Identification Of Cis-regulatory Elements Research Articles

Predmoter-cross-species prediction of plant promoter and enhancer regions.

Identifying cis-regulatory elements (CREs) is crucial for analyzing gene regulatory networks. Next generation sequencing methods were developed to identify CREs but represent a considerable expenditure for targeted analysis of few genomic loci. Thus, predicting the outputs of these methods would significantly cut costs and time investment. We present Predmoter, a deep neural network that predicts base-wise Assay for Transposase Accessible Chromatin using sequencing (ATAC-seq) and histone Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) read coverage for plant genomes. Predmoter uses only the DNA sequence as input. We trained our final model on 21 species for 13 of which ATAC-seq data and for 17 of which ChIP-seq data was publicly available. We evaluated our models on Arabidopsis thaliana and Oryza sativa. Our best models showed accurate predictions in peak position and pattern for ATAC- and histone ChIP-seq. Annotating putatively accessible chromatin regions provides valuable input for the identification of CREs. In conjunction with other in silico data, this can significantly reduce the search space for experimentally verifiable DNA-protein interaction pairs. The source code for Predmoter is available at: https://github.com/weberlab-hhu/Predmoter. Predmoter takes a fasta file as input and outputs h5, and optionally bigWig and bedGraph files.

Genome-Wide Identification of Open Chromatin in Plants Using MH-Seq.

Functional cis-regulatory elements (CREs) act as precise transcriptional switches for fine-tuning gene transcription. Identification of CREs is critical for understanding regulatory mechanisms of gene expression associated with various biological processes in eukaryotes. It is well known that CREs reside in open chromatin that exhibits hypersensitivity to enzyme cleavage and physical shearing. Currently, high-throughput methodologies, such as DNase-seq, ATAC-seq, and FAIRE-seq, have been widely applied in mapping open chromatin in various eukaryotic genomes. More recently, differential MNase (micrococcal nuclease) treatment has been successfully employed to map open chromatin in addition to profiling nucleosome landscape in both mammalian and plant species. We have developed a MNase hypersensitivity sequencing (MH-seq) technique in plants. The MH-seq procedure includes plant nuclei fixation and purification, differential treatments of purified nuclei with MNase, specific recovery of MNase-trimmed small DNA fragments within 20~100bp in length, and MH-seq library construction followed by Illumina sequencing and data analysis. MH-seq has been successfully applied for global identification of open chromatin in both Arabidopsis thaliana and maize. It has been proven to be an attractive alternative for profiling open chromatin. Thus, MH-seq is expected to be valuable in probing chromatin accessibility on a genome-wide scale for other plants with sequenced genomes. Moreover, MHS data allow to implement footprinting assays to unveil binding sites of transcription factors.

Gene expression and chromatin conformation differs between worker bees performing different tasks

BackgroundRevealing the effect of transcriptomic regulation on behavioral differences is a fundamental goal in biology, but the relationship between gene regulatory networks and individual behavior differences remains largely unknown. Honey bees are considered as good models for studying the mechanisms underlying gene expression changes and behavioral differences since they exhibit strong and obvious differences in tasks between individuals. The cis-regulatory regions usually contain the binding sites of diverse transcription factor (TFs) influencing bee behavior. Thus, the identification of cis-regulatory elements in the brains across different behavioral states is important for understanding how genomic and transcriptomic variations affect different tasks in honeybees. MethodsIn this study, we employed transcriptome and genome-wide chromatin accessibility assays to analyze brain tissues of honey bees in different behavioral states for examining the relationship between individual behavior differences and brain gene expression changes. We also used the obtained open chromatin regions to identify cis-motifs associating differentially expressed TFs and genes in order to reveal the transcriptional regulatory mechanism related to the different tasks. ResultsWe identified genetic regulatory modules regulating different tasks that contained key TFs (CTCF, Trl and schlank) associated with open chromatin regions enriched for DNA sequence motifs belonging to the family of the corresponding TFs. The most prominent transcriptomic changes, which correlated with chromatin accessibility modifications within their proximal promoter regions, occurred in nervous system development, and were associated with behavior switch. ConclusionsOur results revealed the regulatory landscape among three behavioral stages in honeybees and identified interactive molecular networks regulating different tasks. These results provide a comprehensive insight into behavioral differences of honeybees, which offers reference for future study.

Detection and identification of cis-regulatory elements using change-point and classification algorithms

BackgroundTranscriptional regulation is primarily mediated by the binding of factors to non-coding regions in DNA. Identification of these binding regions enhances understanding of tissue formation and potentially facilitates the development of gene therapies. However, successful identification of binding regions is made difficult by the lack of a universal biological code for their characterisation.ResultsWe extend an alignment-based method, changept, and identify clusters of biological significance, through ontology and de novo motif analysis. Further, we apply a Bayesian method to estimate and combine binary classifiers on the clusters we identify to produce a better performing composite.ConclusionsThe analysis we describe provides a computational method for identification of conserved binding sites in the human genome and facilitates an alternative interrogation of combinations of existing data sets with alignment data.

The SEEL motif and members of the MYB-related REVEILLE transcription factor family are important for the expression of LORELEI in the synergid cells of the Arabidopsis female gametophyte.

Synergid cells in the micropylar end of the female gametophyte are required for critical cell-cell signaling interactions between the pollen tube and the ovule that precede double fertilization and seed formation in flowering plants. LORELEI (LRE) encodes aputative GPI-anchored protein that is expressed primarily in the synergid cells, and together with FERONIA, a receptor-like kinase, it controls pollen tube reception by the receptive synergid cell. Still, how LRE expression is controlled in synergid cells remains poorly characterized. We identified candidate cis-regulatory elements enriched in LRE and other synergid cell-expressed genes. One of the candidate motifs ('TAATATCT') in the LRE promoter was an uncharacterized variant of the Evening Element motif that we named as the Short Evening Element-like (SEEL) motif. Deletion or point mutations in the SEEL motif of the LRE promoter resulted in decreased reporter expression in synergid cells, demonstrating that the SEEL motif is important for expression of LRE in synergid cells. Additionally, we found that LRE expression is decreased in the loss of function mutants of REVEILLE (RVE) transcription factors, which are clock genes known to bind the SEEL and other closely related motifs. We propose that RVE transcription factors regulate LRE expression in synergid cells by binding to the SEEL motif in the LRE promoter. Identification of cis-regulatory elements and transcription factors involved in the expression of LRE will serve as a foundation to characterize the gene regulatory networks in synergid cells.

Identification and Downstream Analyses of Domains Amplified in Plant Genomes: The Case of StAR-Related Lipid Transfer (START) Domains in Rice.

Plant genomes can withstand small- and large-scale duplications, at a far greater success than any other kingdom in the tree of life, resulting in the existence and evolution of gene families, often with over a hundred members! The gene families, in turn, go through subfunctionalization or neofunctionalization, to form protein domains performing unique or grouped functions in context of the original activity. Due to the large number of such cases in the plant kingdom, it has become a routine task for plant biologists to investigate their specific gene family of interest. In this chapter, we provide a simple and standard pipeline for this effort, taking the example of steroidogenic acute regulatory protein (StAR) related lipid transfer (START) domains in rice, as reference. We describe the extraction, processing, and downstream analysis of Oryza sativa var. japonica proteome towards identification and comparative exploration of START domains. This was done by training profile Hidden Markov Models (HMM) of 35 reported START domains in Arabidopsis, which were then used to search potential homologs in rice. Downstream investigations included domain structure analysis, visualization of exon-intron patterns, chromosomal localization of START genes, and phylogenetic studies, followed by identification of cis-regulatory elements and gene regulatory network construction. Additionally, we have also highlighted various alternative tools and techniques that can be used to perform similar analyses, along with salient features.

Mapping Regulatory Determinants in Plants.

The domestication and improvement of many plant species have frequently involved modulation of transcriptional outputs and continue to offer much promise for targeted trait engineering. The cis-regulatory elements (CREs) controlling these trait-associated transcriptional variants however reside within non-coding regions that are currently poorly annotated in most plant species. This is particularly true in large crop genomes where regulatory regions constitute only a small fraction of the total genomic space. Furthermore, relatively little is known about how CREs function to modulate transcription in plants. Therefore understanding where regulatory regions are located within a genome, what genes they control, and how they are structured are important factors that could be used to guide both traditional and synthetic plant breeding efforts. Here, we describe classic examples of regulatory instances as well as recent advances in plant regulatory genomics. We highlight valuable molecular tools that are enabling large-scale identification of CREs and offering unprecedented insight into how genes are regulated in diverse plant species. We focus on chromatin environment, transcription factor (TF) binding, the role of transposable elements, and the association between regulatory regions and target genes.

Advances in assay for transposase-accessible chromatin with high-throughput sequencing

Assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq) was developed in 2013. It has the advantages of more convenient operation and higher efficiency for DNA recovery than DNase I hypersensitive site sequencing (DNase-seq) and micrococcal nuclease sequencing (MNase-seq). ATAC-seq currently is the most popular technique of genome-wide mapping for chromatin accessibility. It provides information on binding regions of transcription factors and nucleosome localization on the chromatin. Thus, ATAC-seq is of great significance for studying the epigenetics and molecular mechanisms in chromatin structure. In this review, we compare the advantages and disadvantages of multiple techniques for profiling chromatin accessibility, and summarize the principles, main process, development and applications of ATAC-seq. We hope this review will provide a reference for study of genome-wide mapping for chromatin accessibility, identification of cis-regulatory elements, and dissection of the epigenetic and genetic regulatory networks using the ATAC-seq technology in eukaryotes.

Assaying Chromatin Accessibility Using ATAC-Seq in Invertebrate Chordate Embryos.

Cis-regulatory elements (CREs) are non-coding DNA regions involved in the spatio-temporal regulation of gene expression. Gene regulatory changes drive animal development and play major roles during evolution of animal body plans. Therefore, we believe that determining CREs at different developmental stages and across animal lineages is critical to understand how evolution operates through development. The Assay for Transposase-Accessible Chromatin followed by high-throughput sequencing (ATAC-seq) is a powerful technique for the study of CREs that takes advantage of Tn5 transposase activity. Starting from fewer than 105 cells, in a 1-day procedure, it is possible to detect, at a genome-wide level, CREs located in open chromatin regions with high resolution. Here, we describe a detailed step-by-step ATAC-seq protocol for invertebrate chordate marine embryos. We have successfully applied this technique to amphioxus and two species of tunicate embryos. We also show an easy workflow to analyze data generated with this technique. Moreover, we point out that this method and our bioinformatic pipeline are efficient to detect CREs associated with Wnt signaling pathway by simply using embryos treated with a drug that perturbs this pathway. This approach can be extended to other signaling pathways and also to embryo mutants for critical genes. Our results therefore demonstrate the power of ATAC-seq for the identification of CREs that play essential functions during animal development in a wide range of invertebrate or vertebrate animals.

Genome-Wide Analysis for Identifying FOXO Protein-Binding Sites.

Forkhead box O (FOXO) proteins comprise a superfamily of transcription factors that play important roles in controlling various biological processes. Transcriptional control constitutes a crucial component in regulating complex biological processes. The identification of cis-regulatory elements is essential to understand the regulatory mechanism of gene expression. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) is widely used to identify the cis-regulatory elements of transcription factors and other DNA-binding proteins on a genome-wide level. It is a powerful tool to analyze the regulatory networks underlying the biological processes. Here, we describe a detailed protocol for preparingChIP-seq samples that are used for sequencing and subsequent data analyses.

Abiotic stress and genome dynamics: specific genes and transposable elements response to iron excess in rice.

BackgroundIron toxicity is a root related abiotic stress, occurring frequently in flooded soils. It can affect the yield of rice in lowland production systems. This toxicity is associated with high concentrations of reduced iron (Fe2+) in the soil solution. Although the first interface of the element is in the roots, the consequences of an excessive uptake can be observed in several rice tissues. In an original attempt to find both genes and transposable elements involved in the response to an iron toxicity stress, we used a microarray approach to study the transcriptional responses of rice leaves of cv. Nipponbare (Oryza sativa L. ssp. japonica) to iron excess in nutrient solution.ResultsA large number of genes were significantly up- or down-regulated in leaves under the treatment. We analyzed the gene ontology and metabolic pathways of genes involved in the response to this stress and the cis-regulatory elements (CREs) present in the promoter region of up-regulated genes. The majority of genes act in the pathways of lipid metabolic process, carbohydrate metabolism, biosynthesis of secondary metabolites and plant hormones. We also found genes involved in iron acquisition and mobilization, transport of cations and regulatory mechanisms for iron responses, and in oxidative stress and reactive oxygen species detoxification. Promoter regions of 27% of genes up-regulated present at least one significant occurrence of an ABA-responsive CRE. Furthermore, and for the first time, we were able to show that iron stress triggers the up-regulation of many LTR-retrotransposons. We have established a complete inventory of transposable elements transcriptionally activated under iron excess and the CREs which are present in their LTRs.ConclusionThe short-term response of Nipponbare seedlings to iron excess, includes activation of genes involved in iron homeostasis, in particular transporters, transcription factors and ROS detoxification in the leaves, but also many transposable elements. Our data led to the identification of CREs which are associated with both genes and LTR-retrotransposons up-regulated under iron excess. Our results strengthen the idea that LTR-retrotransposons participate in the transcriptional response to stress and could thus confer an adaptive advantage for the plant.Electronic supplementary materialThe online version of this article (doi:10.1186/s12284-015-0045-6) contains supplementary material, which is available to authorized users.

Characterization and Identification of cis-Regulatory Elements in Arabidopsis Based on Single-Nucleotide Polymorphism Information

Identifying regulatory elements and revealing their role in gene expression regulation remains a central goal of plant genome research. We exploited the detailed genomic sequencing information of a large number of Arabidopsis (Arabidopsis thaliana) accessions to characterize known and to identify novel cis-regulatory elements in gene promoter regions of Arabidopsis by relying on conservation as the hallmark signal of functional relevance. Based on the genomic layout and the obtained density profiles of single-nucleotide polymorphisms (SNPs) in sequence regions upstream of transcription start sites, the average length of promoter regions in Arabidopsis could be established at 500 bp. Genes associated with high degrees of variability of their respective upstream regions are preferentially involved in environmental response and signaling processes, while low levels of promoter SNP density are common among housekeeping genes. Known cis-elements were found to exhibit a decreased SNP density than sequence regions not associated with known motifs. For 15 known cis-element motifs, strong positional preferences relative to the transcription start site were detected based on their promoter SNP density profiles. Five novel candidate cis-element motifs were identified as consensus motifs of 17 sequence hexamers exhibiting increased sequence conservation combined with evidence of positional preferences, annotation information, and functional relevance for inducing correlated gene expression. Our study demonstrates that the currently available resolution of SNP data offers novel ways for the identification of functional genomic elements and the characterization of gene promoter sequences.

Motif analysis in DNAse hypersensitivity regions uncovers distal cis elements associated with gene expression.

Reliable identification of cis regulatory elements influencing transcription remains a challenging problem in molecular bioinformatics. This is especially true for enhancer elements which are often located hundreds of kilobases from the gene promoter. High resolution DNase hypersensitivity and connectivity profiling by the ENCODE consortium provides evidence of millions of interacting cis-acting elements in the human genome. This prior knowledge can be incorporated into genome-wide expression analyses, in the form of gene sets sharing regulatory sequence motifs in known DNase hypersensitivity peak regions. High proportions of enrichment among the most extreme differentially transcribed genes from controlled biological experiments may suggest novel hypotheses about signalling pathways. The utility of this approach is demonstrated with the reanalysis of a microarray-derived gene expression data set through the Gene Set Enrichment Analysis pipeline, uncovering new putative distal cis elements in the context of innate immunity. The DNase Hypersensitivity Connectivity informed Motif Enrichment in Gene Expression (DHC-MEGE) method described here has the advantage of identifying distal elements such as enhancers, which are often overlooked with standard promoter motif analysis.AvailabilityThe DHC-MEGE shell script can be obtained from Sourceforge https://sourceforge.net/projects/dhcmege/ and the generated GMT file is attached as supplementary data.

Abstract 4958: Identification of cis-regulatory elements in a BIM deletion polymorphism that regulate splicing and generation of non-apoptotic BIM isoforms

Abstract BIM is a pro-apoptotic member of the BCL-2 family of proteins that trigger apoptosis by either opposing the pro-survival members of the family (BCL-2, BCL-xL, MCL1), or by directly binding the distal apoptotic effectors, BAX and BAK. Importantly, BIM expression is required for sensitivity towards tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML) and EGFR-mutated non-small cell lung cancer (EGFR NSCLC) cell lines, since BIM knockdown in these cells results in TKI resistance. Using paired-end DNA sequencing, we recently discovered a novel 2.9kb deletion polymorphism in intron 2 of the BIM gene that was associated with TKI resistance in CML and EGFR NSCLC cell lines. When present, the deletion polymorphism results in the preferential expression of exon 3 (E3)- vs exon 4 (E4)-containing BIM isoforms in a mutually exclusive manner, since E3 also contains a stop codon and polyadenylation signal sequence. As a result, expression of E4-containing BIM isoforms (e.g. BIMEL, BIML, BIMS) that lack the pro-apoptotic BH3 domain is impaired. This then leads to defective apoptotic signaling in the presence of TKIs, and intrinsic TKI resistance. In this study, we aimed to identify cis-regulatory elements within the deleted fragment that suppress splicing to E3, and to assess the apoptotic activity of the major E3-containing isoform, BIMγ. Using serial deletion analysis, we have mapped the cis-regulatory elements to a 150-nucleotide (nt) region within the deletion. Furthermore, our results also showed that the deletion does not create a novel element that regulates E3 splicing. Ongoing analysis is expected to narrow the region to the 6-7 nt usually required for splicing factor binding, as well as to aid in identifying the relevant splicing factors. Next, we assessed the apoptotic activity of BIMγ, and found that the BIMγ protein was markedly less stable (∼T1/2 <1hr) than E4-containing BIM isoforms (BIML) (∼T1/2 ∼4hrs). Second, forced expression of BIMγ failed to affect cell viability, in contrast to E4-containing isoforms. Lastly, knockdown of BIMγ failed to sensitize a TKI resistant CML cell line harboring the deletion polymorphism (KCL22) to TKI-induced apoptosis. In summary, we have mapped cis-regulatory elements suppressing the splicing of E3 to a 150 nt region within the deletion. We also show that the BIM deletion polymorphism favors the generation of unstable E3-containing BIM isoforms, which, even when overexpressed, lack the pro-apoptotic activity of E4-containing BIM isoforms. Together, these data provide a mechanism by which the deletion polymorphism mediates TKI resistance in CML and EGFR NSCLC cell lines. Importantly, our results identify potential targets for modulating alternative splicing of BIM as a therapeutic approach for overcoming TKI resistance mediated by the BIM deletion polymorphism. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4958. doi:1538-7445.AM2012-4958

Large cliques in Arabidopsis gene coexpression network and motif discovery

Identification of cis-regulatory elements in Arabidopsis is a key step to understanding its transcriptional regulation scheme. In this study, the Arabidopsis gene coexpression network was constructed using the ATTED-II data, and thereafter a subgraph-induced approach and clique-finding algorithm were used to extract gene coexpression groups from the gene coexpression network. A total of 23 large coexpression gene groups were obtained, with each consisting of more than 100 highly correlated genes. Four classical tools were used to predict motifs in the promoter regions of coexpressed genes. Consequently, we detected a large number of candidate biologically relevant regulatory elements, and many of them are consistent with known cis-regulatory elements from AGRIS and AthaMap. Experiments on coexpressed groups, including E2Fa target genes, showed that our method had a high probability of returning the real binding motif. Our study provides the basis for future cis-regulatory module analysis and creates a starting point to unravel regulatory networks of Arabidopsis thaliana.

Identification of cis-Regulatory Elements in the Mammalian Genome: The cREMaG Database

BackgroundA growing number of gene expression-profiling datasets provides a reliable source of information about gene co-expression. In silico analyses of the properties shared among the promoters of co-expressed genes facilitates the identification of transcription factors (TFs) involved in the co-regulation of those genes. Our previous experience with microarray data led to the development of a database suitable for the examination of regulatory motifs in the promoters of co-expressed genes.MethodologyWe introduce the cREMaG (cis-Regulatory Elements in the Mammalian Genome) system designed for in silico studies of the promoter properties of co-regulated mammalian genes. The cREMaG system offers an analysis of data obtained from human, mouse, rat, bovine and canine gene expression-profiling studies. More than eight analysis parameters can be utilized in user-defined combinations. The selection of alternative transcription start sites and information about CpG islands are also available.ConclusionsUsing the cREMaG system, we successfully identified TFs mediating transcriptional responses in reference gene sets. The cREMaG system facilitates in silico studies of mammalian transcriptional gene regulation. The resource is freely available at http://www.cremag.org.

Evolutionary Forces Act on Promoter Length: Identification of Enriched Cis-Regulatory Elements

Transcription factors govern gene expression by binding to short DNA sequences called cis-regulatory elements. These sequences are typically located in promoters, which are regions of variable length upstream of the open reading frames of genes. Here, we report that promoter length and gene function are related in yeast, fungi, and plants. In particular, the promoters for stress-responsive genes are in general longer than those of other genes. Essential genes have, on the other hand, relatively short promoters. We utilize these findings in a novel method for identifying relevant cis-regulatory elements in a set of coexpressed genes. The method is shown to generate more accurate results and fewer false positives compared with other common procedures. Our results suggest that genes with complex transcriptional regulation tend to have longer promoters than genes responding to few signals. This phenomenon is present in all investigated species, indicating that evolution adjust promoter length according to gene function. Identification of cis-regulatory elements in Saccharomyces cerevisiae can be done with the web service located at http://enricher.zool.gu.se.

Identification of cis-regulatory elements from the C. elegans T-box gene mab-9 reveals a novel role for mab-9 in hypodermal function

We have identified Conserved Non-coding Elements (CNEs) in the regulatory region of Caenorhabditis elegans and Caenorhabditis briggsae mab-9, a T-box gene known to be important for cell fate specification in the developing C. elegans hindgut. Two adjacent CNEs (a region 78 bp in length) are both necessary and sufficient to drive reporter gene expression in posterior hypodermal cells. The failure of a genomic mab-9∷gfp construct lacking this region to express in posterior hypodermis correlates with the inability of this construct to completely rescue the mab-9 mutant phenotype. Transgenic males carrying this construct in a mab-9 mutant background exhibit tail abnormalities including morphogenetic defects, altered tail autofluorescence and abnormal lectin-binding properties. Hermaphrodites display reduced susceptibility to the C. elegans pathogen Microbacterium nematophilum. This comparative genomics approach has therefore revealed a previously unknown role for mab-9 in hypodermal function and we suggest that MAB-9 is required for the secretion and/or modification of posterior cuticle.

Identification of cis-regulatory elements and trans-acting proteins of the rat carbohydrate response element binding protein gene

Carbohydrate response element binding protein (ChREBP) is a transcription factor that activates liver glycolytic and lipogenetic enzyme genes in response to high carbohydrate diet. Here we report the transcriptional regulatory mechanisms for the rat ChREBP gene. Firstly, we determined the transcription initiation site and the nucleotide sequences of the rat ChREBP promoter region encompassing approximately 900 bp from the ATG initiation codon. Reporter gene assays demonstrated that the major positive regulatory region exists in the nucleotide sequence between −163 and −32 of the ChREBP gene. This region contains a cluster of putative transcription factor binding elements that consist of two specificity protein 1 (Sp1) binding sites (−66 to −50 and −93 to −78), a sterol regulatory element (−101 to −110), and two nuclear factor-Y (NF-Y) binding sites (−23 to −19 and −131 to −127). Mutations introduced into these sites caused marked reduction of ChREBP promoter activities. Functional synergisms were observed between Sp1/NF-Y and Sp1/sterol regulatory element-binding protein. Additionally, electrophoretic mobility shift assays and chromatin immunoprecipitation assays demonstrated that these factors bound to these elements. Thus, we conclude that functional synergisms between these transcription factors are critical for ChREBP gene transcription.

Identifying gene regulatory elements by genomic microarray mapping of DNaseI hypersensitive sites

The identification of cis-regulatory elements is central to understanding gene transcription. Hypersensitivity of cis-regulatory elements to digestion with DNaseI remains the gold-standard approach to locating such elements. Traditional methods used to identify DNaseI hypersensitive sites are cumbersome and can only be applied to short stretches of DNA at defined locations. Here we report the development of a novel genomic array-based approach to DNaseI hypersensitive site mapping (ADHM) that permits precise, large-scale identification of such sites from as few as 5 million cells. Using ADHM we identified all previously recognized hematopoietic regulatory elements across 200 kb of the mouse T-cell acute lymphocytic leukemia-1 (Tal1) locus, and, in addition, identified two novel elements within the locus, which show transcriptional regulatory activity. We further validated the ADHM protocol by mapping the DNaseI hypersensitive sites across 250 kb of the human TAL1 locus in CD34+ primary stem/progenitor cells and K562 cells and by mapping the previously known DNaseI hypersensitive sites across 240 kb of the human alpha-globin locus in K562 cells. ADHM provides a powerful approach to identifying DNaseI hypersensitive sites across large genomic regions.