Transgenic Mouse Assays Research Articles

Discovery of novel cyclopentane carboxylic acids as potent and selective inhibitors of NaV1.7

Discovery efforts leading to the identification of cyclopentane carboxylic acid 31, a potent inhibitor of NaV1.7 that showed high selectivity over NaV1.5 and exhibited robust analgesic effects in an inherited erythromelalgia (IEM) transgenic mouse assay, are described herein. Key design elements that culminated in the discovery of 31 include exploration of proline substituents, replacement of the proline warhead with cyclopentane carboxylic acid, that led to significantly boosted NaV1.7 potency, and replacement of the metabolically labile adamantane motif with the 2,6-dichlorobenzyl substituted piperidine system, that addressed metabolic instability and led to a significant improvement in PK.

Genetic and phenotypic stability of poliovirus shed from infants who received novel type 2 or Sabin type 2 oral poliovirus vaccines in Panama: an analysis of two clinical trials

Sabin strains used in oral poliovirus vaccines (OPV) can revert to virulence and, in rare instances, cause disease or generate vaccine-derived strains leading to outbreaks in areas of low immunisation coverage. A novel OPV2 (nOPV2) was designed to stabilise the viral genome against reversion and reduce recombination events that might lead to virulent strains. In this study, we evaluated the genetic and phenotypic stability of shed poliovirus following administration of one dose of monovalent OPV2 (mOPV2) or nOPV2 to infants aged 18-22 weeks. In two similarly designed clinical trials (NCT02521974 and NCT03554798) conducted in Panama, infants aged 18-22-weeks, after immunisation with three doses of bivalent OPV (types 1 and 3) and one dose of inactivated poliovirus vaccine, were administered one or two doses of mOPV2 or nOPV2. In this analysis of two clinical trials, faecally shed polioviruses following one dose of mOPV2 or nOPV2 were isolated from stools meeting predetermined criteria related to sample timing and viral presence and quantity and assessed for nucleotide polymorphisms using next-generation sequencing. A transgenic mouse neurovirulence test was adapted to assess the effect of the possible phenotypic reversion of shed mOPV2 and nOPV2 with a logistic regression model. Of the 91 eligible samples, 86 were able to be sequenced, with 72 evaluated in the transgenic mouse assay. Sabin-2 poliovirus reverts rapidly at nucleotide 481, the primary attenuation site in domain V of the 5' untranslated region of the genome. There was no evidence of neurovirulence-increasing polymorphisms in domain V of shed nOPV2. Reversion of shed Sabin-2 virus corresponded with unadjusted paralysis rates of 47·6% at the 4 log10 50% cell culture infectious dose (CCID50) and 76·7% at the 5 log10 CCID50 inoculum levels, with rates of 2·8% for 4 log10 CCID50 and 11·8% for 5 log10 CCID50 observed for shed nOPV2 samples. The estimated adjusted odds ratio at 4·5 log10 of 0·007 (95% CI 0·002-0·023; p<0·0001) indicates significantly reduced odds of mouse paralysis from virus obtained from nOPV2 recipients compared with mOPV2 recipients. The data indicate increased genetic stability of domain V of nOPV2 relative to mOPV2, with significantly lower neurovirulence of shed nOPV2 virus compared with shed mOPV2. While this vaccine is currently being deployed under an emergency use listing, the data on the genetic stability of nOPV2 will support further regulatory and policy decision-making regarding use of nOPV2 in outbreak responses. Bill & Melinda Gates Foundation.

In vivo dissection of a clustered-CTCF domain boundary reveals developmental principles of regulatory insulation

Vertebrate genomes organize into topologically associating domains, delimited by boundaries that insulate regulatory elements from nontarget genes. However, how boundary function is established is not well understood. Here, we combine genome-wide analyses and transgenic mouse assays to dissect the regulatory logic of clustered-CCCTC-binding factor (CTCF) boundaries in vivo, interrogating their function at multiple levels: chromatin interactions, transcription and phenotypes. Individual CTCF binding site (CBS) deletions revealed that the characteristics of specific sites can outweigh other factors such as CBS number and orientation. Combined deletions demonstrated that CBSs cooperate redundantly and provide boundary robustness. We show that divergent CBS signatures are not strictly required for effective insulation and that chromatin loops formed by nonconvergently oriented sites could be mediated by a loop interference mechanism. Further, we observe that insulation strength constitutes a quantitative modulator of gene expression and phenotypes. Our results highlight the modular nature of boundaries and their control over developmental processes.

Single-cell chromatin accessibility maps reveal regulatory programs driving early mouse organogenesis.

During mouse embryonic development, pluripotent cells rapidly divide and diversify, yet the regulatory programs that define the cell repertoire for each organ remain ill-defined. To delineate comprehensive chromatin landscapes during early organogenesis, we mapped chromatin accessibility in 19,453 single nuclei from mouse embryos at 8.25 days post-fertilisation. Identification of cell type-specific regions of open chromatin pinpointed two TAL1-bound endothelial enhancers, which we validated using transgenic mouse assays. Integrated gene expression and transcription factor motif enrichment analyses highlighted cell type-specific transcriptional regulators. Subsequent in vivo experiments in zebrafish revealed a role for the ETS factor FEV in endothelial identity downstream of ETV2 (Etsrp in zebrafish). Concerted in vivo validation experiments in mouse and zebrafish thus illustrate how single-cell open chromatin maps, representative of a mammalian embryo, provide access to the regulatory blueprint for mammalian organogenesis.

Identification of a limb enhancer that is removed by pathogenic deletions downstream of the SHOX gene

Haploinsufficiency of the human SHOX gene causes Léri-Weill dyschondrosteosis (LWD), characterized by shortening of the middle segments of the limbs and Madelung deformity of the wrist. As many as 35% of LWD cases are caused by deletions of non-coding sequences downstream of SHOX that presumably remove an enhancer or enhancers necessary for SHOX expression in developing limbs. We searched for these active sequences using a transgenic mouse assay and identified a 563 basepair (bp) enhancer with specific activity in the limb regions where SHOX functions. This enhancer has previously escaped notice because of its poor evolutionary conservation, although it does contain 100 bp that are conserved in non-rodent mammals. A primary cell luciferase assay confirmed the enhancer activity of the conserved core sequence and demonstrated that putative HOX binding sites are required for its activity. This enhancer is removed in most non-coding deletions that cause LWD. However, we did not identify any likely pathogenic variants of the enhancer in a screen of 124 LWD individuals for whom no causative mutation had been found, suggesting that only larger deletions in the region commonly cause LWD. We hypothesize that loss of this enhancer contributes to the pathogenicity of deletions downstream of SHOX.

Differential analysis of chromatin accessibility and histone modifications for predicting mouse developmental enhancers.

Enhancers are distal cis-regulatory elements that modulate gene expression. They are depleted of nucleosomes and enriched in specific histone modifications; thus, calling DNase-seq and histone mark ChIP-seq peaks can predict enhancers. We evaluated nine peak-calling algorithms for predicting enhancers validated by transgenic mouse assays. DNase and H3K27ac peaks were consistently more predictive than H3K4me1/2/3 and H3K9ac peaks. DFilter and Hotspot2 were the best DNase peak callers, while HOMER, MUSIC, MACS2, DFilter and F-seq were the best H3K27ac peak callers. We observed that the differential DNase or H3K27ac signals between two distant tissues increased the area under the precision-recall curve (PR-AUC) of DNase peaks by 17.5–166.7% and that of H3K27ac peaks by 7.1–22.2%. We further improved this differential signal method using multiple contrast tissues. Evaluated using a blind test, the differential H3K27ac signal method substantially improved PR-AUC from 0.48 to 0.75 for predicting heart enhancers. We further validated our approach using postnatal retina and cerebral cortex enhancers identified by massively parallel reporter assays, and observed improvements for both tissues. In summary, we compared nine peak callers and devised a superior method for predicting tissue-specific mouse developmental enhancers by reranking the called peaks.

GATA-related hematologic disorders.

The transcription factors GATA1 and GATA2 are fundamental regulators of hematopoiesis and have overlapping expression profiles. GATA2 is expressed in hematopoietic stem cells and early erythroid-megakaryocytic progenitors and activates a certain set of early-phase genes, including the GATA2 gene itself. GATA2 also initiates GATA1 gene expression. In contrast, GATA1 is expressed in relatively mature erythroid progenitors and facilitates the expression of genes associated with differentiation, including the GATA1 gene itself; however, GATA1 represses the expression of GATA2. Switching the GATA factors from GATA2 to GATA1 appears to be one of the key regulatory mechanisms underlying erythroid differentiation. Loss-of-function analyses using mice invivo have indicated that GATA2 and GATA1 are functionally nonredundant and that neither can compensate for the absence of the other. However, transgenic expression of GATA2 under the transcriptional regulation of the Gata1 gene rescues lethal dyserythropoiesis in GATA1-deficient mice, illustrating that the dynamic expression profiles of these GATA factors are critically important for the maintenance of hematopoietic homeostasis. Analysis of naturally occurring leukemias in GATA1-knockdown mice revealed that leukemic stem cells undergo functional alterations in response to exposure to chemotherapeutic agents. This mechanism may also underlie the aggravating features of relapsing leukemias. Recent hematologic analyses have suggested that disturbances in the balance of the GATA factors are associated with specific types of hematopoietic disorders. Here, we describe GATA1- and GATA2-related hematologic diseases, focusing on the regulation of GATA factor gene expression.

Sp7/Osterix Is Restricted to Bone-Forming Vertebrates where It Acts as a Dlx Co-factor in Osteoblast Specification

In extant species, bone formation is restricted to vertebrate species. Sp7/Osterix is a key transcriptional determinant of bone-secreting osteoblasts. We performed Sp7 chromatin immunoprecipitation sequencing analysis identifying a large set of predicted osteoblast enhancers and validated a subset of these in cell culture and transgenic mouse assays. Sp family members bind GC-rich target sequences through their zinc finger domain. Several lines of evidence suggest that Sp7 acts differently, engaging osteoblast targets in Dlx-containing regulatory complexes bound to AT-rich motifs. Amino acid differences in the Sp7 zinc finger domain reduce Sp7's affinity for the Sp family consensus GC-box target; Dlx5 binding maps to this domain of Sp7. The data support a model in which Dlx recruitment of Sp7 to osteoblast enhancers underlies Sp7-directed osteoblast specification. Because an Sp7-like zinc finger variant is restricted to vertebrates, the emergence of an Sp7 member within the Sp family was likely closely coupled to the evolution of bone-forming vertebrates.

Structure-function analysis of mouse Sry reveals dual essential roles of the C-terminal polyglutamine tract in sex determination.

The mammalian sex-determining factor SRY comprises a conserved high-mobility group (HMG) box DNA-binding domain and poorly conserved regions outside the HMG box. Mouse Sry is unusual in that it includes a C-terminal polyglutamine (polyQ) tract that is absent in nonrodent SRY proteins, and yet, paradoxically, is essential for male sex determination. To dissect the molecular functions of this domain, we generated a series of Sry mutants, and studied their biochemical properties in cell lines and transgenic mouse embryos. Sry protein lacking the polyQ domain was unstable, due to proteasomal degradation. Replacing this domain with irrelevant sequences stabilized the protein but failed to restore Sry's ability to up-regulate its key target gene SRY-box 9 (Sox9) and its sex-determining function in vivo. These functions were restored only when a VP16 transactivation domain was substituted. We conclude that the polyQ domain has important roles in protein stabilization and transcriptional activation, both of which are essential for male sex determination in mice. Our data disprove the hypothesis that the conserved HMG box domain is the only functional domain of Sry, and highlight an evolutionary paradox whereby mouse Sry has evolved a novel bifunctional module to activate Sox9 directly, whereas SRY proteins in other taxa, including humans, seem to lack this ability, presumably making them dependent on partner proteins(s) to provide this function.

Tissue-specific SMARCA4 binding at active and repressed regulatory elements during embryogenesis.

The SMARCA4 (also known as BRG1 in humans) chromatin remodeling factor is critical for establishing lineage-specific chromatin states during early mammalian development. However, the role of SMARCA4 in tissue-specific gene regulation during embryogenesis remains poorly defined. To investigate the genome-wide binding landscape of SMARCA4 in differentiating tissues, we engineered a Smarca4FLAG knock-in mouse line. Using ChIP-seq, we identified ∼51,000 SMARCA4-associated regions across six embryonic mouse tissues (forebrain, hindbrain, neural tube, heart, limb, and face) at mid-gestation (E11.5). The majority of these regions was distal from promoters and showed dynamic occupancy, with most distal SMARCA4 sites (73%) confined to a single or limited subset of tissues. To further characterize these regions, we profiled active and repressive histone marks in the same tissues and examined the intersection of informative chromatin states and SMARCA4 binding. This revealed distinct classes of distal SMARCA4-associated elements characterized by activating and repressive chromatin signatures that were associated with tissue-specific up- or down-regulation of gene expression and relevant active/repressed biological pathways. We further demonstrate the predicted active regulatory properties of SMARCA4-associated elements by retrospective analysis of tissue-specific enhancers and direct testing of SMARCA4-bound regions in transgenic mouse assays. Our results indicate a dual active/repressive function of SMARCA4 at distal regulatory sequences in vivo and support its role in tissue-specific gene regulation during embryonic development.

A survey of ancient conserved non-coding elements in the PAX6 locus reveals a landscape of interdigitated cis-regulatory archipelagos

Biological differences between cell types and developmental processes are characterised by differences in gene expression profiles. Gene-distal enhancers are key components of the regulatory networks that specify the tissue-specific expression patterns driving embryonic development and cell fate decisions, and variations in their sequences are a major contributor to genetic disease and disease susceptibility. Despite advances in the methods for discovery of putative cis-regulatory sequences, characterisation of their spatio-temporal enhancer activities in a mammalian model system remains a major bottle-neck. We employed a strategy that combines gnathostome sequence conservation with transgenic mouse and zebrafish reporter assays to survey the genomic locus of the developmental control gene PAX6 for the presence of novel cis-regulatory elements. Sequence comparison between human and the cartilaginous elephant shark (Callorhinchus milii) revealed several ancient gnathostome conserved non-coding elements (agCNEs) dispersed widely throughout the PAX6 locus, extending the range of the known PAX6 cis-regulatory landscape to contain the full upstream PAX6-RCN1 intergenic region. Our data indicates that ancient conserved regulatory sequences can be tested effectively in transgenic zebrafish even when not conserved in zebrafish themselves. The strategy also allows efficient dissection of compound regulatory regions previously assessed in transgenic mice. Remarkable overlap in expression patterns driven by sets of agCNEs indicates that PAX6 resides in a landscape of multiple tissue-specific regulatory archipelagos.

Rapid and Pervasive Changes in Genome-wide Enhancer Usage during Mammalian Development

Enhancers are distal regulatory elements that canactivate tissue-specific gene expression and areabundant throughout mammalian genomes. Although substantial progress has been made toward genome-wide annotation of mammalian enhancers, their temporal activity patterns and global contributions in the context of developmental invivo processes remain poorly explored. Here we used epigenomic profiling for H3K27ac, a mark of active enhancers, coupled to transgenic mouse assays to examine the genome-wide utilization of enhancers in three different mouse tissues across seven developmental stages. The majority of the ∼90,000 enhancers identified exhibited tightly temporally restricted predicted activity windows and were associated with stage-specific biological functions and regulatory pathways in individual tissues. Comparative genomic analysis revealed that evolutionary conservation of enhancers decreases following midgestation across all tissues examined. The dynamic enhancer activities uncovered in this study illuminate rapid and pervasive temporal invivo changes in enhancer usage that underlie processes central to development and disease.

Verification of the in vivo activity of three distinct cis‐acting elements within the Gata1 gene promoter‐proximal enhancer in mice

The transcription factor GATA1 is essential for erythroid and megakaryocytic cell differentiation. Gata1 hematopoietic regulatory domain (G1HRD) has been shown to recapitulate endogenous Gata1 gene expression in transgenic mouse assays in vivo. G1HRD contains a promoter-proximal enhancer composed of a GATA-palindrome motif, four CP2-binding sites and two CACCC boxes. We prepared transgenic reporter mouse lines in which green fluorescent protein and β-galactosidase expression are driven by wild-type G1HRD (as a positive control) and the G1HRD harboring mutations within these cis-acting elements (as the experimental conditions), respectively. Exploiting this transgenic dual reporter (TDR) assay, we show here that in definitive erythropoiesis, G1HRD activity was markedly affected by individual mutations in the GATA-palindrome motif and the CACCC boxes. Mutation of CP2-binding sites also moderately decreased G1HRD activity. The combined mutation of the CP2-binding sites and the GATA-palindrome motif resulted in complete loss of G1HRD activity. In contrast, in primitive erythroid cells, individual mutations of each element did not affect G1HRD activity; G1HRD activity was abolished only when these three mutations were combined. These results thus show that all three elements independently and cooperatively contribute to G1HRD activity in vivo in definitive erythropoiesis, although these are contributing redundantly to primitive erythropoiesis.

Carcinogenicity and Chronic Rodent Toxicity of the Selective Progesterone Receptor Modulator Ulipristal Acetate

Carcinogenic properties of ulipristal acetate (UPA), a selective progesterone receptor modulator developed for the treatment of benign gynecological conditions such as uterine fibroids, were assessed in a 26-week carcinogenicity study in transgenic TgRasH2 mice and a 104-week study in Sprague Dawley rats. Dose levels used in the mouse study were 15, 45, or 130 mg/kg/day and for the ratstudy the doses used were 1, 3, or 10 mg/kg/day. Vehicle and water controls were part of both studies and a positive control, N-Nitroso-N-methylurea intraperitoneally, was included in the transgenic mouse assay. Survival at all dose levels was similar to vehicle controls in both sexes of both species and there was no evidence of any UPA-induced carcinogenicity in either species. Rats receiving UPA had decreased incidences of fibroadenomas and adenocarcinomas in the mammary gland in all treated groups. UPA exposure [AUC(0-24h)] at the highest dose in rats was 67 times human therapeutic exposure at 10 mg/day. In mice, no tumor of any type increased at UPA exposures up to 313 times of therapeutic exposure. UPA-related findings in mice were limited to organ weight changes in the liver, pituitary, thyroid/parathyroid glands, and epididymis as well as minimal panlobular hepatocellular hypertrophy in male and female mice receiving 130 mg/kg/day. Rats had UPA-related non-neoplastic findings in the reproductive system (mammary gland, ovary, uterus, vagina, seminal vesicle, prostate), endocrine system (adrenal, pituitary), thymus, muscle, liver, pancreas and lungs most of which are considered to be due to exaggerated pharmacological action of the compound.

DEHP: Genotoxicity and potential carcinogenic mechanisms—A review

Di(ethylhexyl) phthalate (DEHP) is a manufactured chemical commonly added to plastics: it is a ubiquitous environmental contaminant to which humans are exposed through multiple routes. DEHP is a rodent carcinogen with an extensive data base on genotoxicity and related effects spanning several decades. Although DEHP has been reported to be negative in most non-mammalian in vitro mutation assays, most studies were performed under conditions of concurrent cytotoxicity, precipitation, or irrelevant metabolic activation. However, a number of in vitro rodent tissue assays have reported DEHP to be positive for effects on chromosomes, spindle, and mitosis. A robust database shows that DEHP increases transformation and inhibits apoptosis in Syrian hamster embryo cells. In a transgenic mouse assay, in vivo DEHP exposure increased the mutation frequency only in the liver, which is the target organ for cancer. In vitro exposure of human cells or tissues to DEHP induced DNA damage; altered mitotic rate, apoptosis, and cell proliferation; increased proliferation, tumor mobility, and invasiveness of tumor cell lines; and activated a number of nuclear receptors. DEHP has been shown to be an agonist for CAR2, a novel constitutive androstane receptor occurring only in humans. Environmental exposures of humans to DEHP have been associated with DNA damage. After taking into account study context and relevant issues affecting interpretation, in vitro studies reported that a similar DEHP concentration range induced both mutagenic and non-mutagenic effects in human tissues and, using a much more limited rodent database, transformation of embryonic rodent tissues. The human and rodent data suggest that DEHP induces cancer through multiple molecular signals, including DNA damage. The analyses presented here may provide guidance for similar data sets used in structure-activity relationships, computational-toxicology extrapolations, and attempts to extrapolate in vitro results to predict in vivo effects for hazard characterization.

Identification of an Enhancer in the Ad4BP/SF-1 Gene Specific for Fetal Leydig Cells

Adrenal 4 binding protein/steroidogenic factor 1 (Ad4BP/SF-1) (Nr5a1) is a nuclear receptor essential for reproductive tissue development and endocrine regulation. This factor is expressed in steroidogenic tissues (e.g. adrenal glands and gonads), and expression of this factor is tightly regulated in a tissue and cell type-specific manner. Our previous studies have identified tissue and cell type-specific enhancers in the introns of the Ad4BP/SF-1 gene in fetal adrenal glands, ventromedial hypothalamus, and pituitary gonadotrope. Characterization of the enhancers had provided new insights into tissue and cell development. However, these studies have failed to identify any gonad-specific enhancer. Here, we identified a fetal Leydig cell-specific enhancer in the upstream region of the mouse Ad4BP/SF-1 gene using transgenic mouse assays. Alignment of the upstream regions among vertebrate animal species demonstrated that the enhancer consisted of three conserved regions, whereby the most highly conserved region contained an Ad4BP/SF-1 binding sequence and an E-box. Mutation of each sequence abolished the enhancer activity and led to a loss of reporter gene expression. These results suggested that Ad4BP/SF-1 gene expression in the fetal Leydig cell is regulated by a yet unidentified E-box binding protein(s) and by an autoregulatory loop formed by Ad4BP/SF-1. Although fetal Leydig cells have been thought to play crucial roles for masculinization of various fetal tissues through androgen production, other functions have remained elusive. Our identification of a fetal Leydig cell-specific enhancer in the Ad4BP/SF-1 gene would be a powerful tool to address these gaps in the knowledge base.

Re‐evaluation of the big blue® mouse assay of propiconazole suggests lack of mutagenicity

Propiconazole (PPZ) is a conazole fungicide that is not mutagenic, clastogenic, or DNA damaging in standard in vitro and in vivo genetic toxicity tests for gene mutations, chromosome aberrations, DNA damage, and cell transformation. However, it was demonstrated to be a male mouse liver carcinogen when administered in food for 24 months only at a concentration of 2,500 ppm that exceeded the maximum tolerated dose based on increased mortality, decreased body weight gain, and the presence of liver necrosis. PPZ was subsequently tested for mutagenicity in the Big Blue® transgenic mouse assay at the 2,500 ppm dose, and the result was reported as positive by Ross et al. ([2009]: Mutagenesis 24:149-152). Subsets of the mutants from the control and PPZ-exposed groups were sequenced to determine the mutation spectra and a multivariate clustering analysis method purportedly substantiated the increase in mutant frequency with PPZ (Ross and Leavitt. [2010]: Mutagenesis 25:231-234). However, as reported here, the results of the analysis of the mutation spectra using a conventional method indicated no treatment-related differences in the spectra. In this article, we re-examine the Big Blue® mouse findings with PPZ and conclude that the compound does not act as a mutagen in vivo.

Loss-of-Function Mutation in a Repressor Module of Human-Specifically Activated Enhancer HACNS1

The cis-regulatory element contributed to gaining humanness is of great interest in human evolutionary studies. A human-accelerated region exceeding neutral evolutionary rates, termed HACNS1, was recently reported as a positively selected sequence acquiring novel TF-binding sites responsible for human-specific gain of limb enhancer function. However, another possibility is loss of function in repressor element in HACNS1. Signature of the human substitutions in the 81-bp region infers that a GC-biased gene conversion (BGC) might create these seemingly excessive substitutions. To evaluate the 81-bp function, we performed transgenic mouse assay of the HACNS1 construct lacking the 81-bp region. The deleted construct showed similar enhancer activity to the intact human HACNS1, suggesting that the function of the human 81-bp region is not an activating enhancer but rather a disrupted repressor. This result infers that loss of function in the HACNS1 81-bp region, possibly via a BGC, played an important role in human-specific evolution.

A compendium of genome-wide hematopoietic transcription factor maps supports the identification of gene regulatory control mechanisms

Key regulators of blood stem cell differentiation into the various mature hematopoietic lineages are commonly encoded by transcription factor genes. Elucidation of transcriptional regulatory mechanisms therefore holds great promise in advancing our understanding of both normal and malignant hematopoiesis. Recent technological advances have enabled the generation of genome-wide transcription factor binding maps using chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq). However, transcription factors operate in a combinatorial fashion suggesting that integrated analysis of genome-wide maps for multiple transcription factors will be essential to fully exploit these new genome-scale data sets. Here we have generated a compendium that integrates 53 ChIP-Seq studies covering 30 factors across all major hematopoietic lineages with a total of 754,380 binding peaks. We also used transgenic mouse assays to validate a newly predicted transcriptional enhancer. Integrated analysis of all 53 ChIP-Seq studies demonstrated that cell-type identity exerts a larger influence on global transcription factor binding patterns than the nature of the individual transcription factors. Furthermore, regions highlighted by multifactor binding within specific gene loci overlap with known regulatory elements and also provide a useful guide for identifying novel elements, as demonstrated by transgenic analysis of a previously unrecognized enhancer in the Maml3 gene locus. The ChIP-Seq compendium described here provides a valuable resource for the wider research community by accelerating the discovery of transcriptional mechanisms operating in the hematopoietic system.

ChIP-Seq identification of weakly conserved heart enhancers

Accurate control of tissue-specific gene expression plays a pivotal role in heart development, but few cardiac transcriptional enhancers have thus far been identified. Extreme noncoding-sequence conservation has successfully predicted enhancers that are active in many tissues but has failed to identify substantial numbers of heart-specific enhancers. Here, we used ChIP-Seq with the enhancer-associated protein p300 from mouse embryonic day 11.5 heart tissue to identify over 3,000 candidate heart enhancers genome wide. Compared to enhancers active in other tissues we studied at this time point, most candidate heart enhancers were less deeply conserved in vertebrate evolution. Nevertheless, transgenic mouse assays of 130 candidate regions revealed that most function reproducibly as enhancers active in the heart, irrespective of their degree of evolutionary constraint. These results provide evidence for a large population of poorly conserved heart enhancers and suggest that the evolutionary conservation of embryonic enhancers can vary depending on tissue type.