Rachel Byron

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. DNase I footprinting in 41 cell and tissue types reveals millions of short sequence elements encoding an expansive repertoire of conserved recognition sequences for DNA-binding proteins. DNaseI footprinting detects DNA sequences that are protected from cleavage by DNaseI because they are bound by regulatory factors. Studying these footprints in 41 diverse cell and tissue types, the authors describe millions of short sequence elements that are conserved recognition sequences for DNA-binding proteins. The effort nearly doubles the size of the human cis-regulatory lexicon and provides insight into chromatin states and levels of evolutionary conservation. A large collection of novel regulatory-factor recognition motifs that closely parallel major regulators of development, differentiation and pluripotency is also described.

e laboratory mouse is the premier mammalian model organism for the study of human disease, and it has played a vital role in both the annotation of the human genome and the study of gene function and regulation. Similar to humans, mice naturally develop diverse diseases that affect the hematologic, nervous, cardiovascular, endocrine, musculoskeletal, renal and other systems, providing excellent experimental paradigms for studying the pathogenesis of cancer, autoimmune disease, diabetes, obesity, atherosclerosis, hypertension, gastrointestinal disorders and diverse neurodegenerative states. Mouse models are currently available for hundreds of human disorders [1-4], spanning diverse quantitative and behavioral phenotypes and physiological systems. ese comprise both inbred strains and genetically engineered mutants, many of which have been extensively characterized. For these reasons, the mouse has emerged as a premier system for translating basic human genetic, genomic and physiologic research into paradigms for therapeutic development.

We have examined the relationship between nuclear localization and transcriptional activity of the endogenous murine beta-globin locus during erythroid differentiation. Murine fetal liver cells were separated into distinct erythroid maturation stages by fluorescence-activated cell sorting, and the nuclear position of the locus was determined at each stage. We find that the beta-globin locus progressively moves away from the nuclear periphery with increasing maturation. Contrary to the prevailing notion that the nuclear periphery is a repressive compartment in mammalian cells, beta(major)-globin expression begins at the nuclear periphery prior to relocalization. However, relocation of the locus to the nuclear interior with maturation is accompanied by an increase in beta(major)-globin transcription. The distribution of nuclear polymerase II (Pol II) foci also changes with erythroid differentiation: Transcription factories decrease in number and contract toward the nuclear interior. Moreover, both efficient relocalization of the beta-globin locus from the periphery and its association with hyperphosphorylated Pol II transcription factories require the locus control region (LCR). These results suggest that the LCR-dependent association of the beta-globin locus with transcriptionally engaged Pol II foci provides the driving force for relocalization of the locus toward the nuclear interior during erythroid maturation.

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.