high percentage of cardiomyocytes. Cell-specific depletion of over 60% of the ventricular myocardium triggered signs of cardiac failure that were not observed after partial ventricular resection, including reduced animal exercise tolerance and sudden death in the setting of stressors. Massive myocardial loss activated robust cellular and molecular responses by endocardial, immune, epicardial and vascular cells. Destroyed cardiomyocytes fully regenerated within several days, restoring cardiac anatomy, physiology and performance. Regenerated muscle originated from spared cardiomyocytes that acquired ultrastructural and electrophysiological characteristics of de-differentiation and underwent vigorous proliferation. Our study indicates that genetic depletion of cardiomyocytes, even at levels so extreme as to elicit signs of cardiac failure, can be reversed by natural regenerative capacity in lower vertebrates such as zebrafish.


Subject(s)
Heart Failure/genetics , Heart Failure/pathology , Heart/physiology , Myocytes, Cardiac/cytology , Regeneration , Zebrafish/genetics , Zebrafish/physiology , Animals , Cell Death
16.
Genome Res ; 21(7): 1055-64, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21527551

ABSTRACT

The pluripotency control regions (PluCRs) are defined as genomic regions that are bound by POU5F1, SOX2, and NANOG in vivo. We utilized a high-throughput binding assay to record more than 270,000 different DNA/protein binding measurements along incrementally tiled windows of DNA within these PluCRs. This high-resolution binding map is then used to systematically define the context of POU factor binding, and reveals patterns of cooperativity and competition in the pluripotency network. The most prominent pattern is a pervasive binding competition between POU5F1 and the forkhead transcription factors. Like many transcription factors, POU5F1 is co-expressed with a paralog, POU2F1, that shares an apparently identical binding specificity. By analyzing thousands of binding measurements, we discover context effects that discriminate POU2F1 from POU5F1 binding. Proximal NANOG binding promotes POU5F1 binding, whereas nearby SOX2 binding favors POU2F1. We demonstrate by cross-species comparison and by chromatin immunoprecipitation (ChIP) that the contextual sequence determinants learned in vitro are sufficient to predict POU2F1 binding in vivo.


Subject(s)
Genome , Homeodomain Proteins/genetics , Octamer Transcription Factor-1/genetics , Octamer Transcription Factor-3/genetics , Animals , Binding Sites/genetics , Blotting, Western , Cells, Cultured , Chromatin Immunoprecipitation , Chromosome Mapping , Electrophoresis, Polyacrylamide Gel , Gene Expression Regulation, Developmental , High-Throughput Nucleotide Sequencing , Homeodomain Proteins/metabolism , Humans , Octamer Transcription Factor-1/metabolism , Octamer Transcription Factor-3/metabolism , Promoter Regions, Genetic , Protein Binding/genetics , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolism
17.
Genes Dev ; 23(2): 208-22, 2009 Jan 15.
Article in English | MEDLINE | ID: mdl-19171782

ABSTRACT

Oct1 and Oct4 are homologous transcription factors with similar DNA-binding specificities. Here we show that Oct1 is dynamically phosphorylated in vivo following exposure of cells to oxidative and genotoxic stress. We further show that stress regulates the selectivity of both proteins for specific DNA sequences. Mutation of conserved phosphorylation target DNA-binding domain residues in Oct1, and Oct4 confirms their role in regulating binding selectivity. Using chromatin immunoprecipitation, we show that association of Oct4 and Oct1 with a distinct group of in vivo targets is inducible by stress, and that Oct1 is essential for a normal post-stress transcriptional response. Finally, using an unbiased Oct1 target screen we identify a large number of genes targeted by Oct1 specifically under conditions of stress, and show that several of these inducible Oct1 targets are also inducibly bound by Oct4 in embryonic stem cells following stress exposure.


Subject(s)
DNA Damage/physiology , Gene Expression Regulation , Octamer Transcription Factor-1/metabolism , Octamer Transcription Factor-3/metabolism , Oxidative Stress/physiology , Amino Acid Sequence , Animals , Dimerization , HeLa Cells , Humans , Inverted Repeat Sequences/genetics , Mice , Models, Molecular , Molecular Sequence Data , Mutation , Octamer Transcription Factor-1/chemistry , Octamer Transcription Factor-1/genetics , Octamer Transcription Factor-3/chemistry , Octamer Transcription Factor-3/genetics , Phosphorylation , Protein Binding , Protein Structure, Tertiary
18.
Genome Res ; 18(4): 631-9, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18212089

ABSTRACT

The transcription factor POU5F1 is a key regulator of embryonic stem (ES) cell pluripotency and a known oncoprotein. We have developed a novel high-throughput binding assay called MEGAshift (microarray evaluation of genomic aptamers by shift) that we use to pinpoint the exact location, affinity, and stoichiometry of the DNA-protein complexes identified by chromatin immunoprecipitation studies. We consider all genomic regions identified as POU5F1-ChIP-enriched in both human and mouse. Compared with regions that are ChIP-enriched in a single species, we find these regions more likely to be near actively transcribed genes in ES cells. We resynthesize these genomic regions as a pool of tiled 35-mers. This oligonucleotide pool is then assayed for binding to recombinant POU5F1 by gel shift. The degree of binding for each oligonucleotide is accurately measured on a custom oligonucleotide microarray. We explore the relationship between experimentally determined and computationally predicted binding strengths, find many novel functional combinations of POU5F1 half sites, and demonstrate efficient motif discovery by incorporating binding information into a motif finding algorithm. In addition to further refining location studies for transcription factors, this method holds promise for the high-throughput screening of promoters, SNP regions, and epigenetic modifications for factor binding.


Subject(s)
Electrophoretic Mobility Shift Assay/methods , Octamer Transcription Factor-3/metabolism , Oligonucleotide Array Sequence Analysis/methods , Regulatory Elements, Transcriptional , Animals , Binding Sites , Cell Line , Chromatin Immunoprecipitation , DNA/chemistry , DNA/metabolism , Embryonic Stem Cells/metabolism , Genomics , Humans , Mice , Oligonucleotide Probes , Promoter Regions, Genetic
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