Audra K. Johnson

Systematic Localization of Common Disease-Associated Variation in Regulatory DNA

Predictions of Genetic Disease Many genome-wide association studies (GWAS) have identified loci and variants associated with disease, but the ability to predict disease on the basis of these genetic variants remains small. Maurano et al. (p. 1190; see the Perspective by Schadt and Chang; see the cover) characterize the location of GWAS variants in the genome with respect to their proximity to regulatory DNA [marked by deoxyribonuclease I (DNase I) hypersensitive sites] by tissue type, disease, and enrichments in physiologically relevant transcription factor binding sites and networks. They found many noncoding disease associations in regulatory DNA, indicating tissue and developmental-specific regulatory roles for many common genetic variants and thus enabling links to be made between gene regulation and adult-onset disease. Genetic variants that have been associated with diseases are concentrated in regulatory regions of the genome. Genome-wide association studies have identified many noncoding variants associated with common diseases and traits. We show that these variants are concentrated in regulatory DNA marked by deoxyribonuclease I (DNase I) hypersensitive sites (DHSs). Eighty-eight percent of such DHSs are active during fetal development and are enriched in variants associated with gestational exposure–related phenotypes. We identified distant gene targets for hundreds of variant-containing DHSs that may explain phenotype associations. Disease-associated variants systematically perturb transcription factor recognition sequences, frequently alter allelic chromatin states, and form regulatory networks. We also demonstrated tissue-selective enrichment of more weakly disease-associated variants within DHSs and the de novo identification of pathogenic cell types for Crohn’s disease, multiple sclerosis, and an electrocardiogram trait, without prior knowledge of physiological mechanisms. Our results suggest pervasive involvement of regulatory DNA variation in common human disease and provide pathogenic insights into diverse disorders.

The accessible chromatin landscape of the human genome.

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

An expansive human regulatory lexicon encoded in transcription factor footprints

Regulatory factor binding to genomic DNA protects the underlying sequence from cleavage by DNase I, leaving nucleotide-resolution footprints. Using genomic DNase I footprinting across 41 diverse cell and tissue types, we detected 45 million transcription factor occupancy events within regulatory regions, representing differential binding to 8.4 million distinct short sequence elements. Here we show that this small genomic sequence compartment, roughly twice the size of the exome, encodes an expansive repertoire of conserved recognition sequences for DNA-binding proteins that nearly doubles the size of the human cis–regulatory lexicon. We find that genetic variants affecting allelic chromatin states are concentrated in footprints, and that these elements are preferentially sheltered from DNA methylation. High-resolution DNase I cleavage patterns mirror nucleotide-level evolutionary conservation and track the crystallographic topography of protein–DNA interfaces, indicating that transcription factor structure has been evolutionarily imprinted on the human genome sequence. We identify a stereotyped 50-base-pair footprint that precisely defines the site of transcript origination within thousands of human promoters. Finally, we describe a large collection of novel regulatory factor recognition motifs that are highly conserved in both sequence and function, and exhibit cell-selective occupancy patterns that closely parallel major regulators of development, differentiation and pluripotency.

An encyclopedia of mouse DNA elements (Mouse ENCODE)

To complement the human Encyclopedia of DNA Elements (ENCODE) project and to enable a broad range of mouse genomics efforts, the Mouse ENCODE Consortium is applying the same experimental pipelines developed for human ENCODE to annotate the mouse genome.

BEDOPS: high-performance genomic feature operations

UNLABELLED The large and growing number of genome-wide datasets highlights the need for high-performance feature analysis and data comparison methods, in addition to efficient data storage and retrieval techniques. We introduce BEDOPS, a software suite for common genomic analysis tasks which offers improved flexibility, scalability and execution time characteristics over previously published packages. The suite includes a utility to compress large inputs into a lossless format that can provide greater space savings and faster data extractions than alternatives. AVAILABILITY http://code.google.com/p/bedops/ includes binaries, source and documentation.

Conservation of trans-acting circuitry during mammalian regulatory evolution

The basic body plan and major physiological axes have been highly conserved during mammalian evolution, yet only a small fraction of the human genome sequence appears to be subject to evolutionary constraint. To quantify cis- versus trans-acting contributions to mammalian regulatory evolution, we performed genomic DNase I footprinting of the mouse genome across 25 cell and tissue types, collectively defining ∼8.6 million transcription factor (TF) occupancy sites at nucleotide resolution. Here we show that mouse TF footprints conjointly encode a regulatory lexicon that is ∼95% similar with that derived from human TF footprints. However, only ∼20% of mouse TF footprints have human orthologues. Despite substantial turnover of the cis-regulatory landscape, nearly half of all pairwise regulatory interactions connecting mouse TF genes have been maintained in orthologous human cell types through evolutionary innovation of TF recognition sequences. Furthermore, the higher-level organization of mouse TF-to-TF connections into cellular network architectures is nearly identical with human. Our results indicate that evolutionary selection on mammalian gene regulation is targeted chiefly at the level of trans-regulatory circuitry, enabling and potentiating cis-regulatory plasticity.

Mapping and dynamics of regulatory DNA and transcription factor networks in A. thaliana.

Our understanding of gene regulation in plants is constrained by our limited knowledge of plant cis-regulatory DNA and its dynamics. We mapped DNase I hypersensitive sites (DHSs) in A. thaliana seedlings and used genomic footprinting to delineate ∼ 700,000 sites of in vivo transcription factor (TF) occupancy at nucleotide resolution. We show that variation associated with 72 diverse quantitative phenotypes localizes within DHSs. TF footprints encode an extensive cis-regulatory lexicon subject to recent evolutionary pressures, and widespread TF binding within exons may have shaped codon usage patterns. The architecture of A. thaliana TF regulatory networks is strikingly similar to that of animals in spite of diverged regulatory repertoires. We analyzed regulatory landscape dynamics during heat shock and photomorphogenesis, disclosing thousands of environmentally sensitive elements and enabling mapping of key TF regulatory circuits underlying these fundamental responses. Our results provide an extensive resource for the study of A. thaliana gene regulation and functional biology.

Mouse regulatory DNA landscapes reveal global principles of cis-regulatory evolution

To study the evolutionary dynamics of regulatory DNA, we mapped >1.3 million deoxyribonuclease I–hypersensitive sites (DHSs) in 45 mouse cell and tissue types, and systematically compared these with human DHS maps from orthologous compartments. We found that the mouse and human genomes have undergone extensive cis-regulatory rewiring that combines branch-specific evolutionary innovation and loss with widespread repurposing of conserved DHSs to alternative cell fates, and that this process is mediated by turnover of transcription factor (TF) recognition elements. Despite pervasive evolutionary remodeling of the location and content of individual cis-regulatory regions, within orthologous mouse and human cell types the global fraction of regulatory DNA bases encoding recognition sites for each TF has been strictly conserved. Our findings provide new insights into the evolutionary forces shaping mammalian regulatory DNA landscapes. Mouse-to-human genomic comparisons illuminate conserved transcriptional programs despite regulatory rewiring. Rewiring the gene regulatory landscape DNAse I hypersensitive sites (DHSs) correlate with genomic locations that control where messenger RNA is to be produced. DHSs differ, depending on the cell type, developmental stage, and species. Viestra et al. compared mouse and human genome-wide DHS maps. Approximately one-third of the DHSs are conserved between the species, which separated approximately 550 million years ago. Most DHSs fell into tissue-specific cohorts; however, these were generally not conserved between the human and mouse. It seems that the majority of DHSs evolve because of changes in the sequence that gradually change how the region is regulated. Science, this issue p. 1007

Enhancing GTEx by bridging the gaps between genotype, gene expression, and disease

Genetic variants have been associated with myriad molecular phenotypes that provide new insight into the range of mechanisms underlying genetic traits and diseases. Identifying any particular genetic variants cascade of effects, from molecule to individual, requires assaying multiple layers of molecular complexity. We introduce the Enhancing GTEx (eGTEx) project that extends the GTEx project to combine gene expression with additional intermediate molecular measurements on the same tissues to provide a resource for studying how genetic differences cascade through molecular phenotypes to impact human health.

Cryptic Promoter Activation Drives POU5F1 (OCT4) Expression in Renal Cell Carcinoma

Transcriptional dysregulation drives cancer formation but the underlying mechanisms are still poorly understood. As a model system, we used renal cell carcinoma (RCC), the most common malignant kidney tumor which canonically activates the hypoxia-inducible transcription factor (HIF) pathway. We performed genome-wide chromatin accessibility and transcriptome profiling on paired tumor/normal samples and found that numerous transcription factors with a RCC-selective expression pattern also demonstrated evidence of HIF binding in the vicinity of their gene body. Some of these transcription factors influenced the tumor’s regulatory landscape, notably the stem cell transcription factor POU5F1 (OCT4). Unexpectedly, we discovered a HIF-pathway-responsive cryptic promoter embedded within a human-specific retroviral repeat element that drives POU5F1 expression in RCC via a novel transcript. Elevat POU5F1 expression levels were correlated with advanced tumor stage and poorer overall survival in RCC patients. Thus, integrated transcriptomic and epigenomic analysis of even a small number of primary patient samples revealed remarkably convergent shared regulatory landscapes and a novel mechanism for dysregulated expression of POU5F1 in RCC.