DNase I-hypersensitive Regions Research Articles

Genetic architecture of asthma in African American patients

Asthma is a chronic inflammatory disorder with a strong genetic inheritance. Although more than 100 loci were reported through the genome-wide association study of European populations, the genetic underpinning of asthma in African American individuals remains largely elusive. We aimed to identify genetic loci associated with asthma in African American individuals. Three cohorts were genotyped at the Children's Hospital of Philadelphia by using the Illumina single-nucleotide polymorphism array platform. Genotype imputation was performed by using the Trans-Omics for Precision Medicine (TOPMed) reference panel, which includes whole genome sequencing data from more than 100,000 individuals. Ameta-analysis of 3 Children's Hospital of Philadelphia cohorts and 10 Consortium on Asthma among African Ancestry Populations in the Americas cohorts, totaling 19,628 subjects, was conducted to identify genetic loci associated with asthma in African American individuals. Our study identified 12 loci surpassing the classical genome-wide significance threshold (5×10-8). Of those loci, 8 reached the stricter significance threshold (3×10-8). The 9p24.1 locus (rs10975467 [P= 1.63×10-8]) has previously been associated with asthma in European individuals. Six loci are associated with enhancer activities, 2 loci are in DNase I-hypersensitive regions, and all of them are associated with regulatory motifs. Moreover, the locus 11q13.4 (rs7480008) isanexpression quantitative trait locus of XRRA1 in lung(P= 9.4×10-10), and the locus 13q14.3 (rs1543525) is a splicing quantitative trait locus of DHRS12 in lung (P= 1.1×10-13). Our findings provide candidate genetic loci for therapeutic target identification and prioritization for African populations.

Genomic map of candidate human imprint control regions: the imprintome

ABSTRACT Imprinted genes – critical for growth, metabolism, and neuronal function – are expressed from one parental allele. Parent-of-origin-dependent CpG methylation regulates this expression at imprint control regions (ICRs). Since ICRs are established before tissue specification, these methylation marks are similar across cell types. Thus, they are attractive for investigating the developmental origins of adult diseases using accessible tissues, but remain unknown. We determined genome-wide candidate ICRs in humans by performing whole-genome bisulphite sequencing (WGBS) of DNA derived from the three germ layers and from gametes. We identified 1,488 hemi-methylated candidate ICRs, including 19 of 25 previously characterized ICRs (https://humanicr.org/). Gamete methylation approached 0% or 100% in 332 ICRs (178 paternally and 154 maternally methylated), supporting parent-of-origin-specific methylation, and 65% were in well-described CTCF-binding or DNaseI hypersensitive regions. This draft of the human imprintome will allow for the systematic determination of the role of early-acquired imprinting dysregulation in the pathogenesis of human diseases and developmental and behavioural disorders.

Multiple SCN5A variant enhancers modulate its cardiac gene expression and the QT interval

The rationale for genome-wide association study (GWAS) results is sequence variation in cis-regulatory elements (CREs) modulating a target gene's expression as the major cause of trait variation. To understand the complete molecular landscape of one of these GWAS loci, we performed in vitro reporter screens in cardiomyocyte cell lines for CREs overlapping nearly all common variants associated with any of five independent QT interval (QTi)-associated GWAS hits at the SCN5A-SCN10A locus. We identified 13 causal CRE variants using allelic reporter activity, cardiomyocyte nuclear extract-based binding assays, overlap with human cardiac tissue DNaseI hypersensitive regions, and predicted impact of sequence variants on DNaseI sensitivity. Our analyses identified at least one high-confidence causal CRE variant for each of the five sentinel hits that could collectively predict SCN5A cardiac gene expression and QTi association. Although all 13 variants could explain SCN5A gene expression, the highest statistical significance was obtained with seven variants (inclusive of the five above). Thus, multiple, causal, mutually associated CRE variants can underlie GWAS signals.

Information-dense transcription factor binding site clusters identify target genes with similar tissue-wide expression profiles and serve as a buffer against mutations

Background: The distribution and composition of cis-regulatory modules (e.g. transcription factor binding site (TFBS) clusters) in promoters substantially determine gene expression patterns and TF targets, whose expression levels are significantly regulated by TF binding. TF knockdown experiments have revealed correlations between TF binding profiles and gene expression levels. We present a general framework capable of predicting genes with similar tissue-wide expression patterns from activated or repressed TF targets using machine learning to combine TF binding and epigenetic features. Methods: Genes with correlated expression patterns across 53 tissues were identified according to their Bray-Curtis similarity. DNase I-accessible promoter intervals of direct TF target genes were scanned with previously derived information theory-based position weight matrices (iPWMs) of 82 TFs. Features from information density-based TFBS clusters were used to predict target genes with machine learning classifiers. The accuracy, specificity and sensitivity of the classifiers were determined for different feature sets. Mutations in TFBSs were also introduced to in silico examine their impact on cluster densities and the regulatory states of target genes. Results: We initially chose the glucocorticoid receptor gene (NR3C1), whose regulation has been extensively studied, to test this approach. SLC25A32 and TANK were found to exhibit the most similar expression patterns to this gene across 53 tissues. A Decision Tree classifier exhibited the largest area (0.9987 and 0.9956 respectively before and after eliminating inaccessible promoter intervals based on DNase I HyperSensitive Regions (DHSs)) under the Receiver Operating Characteristic (ROC) curve in detecting such coordinately regulated genes. Target gene prediction was confirmed using siRNA knockdown data of TFs, which was more accurate than CRISPR inactivation. In-silico mutation analyses of TFBSs also revealed that one or more information-dense TFBS clusters in promoters are required for accurate target gene prediction. Conclusions: Machine learning based on TFBS information density, organization, and chromatin accessibility accurately identifies gene targets with comparable tissue-wide expression patterns. Multiple, information-dense TFBS clusters in promoters appear to protect promoters from the effects of deleterious binding site mutations in a single TFBS that would effectively alter the expression state of these genes.

Abstract 1574: Computational approach for discovery of regulatory noncoding variants in cancer

Abstract Noncoding mutations in the cancer genome have historically been poorly understood. Recent studies have shown however that mutations in intergenic regions may play an important role in cancer by introducing de novo regulatory elements and thus cis-regulating target gene expression. While mono-allelic expression of the dysregulated gene is an important signature of regulatory noncoding mutations, recognition of this feature has not been implemented by any methods evaluating noncoding mutations to date. To perform genome-wide detection of noncoding mutations with a potential regulatory function, we developed a novel computational approach implementing this allelic specific expression signature (ASE) as a core component, using WGS and RNAseq data as input. We first searched for genes showing ASE across the tumor genome by comparing the minor allele frequency of heterozygous markers between WGS and RNAseq data. We excluded genes known to be in an imprinted locus, as well as sites matching a precomputed set of 5,576,857 problematic polymorphism markers (estimated by analyzing WGS data from 625 normal genomes). Next, the somatic mutations were filtered for regulatory candidates that could introduce a transcription factor (TF) binding motif absent from the reference sequence. We included 614 TF binding motifs from the HOCOMOCO database and performed motif analysis with the FIMO package. Finally, the somatic mutations were assigned to dysregulated genes in the same topologically associating domains in human defined by public Hi-C data and prioritized by open chromatin state defined by DNaseI hypersensitivity regions (ENCODE project). We applied our approach to 22 T-ALL tumor samples collected at Shanghai Children’s Medical Center in a pilot study consisting of 13 diagnosis and 9 relapse samples (9 of these were trios). WGS was performed on both tumor and normal samples and RNAseq was performed for the tumor sample only. We predicted 213 regulatory noncoding mutations (157 unique variants) in total from this cohort, potentially targeting 119 genes. Among the dysregulated targets we found TAL1 and LMO2, which are known TFs playing a key role in T-ALL. TAL1 mutations were identified in a total of 6 tumors (3 trios). Besides the known oncogenic insertion at ~7kb upstream of TAL1 (1 trio), we also discovered a novel mutation in the first intron of TAL1 (2 trios) that may potentially drive ectopic TAL1 expression. Novel LMO2 mutations were found in 6 tumors (3 trios) at 3 different sites. This approach also identified novel targets such as GPSM1 and PTPRN2, shedding light on heretofore unknown players in T-ALL targeted by noncoding mutations. By integrating WGS and RNAseq data, our novel computational framework can identify somatic noncoding mutations in cancer that may introduce de novo regulatory elements, thus driving target gene expression. Wider application of this approach will expand our understanding of the role of noncoding mutations in the cancer genome. Citation Format: Yu Liu, Michael Edmonson, Xiaotu Ma, Michael Rusch, Yongjin Li, Benshang Li, Shuhong Shen, A. Thomas Look, Jinghui Zhang. Computational approach for discovery of regulatory noncoding variants in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1574. doi:10.1158/1538-7445.AM2017-1574

Abstract 287: Characterizing the genetic basis of methylome diversity in histologically normal human lung tissue

Abstract The genetic regulation of the human epigenome is not fully appreciated. Here we describe the effects of genetic variants on the DNA methylome in human lung based on methylation-quantitative trait loci (meQTL) analyses. We report 34,304 cis- and 585 trans-meQTLs, a genetic-epigenetic interaction of surprising magnitude, including a regulatory hotspot. These findings are replicated in both breast and kidney tissues and show distinct patterns: cis-meQTLs mostly localize to CpG sites outside of genes, promoters, and CpG islands (CGIs) while trans-meQTLs are over-represented in promoter CGIs. meQTL SNPs are enriched in CTCF binding sites, DNaseI hypersensitivity regions and histone marks. Importantly, 4 of the 5 established lung cancer risk loci in European ancestry are cis-meQTLs and, in aggregate, cis-meQTLs are enriched for lung cancer risk in a genome-wide analysis of 11,587 subjects. Thus, inherited genetic variation may affect lung carcinogenesis by regulating the human methylome. Citation Format: Jianxin Shi, Crystal Marconett, Jubao Duan, Paula Hyland, Peng Li, Zhaoming Wang, William Wheeler, Mihaela Campan, Jing Huang, Weiyin Zhou, Tim Triche, Laufey Amundadottir, Amy Hutchinson, Po-Han Chen, Beiyun Zhou, Brian Chung, Pier Alberto Bertazzi, Andrew W. Bergen, Mathew Freedman, Diane Lee, Kim Siegmund, Andrew Clay Warner, Ben Berman, Angela C. Pesatori, Zea Borok, Dario Consonni, Nilanjan Chatterjee, Margaret Tucker, Neil Caporaso, Stephen J. Chanock, Ite A. Laird-Offringa, Maria Teresa Landi. Characterizing the genetic basis of methylome diversity in histologically normal human lung tissue. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 287. doi:10.1158/1538-7445.AM2014-287

Nucleosome organization in the vicinity of transcription factor binding sites in the human genome.

BackgroundThe binding of transcription factors (TFs) to specific DNA sequences is an initial and crucial step of transcription. In eukaryotes, this process is highly dependent on the local chromatin state, which can be modified by recruiting chromatin remodelers. However, previous studies have focused mainly on nucleosome occupancy around the TF binding sites (TFBSs) of a few specific TFs. Here, we investigated the nucleosome occupancy profiles around computationally inferred binding sites, based on 519 TF binding motifs, in human GM12878 and K562 cells.ResultsAlthough high nucleosome occupancy is intrinsically encoded at TFBSs in vitro, nucleosomes are generally depleted at TFBSs in vivo, and approximately a quarter of TFBSs showed well-positioned in vivo nucleosomes on both sides. RNA polymerase near the transcription start site (TSS) has a large effect on the nucleosome occupancy distribution around the binding sites located within one kilobase to the nearest TSS; fuzzier nucleosome positioning was thus observed around these sites. In addition, in contrast to yeast, repressors, rather than activators, were more likely to bind to nucleosomal DNA in the human cells, and nucleosomes around repressor sites were better positioned in vivo. Genes with repressor sites exhibiting well-positioned nucleosomes on both sides, and genes with activator sites occupied by nucleosomes had significantly lower expression, suggesting that actions of activators and repressors are associated with the nucleosome occupancy around their binding sites. It was also interesting to note that most of the binding sites, which were not in the DNase I-hypersensitive regions, were cell-type specific, and higher in vivo nucleosome occupancy were observed at these binding sites.ConclusionsThis study demonstrated that RNA polymerase and the functions of bound TFs affected the local nucleosome occupancy around TFBSs, and nucleosome occupancy patterns around TFBSs were associated with the expression levels of target genes.Electronic supplementary materialThe online version of this article (doi: 10.1186/1471-2164-15-493) contains supplementary material, which is available to authorized users.

Characterizing the genetic basis of methylome diversity in histologically normal human lung tissue.

The genetic regulation of the human epigenome is not fully appreciated. Here we describe the effects of genetic variants on the DNA methylome in human lung based on methylation-quantitative trait loci (meQTL) analyses. We report 34,304 cis- and 585 trans-meQTLs, a genetic-epigenetic interaction of surprising magnitude, including a regulatory hotspot. These findings are replicated in both breast and kidney tissues and show distinct patterns: cis-meQTLs mostly localize to CpG sites outside of genes, promoters, and CpG islands (CGIs), while trans-meQTLs are over-represented in promoter CGIs. meQTL SNPs are enriched in CTCF binding sites, DNaseI hypersensitivity regions and histone marks. Importantly, 4 of the 5 established lung cancer risk loci in European ancestry are cis-meQTLs and, in aggregate, cis-meQTLs are enriched for lung cancer risk in a genome-wide analysis of 11,587 subjects. Thus, inherited genetic variation may affect lung carcinogenesis by regulating the human methylome.

Three promoters regulate the transcriptional activity of the human holocarboxylase synthetase gene

Holocarboxylase synthetase (HLCS) is the only protein biotin ligase in the human proteome. HLCS-dependent biotinylation of carboxylases plays crucial roles in macronutrient metabolism. HLCS appears to be an essential part of multiprotein complexes in the chromatin that cause gene repression and contribute toward genome stability. Consistent with these essential functions, HLCS knockdown causes strong phenotypes including shortened life span and low stress resistance in Drosophila melanogaster, and de-repression of long-terminal repeats in humans, other mammalian cell lines and Drosophila. Despite previous observations that the expression of HLCS depends on biotin status in rats and in human cell lines, little is known about the regulation of HLCS expression. The goal of this study was to identify promoters that regulate the expression of the human HLCS gene. Initially, the human HLCS locus was interrogated in silico using predictors of promoters including sequences of HLCS mRNA and expressed sequence tags, CpG islands, histone marks denoting transcriptionally poised chromatin, transcription factor binding sites and DNaseI hypersensitive regions. Our predictions revealed three putative HLCS promoters, denoted P1, P2 and P3. Promoters lacked a TATA box, which is typical for housekeeping genes. When the three promoters were cloned into a luciferase reporter plasmid, reporter gene activity was at least three times background noise in human breast, colon and kidney cell lines; activities consistently followed the pattern P1>>P3>P2. Promoter activity depended on the concentration of biotin in culture media, but the effect was moderate. We conclude that we have identified promoters in the human HLCS gene.

A closer look into DNase I hypersensitivity

DNase I hypersensitivity (DHS) combined with next generation sequencing (DNase-seq) is an efficient way of observing, in a single experiment, the genome-wide chromatin effects associated with the binding of multiple transcription factors. Using quantitative contrasts of DHS before and after estrogen and androgen stimulation in breast and prostate cancer cell lines, we have shown that differential DHS can accurately predict hormone induced transcription factor binding. Despite its effectiveness, the DHS assay can vary significantly depending on the experimental parameters. To increase the robustness of this assay, we have systematically evaluated two major parameters, digestion level and fragment size. We found that while there is a broad range of suitable digestion level, over-digestion dramatically decreases the efficiency of detecting DHS regions. More interestingly, we found that different fragment sizes capture distinct chromatin elements, and thus represent different chromatin structures. We were able to classify different combinations of estrogen receptor coregulators that resulted in different local chromatin structures.

Genomic occupancy of HLH, AP1 and Runx2 motifs within a nuclease sensitive site of the Runx2 gene

Epigenetic mechanisms mediating expression of the Runt-related transcription factor Runx2 are critical for controlling its osteogenic activity during skeletal development. Here, we characterized bona fide regulatory elements within 120 kbp of the endogenous bone-related Runx2 promoter (P1) in osteoblasts by genomic DNase I footprinting and chromatin immuno-precipitations (ChIPs). We identified a ~10 kbp genomic domain spanning the P1 promoter that interacts with acetylated histones H3 and H4 reflecting an open chromatin conformation in MC3T3 osteoblasts. This large chromatin domain contains a single major DNaseI hypersensitive (DHS) region that defines a 0.4 kbp "basal core" promoter. This region encompasses two endogenous genomic protein/DNA interaction sites (i.e., footprints at Activating Protein 1 [AP1], E-box and Runx motifs). Helix-Loop-Helix (HLH)/E-box occupancy and presence of the DHS region persists in several mesenchymal cell types, but AP1 site occupancy occurs only during S phase when Runx2 expression is minimal. Point-mutation of the HLH/E box dramatically reduces basal promoter activity. Our results indicate that the Runx2 P1 promoter utilizes two stable principal protein/DNA interaction domains associated with AP1 and HLH factors. These sites function together with dynamic and developmentally responsive sites in a major DHS region to support epigenetic control of bone-specific transcription when osteoblasts transition into a quiescent or differentiated state.

I-cisTarget: an integrative genomics method for the prediction of regulatory features and cis-regulatory modules

The field of regulatory genomics today is characterized by the generation of high-throughput data sets that capture genome-wide transcription factor (TF) binding, histone modifications, or DNAseI hypersensitive regions across many cell types and conditions. In this context, a critical question is how to make optimal use of these publicly available datasets when studying transcriptional regulation. Here, we address this question in Drosophila melanogaster for which a large number of high-throughput regulatory datasets are available. We developed i-cisTarget (where the ‘i’ stands for integrative), for the first time enabling the discovery of different types of enriched ‘regulatory features’ in a set of co-regulated sequences in one analysis, being either TF motifs or ‘in vivo’ chromatin features, or combinations thereof. We have validated our approach on 15 co-expressed gene sets, 21 ChIP data sets, 628 curated gene sets and multiple individual case studies, and show that meaningful regulatory features can be confidently discovered; that bona fide enhancers can be identified, both by in vivo events and by TF motifs; and that combinations of in vivo events and TF motifs further increase the performance of enhancer prediction.

The immunoglobulin heavy chain gene 3′ enhancers induce Bcl2 deregulation and lymphomagenesis in murine B cells

Human follicular B-cell lymphoma is associated with the t(14;18) chromosomal translocation that juxtaposes the Bcl2 proto-oncogene with the immunoglobulin heavy chain (Igh) locus, resulting in the deregulated expression of Bcl2. Our previous studies have shown that the Igh 3’ enhancers deregulate Bcl2 expression in vitro. However, the effects of the Igh 3’ enhancer elements on Bcl2 expression in vivo are not known. To investigate the role of the Igh 3’ enhancers in Bcl2 deregulation, we used gene targeting to generate knock-in mice in which four DNase I hypersensitive regions from the murine Igh 3’ region were integrated 3’ of the Bcl2 locus. Increased levels of Bcl2 mRNA and protein were observed in the B cells of Igh-3’E-bcl2 mice. B cells from Igh-3’E-bcl2 mice demonstrated an extended survival in vitro compared with B cells from wild-type mice. The Bcl2 promoter shift from P1 (the 5’ promoter) to P2 (the 3’ promoter) was observed in B cells from Igh-3’E-bcl2 mice, similar to human t(14;18) lymphomas. The IgH-3’E-bcl2 mice developed monoclonal B-cell follicular lymphomas, which were slowly progressive. These studies demonstrate that the Igh 3’ enhancers play an important role in the deregulation of Bcl2 and B-cell lymphomagenesis in vivo.

Epigenetic control of expression of the human L‐ and M‐ pigment genes

Epigenetics alters gene expression by chromatin modification without changing the sequence of DNA. DNA methylation is an essential signal for epigenetic gene regulation. Methylation of cytosine bases at CpG dinucleotides in DNA results in chromatin condensation resulting in suppression of gene expression. DNA methylation has been shown to play important roles in cell differentiation, genomic imprinting and X-chromosome inactivation. We compared the CpG methylation patterns of the promoters of the L-opsin gene (OPN1LW) and the M-opsin gene (OPN1MW), plus a DNase I hypersensitive (DHS) site located about 8 kb (kilobases) upstream of the OPN1LW gene. Comparisons were made using the human retinoblastoma cell line WERI, which expresses the L and M opsin genes when treated with thyroid hormone (T3), and a lymphoblastoid cell line GM06990 that does not express these genes. The results showed that the great majority of the 14 CpGs located within the proximal 200 bp (base pairs) of each promoter, plus 20 bp of the 5'-untranslated region, were hypo-methylated in WERI-Rb-1 cells, whether or not treated with T3, but almost totally methylated in the lymphoblastoid cell line. Three of the CpGs that are located beyond 200 bp from the transcription start site of OPN1LW were hyper-methylated in both WERI and lymphoblastoid cells. Significant differential methylation was also observed within the DHS region (24 CpGs). This DHS region contains a highly conserved motif that binds CCCTC-binding factor (CTCF), referred to as a 'chromatin insulator or boundary element', that has been shown to regulate gene expression at several genome locations. The results suggest that DNA methylation is likely to contribute to regulation of expression of the L- and M-opsin genes during differentiation, as well as to the retinal L:M cone ratio. In addition, thyroid hormone induction of the opsin genes does not appear to alter DNA methylation.

A Helix‐Loop‐Helix Motif within a Genomic DNaseI Footprint of the Runx2 Gene Promoter is Essential for Basal Bone Tissue‐Specific Transcription

Transcriptional mechanisms controlling Runx2 levels are critical for its osteogenic function during osteoblast differentiation. Here we examine the chromatin organization of the genomic Runx2 locus in intact cells. Chromatin immuno‐precipitations were performed with antibodies of modified histones and PCR probes spanning 120 kbp surrounding its bone‐specific (P1) promoter. The results show that a ~10 kbp genomic segment around the P1 promoter interacts with highly acetylated histones H3 and H4 reflecting an open chromatin conformation. Within this acetylated domain, we find a single major DNaseI hypersensitive (DHS) region that resolves into a cluster of smaller sites (−0.4 ~0.0 kbp) and coincides with the promoter segment that controls basal Runx2 transcription in osteoblasts. Genomic DNase I footprinting indicates that the DHS region contains several protein/DNA interaction sites (e.g., at AP1, HLH/E‐box and Runx motifs). HLH/E‐box occupancy of the DHS region persists in all mesenchymal cell types examined. Occupancy of the AP1 binding site occurs only during S phase when Runx2 expression is minimal. Point‐mutation of the HLH/E box causes a dramatic reduction of the P1 promoter activity. We propose that protein/DNA interactions at the AP1 and HLH/E box motifs may control Runx2 transcription when osteoblasts transit into a quiescent and differentiated state. Supported by NIH grant AR039588.

Rapid high-throughput analysis of DNaseI hypersensitive sites using a modified Multiplex Ligation-dependent Probe Amplification approach

BackgroundMapping DNaseI hypersensitive sites is commonly used to identify regulatory regions in the genome. However, currently available methods are either time consuming and laborious, expensive or require large numbers of cells. We aimed to develop a quick and straightforward method for the analysis of DNaseI hypersensitive sites that overcomes these problems.ResultsWe have developed a modified Multiplex Ligation-dependent Probe Amplification (MLPA) approach for the identification and analysis of genomic regulatory regions. The utility of this approach was demonstrated by simultaneously analysing 20 loci from the ENCODE project for DNaseI hypersensitivity in a range of different cell lines. We were able to obtain reproducible results with as little as 5 × 104 cells per DNaseI treatment. Our results broadly matched those previously reported by the ENCODE project, and both technical and biological replicates showed high correlations, indicating the sensitivity and reproducibility of this method.ConclusionThis new method will considerably facilitate the identification and analysis of DNaseI hypersensitive sites. Due to the multiplexing potential of MLPA (up to 50 loci can be examined) it is possible to analyse dozens of DNaseI hypersensitive sites in a single reaction. Furthermore, the high sensitivity of MLPA means that fewer than 105 cells per DNaseI treatment can be used, allowing the discovery and analysis of tissue specific regulatory regions without the need for pooling. This method is quick and easy and results can be obtained within 48 hours after harvesting of cells or tissues. As no special equipment is required, this method can be applied by any laboratory interested in the analysis of DNaseI hypersensitive regions.

Sequences in Intron 51 of the von Willebrand Factor Gene Target Promoter Activation to a Subset of Lung Endothelial Cells in Transgenic Mice

In vivo analyses of the VWF promoter previously demonstrated that a fragment spanning sequences -487 to +247 targets promoter activation to brain vascular endothelial cells, whereas a longer fragment including 2182 bp of the 5'-flanking sequences, the first exon, and the first intron activated expression in endothelial cells of the heart and muscles as well as the brain of transgenic mice. These results suggested that additional VWF gene sequences were required for expression in other vascular endothelial cells in vivo. We have now identified a region within intron 51 of the VWF gene that is DNase I-hypersensitive (HSS) specifically in non-endothelial cells and interacts with endothelial and non-endothelial specific complexes that contain YY1. We demonstrate that beta-actin is associated with YY1 specifically in the nucleus of non-endothelial cells and is a component of the nuclear protein complexes that interact with the DNase I-hypersensitive region. In vitro transfection analyses demonstrated that HSS sequences containing this YY1-binding site do not significantly affect VWF promoter activity. However, in vivo analyses demonstrated that addition of these sequences to the VWF promoter (-487 to +247) results in promoter activation in lung and brain vascular endothelial cells. These results demonstrate that the HSS sequences in intron 51 of the VWF gene contain cis-acting elements that are necessary for the VWF gene transcription in a subset of lung endothelial cells in vivo.

Role of a 5′-enhancer in the transcriptional regulation of the human endothelial cell protein C receptor gene

AbstractThe endothelial cell protein C receptor (EPCR) is expressed by endothelial cells of large blood vessels and by hematopoietic stem cells. DNaseI hypersensitive (DH) site mapping across 38 kb of the human EPCR gene (hEPCR) locus identified 3 potential regulatory elements. By itself, the DH region spanning the proximal promoter (PP) was unable to direct cell-specific transcription in transgenic mice. A second DH element, located upstream of PP and termed –5.5HS was hypersensitive only in endothelial cells (ECs) and immature hematopoietic cell lines. Transgenes expressing LacZ under the control of –5.5HS coupled to either PP or the SV40 promoter were able to direct β-galactosidase activity to the endothelium of large vessels during embryogenesis and adulthood. The –5.5HS exhibited enhancer activity that was conferred by the interplay of transcription factors interacting with conserved Ets and composite GATA/Tal1 motifs. The third DH element, located in intron 2, was primarily hypersensitive in EPCR-negative cells, and capable of initiating antisense transcription, suggesting a role in hEPCR silencing. This study identifies critical elements required for the tissue specificity of hEPCR and suggests a mechanism for endothelial and hematopoietic stem cell–specific transcriptional regulation that reflects the common origin of these cell types.

The ups and downs of gene expression and retroviral DNA integration

Unique among animal viruses, retroviral replication proceeds through an obligate recombination step with host cell DNA. Although this step makes it extremely difficult to eradicate a retrovirus from a cell, it also confers a desirable trait to gene therapy vectors. Yet stable integration can yield unwanted side effects, as highlighted in a recent gene therapy trial in which 2 of 11 patients developed leukemia-like illness because of the integration of a murine leukemia virus (MLV)-based vector in the vicinity of the LMO2 protooncogene (1). Deciphering the mechanism(s) of integration site selection not only will shed light on a basic step(s) in retroviral biology but also holds promise for the design of more effective vectors. Although different cloning strategies have indicated an overall preference for transcription units, the mechanism of retroviral integration site selection is for the most part unknown. In this issue of PNAS, Maxfield et al . (2) assayed RAV-1 (a subgroup A Alpharetrovirus ) integration into the quail metallothionein (MT) gene with and without transcriptional activation. A 100-fold increase in gene expression reduced integration 6-fold. Taken alongside a previous study that demonstrated that more modest levels of induction also inhibited integration (3), the active transcription complex appears unlikely to help guide retroviruses to their sites of integration. Retroviruses encode their own integrase (IN) protein that is carried into the cell as part of the virus. After reverse transcription the viral cDNA, IN and other proteins comprise an integration-competent nucleoprotein complex known as the preintegration complex (PIC) (reviewed in ref. 4). Early studies indicated that transcriptionally active and/or DNase I-hypersensitive regions were preferred sites, but it was unclear to what extent relatively small sample size and the selective outgrowth of certain integrants before cloning may have influenced those results (see ref. 5 …

Expression of Pax2 in the intermediate mesoderm is regulated by YY1

The transcription factor Pax2 is essential for the development of the urogenital system. Pax2 expression can be detected by mouse embryonic day 8.5 (E8.5) in the region of intermediate mesoderm fated to become the nephric duct, pronephros, and mesonephros. Elements that direct Pax2 expression to nephrogenic precursor cells must be responding to positional information that controls nephrogenic fate. A 4.1-kb Pax2 promoter/enhancer fragment directs expression of a lacZ reporter to the nephrogenic region and the midbrain–hindbrain junction in transgenic mice. As kidney development proceeds, transgene expression is limited to the nephric duct and its derivatives, but not the metanephric mesenchyme. The early expression driven by the 4.1 promoter does not require the Pax2 protein, demonstrating that it receives positional information in the absence of a developing pro- or mesonephros. We have identified two DNAseI hypersensitive regions within this promoter, one at the start site of transcription initiation and a second approximately 2-kb upstream. Deletion of the more distal site significantly attenuates lacZ expression in the nephrogenic region but not in the midbrain–hindbrain region. DNA footprinting of this fragment revealed a highly conserved sequence between mouse and human Pax2 promoter sequences. Using fractionated nuclear extracts, we identified the transcription factor Yin Yang 1 (YY1) as the protein that binds to this conserved sequence. Deletion of the YY1 element significantly attenuated expression of a full-length 4.1 promoter. Moreover, inclusion of this YY1 binding element significantly enhanced expression of a minimal Pax2 promoter/enhancer transgene in E12 embryos. These data point to a novel role for YY1 in the establishment of high level tissue-specific expression within the intermediate mesoderm.