SummaryAs the premier model organism in biomedical research, the laboratory mouse shares the majority of protein-coding genes with humans, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications, and replication domains throughout the mouse genome in diverse cell and tissue types. By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of other sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases.

Regulatory factor binding to genomic DNA protects the underlying sequence from cleavage by DNaseI, leaving nucleotide-resolution footprints. Using genomic DNaseI footprinting across 41 diverse cell and tissue types, we detected 45 million 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 DNaseI 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.

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.

The fundamental body plan and major physiological axes have been highly conserved during mammalian evolution, despite constraint of only a fraction of the human genome sequence. To quantify cis- vs. trans-regulatory 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 TF occupancy sites at nucleotide resolution. Here we show that mouse TF footprints encode a regulatory lexicon of >600 motifs that is >95% similar with that recognized in vivo by human TFs. However, only ~20% of mouse TF footprints have human orthologues. Despite substantial turnover of the cis-regulatory landscape around each TF gene, 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. Strikingly, the higher-level organization of mouse TF-to-TF connections into cellular network architectures is nearly identical with human. Our results suggest that evolutionary selection on mammalian gene regulation is targeted chiefly at the level of trans-regulatory circuitry.

We have previously reported that DNase I hypersensitive site 5 (5'HS5) of the human beta-globin locus control region functions as a chromatin insulator in stable transfection assays. In this report we show that a 3.2 kb DNA fragment containing the entire 5'HS5 region can protect a position-sensitive (A)gamma-globin gene against position effects in transgenic mice. Bracketing is required for function of 5'HS5 as an insulator. The 5'HS5 insulator operates in adult as well as in embryonic murine erythroid cells. The insulator has no significant stimulatory effects of its own. These results indicate that 5'HS5 can function as a chromatin insulator in vivo.