Summary Distant-acting tissue-specific enhancers vastly outnumber protein-coding genes in mammalian genomes, but the functional significance of this regulatory complexity remains insufficiently understood1,2. Here we show that the pervasive presence of multiple enhancers with similar activities near the same gene confers phenotypic robustness to loss-of-function mutations in individual enhancers. We used genome editing to create 23 mouse deletion lines and inter-crosses, including both single and combinatorial enhancer deletions at seven distinct loci required for limb development. Surprisingly, none of ten deletions of individual enhancers caused noticeable changes in limb morphology. In contrast, removal of pairs of limb enhancers near the same gene resulted in discernible phenotypes, indicating that enhancers function redundantly in establishing normal morphology. In a genetic background sensitized by reduced baseline expression of the target gene, even single enhancer deletions caused limb abnormalities, suggesting that functional redundancy is conferred by additive effects of enhancers on gene expression levels. A genome-wide analysis integrating epigenomic and transcriptomic data from 29 developmental mouse tissues revealed that mammalian genes are very commonly associated with multiple enhancers that have similar spatiotemporal activity. Systematic exploration of three representative developmental structures (limb, brain, heart) uncovered more than a thousand cases in which five or more enhancers with redundant activity patterns were found near the same gene. Taken together, our data indicate that enhancer redundancy is a remarkably widespread feature of mammalian genomes and provides an effective regulatory buffer preventing deleterious phenotypic consequences upon loss of individual enhancers.

The Encyclopedia of DNA Elements (ENCODE) project has established a genomic resource for mammalian development, profiling a diverse panel of mouse tissues at 8 developmental stages from 10.5 days after conception until birth, including transcriptomes, methylomes and chromatin states. Here we systematically examined the state and accessibility of chromatin in the developing mouse fetus. In total we performed 1,128 chromatin immunoprecipitation with sequencing (ChIP–seq) assays for histone modifications and 132 assay for transposase-accessible chromatin using sequencing (ATAC–seq) assays for chromatin accessibility across 72 distinct tissue-stages. We used integrative analysis to develop a unified set of chromatin state annotations, infer the identities of dynamic enhancers and key transcriptional regulators, and characterize the relationship between chromatin state and accessibility during developmental gene regulation. We also leveraged these data to link enhancers to putative target genes and demonstrate tissue-specific enrichments of sequence variants associated with disease in humans. The mouse ENCODE data sets provide a compendium of resources for biomedical researchers and achieve, to our knowledge, the most comprehensive view of chromatin dynamics during mammalian fetal development to date.

DNA methylation is implicated in a surprising diversity of regulatory, evolutionary processes and diseases in eukaryotes. The introduction of whole-genome bisulfite sequencing has enabled the study of DNA methylation at a single-base resolution, revealing many new aspects of DNA methylation and highlighting the usefulness of methylome data in understanding a variety of genomic phenomena. As the number of publicly available whole-genome bisulfite sequencing studies reaches into the hundreds, reliable and convenient tools for comparing and analyzing methylomes become increasingly important. We present MethPipe, a pipeline for both low and high-level methylome analysis, and MethBase, an accompanying database of annotated methylomes from the public domain. Together these resources enable researchers to extract interesting features from methylomes and compare them with those identified in public methylomes in our database.

We present an open-source web platform, Ginkgo (http://qb.cshl.edu/ginkgo), for the analysis and assessment of single-cell copy-number variations (CNVs). Ginkgo automatically constructs copy-number profiles of cells from mapped reads and constructs phylogenetic trees of related cells. We validate Ginkgo by reproducing the results of five major studies and examine the characteristics of three commonly used single-cell amplification techniques to conclude degenerate oligonucleotide-primed PCR to be the most consistent for CNV analysis.

The evolution of body shape is thought to be tightly coupled to changes in regulatory sequences, but specific molecular events associated with major morphological transitions in vertebrates have remained elusive. We identified snake-specific sequence changes within an otherwise highly conserved long-range limb enhancer of Sonic hedgehog (Shh). Transgenic mouse reporter assays revealed that the in vivo activity pattern of the enhancer is conserved across a wide range of vertebrates including fish, but not in snakes. Genomic substitution of the mouse enhancer with its human or fish ortholog results in normal limb development. In contrast, replacement with snake orthologs caused severe limb reduction. Synthetic restoration of a single transcription factor binding site lost in the snake lineage reinstated full in vivo function to the snake enhancer. Our results demonstrate changes in a regulatory sequence associated with a major body plan transition and highlight the role of enhancers in morphological evolution.