Chromatin-based functional genomic analyses and genomewide association studies (GWASs) together implicate enhancers as critical elements influencing gene expression and risk for common diseases. Here, we performed systematic chromatin and transcriptome profiling in human pancreatic islets. Integrated analysis of islet data with those from nine cell types identified specific and significant enrichment of type 2 diabetes and related quantitative trait GWAS variants in islet enhancers. Our integrated chromatin maps reveal that most enhancers are short (median = 0.8 kb). Each cell type also contains a substantial number of more extended (≥3 kb) enhancers. Interestingly, these stretch enhancers are often tissue-specific and overlap locus control regions, suggesting that they are important chromatin regulatory beacons. Indeed, we show that (i) tissue specificity of enhancers and nearby gene expression increase with enhancer length; (ii) neighborhoods containing stretch enhancers are enriched for important cell type-specific genes; and (iii) GWAS variants associated with traits relevant to a particular cell type are more enriched in stretch enhancers compared with short enhancers. Reporter constructs containing stretch enhancer sequences exhibited tissue-specific activity in cell culture experiments and in transgenic mice. These results suggest that stretch enhancers are critical chromatin elements for coordinating cell type-specific regulatory programs and that sequence variation in stretch enhancers affects risk of major common human diseases.

HIV-1-neutralizing antibodies develop in most HIV-1-infected individuals, although highly effective antibodies are generally observed only after years of chronic infection. Here we characterize the rate of maturation and extent of diversity for the lineage that produced the broadly neutralizing antibody VRC01 through longitudinal sampling of peripheral B cell transcripts over 15 years and co-crystal structures of lineage members. Next-generation sequencing identified VRC01-lineage transcripts, which encompassed diverse antibodies organized into distinct phylogenetic clades. Prevalent clades maintained characteristic features of antigen recognition, though each evolved binding loops and disulfides that formed distinct recognition surfaces. Over the course of the study period, VRC01-lineage clades showed continuous evolution, with rates of ~2 substitutions per 100 nucleotides per year, comparable to that of HIV-1 evolution. This high rate of antibody evolution provides a mechanism by which antibody lineages can achieve extraordinary diversity and, over years of chronic infection, develop effective HIV-1 neutralization.

Next-generation sequencing of antibody transcripts from HIV-1-infected individuals with broadly neutralizing antibodies could provide an efficient means for identifying somatic variants and characterizing their lineages. Here, we used 454 pyrosequencing and identity/divergence grid sampling to analyze heavy-and lightchain sequences from donor N152, the source of the broadly neutralizing antibody 10E8. We identified variants with up to 28% difference in amino acid sequence. Heavy-and light-chain phylogenetic trees of identified 10E8 variants displayed similar architectures, and 10E8 variants reconstituted from matched and unmatched phylogenetic branches displayed significantly lower autoreactivity when matched. To test the generality of phylogenetic pairing, we analyzed donor International AIDS Vaccine Initiative 84, the source of antibodies PGT141-145. Heavy-and light-chain phylogenetic trees of PGT141-145 somatic variants also displayed remarkably similar architectures; in this case, branch pairings could be anchored by known PGT141-145 antibodies. Altogether, our findings suggest that phylogenetic matching of heavy and light chains can provide a means to approximate natural pairings.antibody-affinity maturation | antibodyomics | B-cell ontogeny | DNA sequencing | immunological tolerance

Acinetobacter baumannii is an emerging human pathogen and a significant cause of nosocomial infections among hospital patients worldwide. The enormous increase in multidrug resistance among hospital isolates and the recent emergence of pandrug-resistant strains underscores the urgency to understand how A. baumannii evolves in hospital environments. To this end, we undertook a genomic study of a polyclonal outbreak of multidrugresistant A. baumannii at the research-based National Institutes of Health Clinical Center. Comparing the complete genome sequences of the three dominant outbreak strain types enabled us to conclude that, despite all belonging to the same epidemic lineage, the three strains diverged before their arrival at the National Institutes of Health. The simultaneous presence of three divergent strains from this lineage supports its increasing prevalence in international hospitals and suggests an ongoing adaptation to the hospital environment. Further genomic comparisons uncovered that much of the diversification that occurred since the divergence of the three outbreak strains was mediated by homologous recombination across 20% of their genomes. Inspection of recombinant regions revealed that several regions were associated with either the loss or swapping out of genes encoding proteins that are exposed to the cell surface or that synthesize cell-surface molecules. Extending our analysis to a larger set of international clinical isolates revealed a previously unappreciated ability of A. baumannii to vary surface molecules through horizontal gene transfer, with subsequent intraspecies dissemination by homologous recombination. These findings have immediate implications in surveillance, prevention, and treatment of A. baumannii infections. bacterial evolution | microbial genomics | clinical microbiology | hospital epidemiology | hospital infection

Somatic hypermutation (SHM) plays a critical role in the maturation of antibodies, optimizing recognition initiated by recombination of V(D)J genes. Previous studies have shown that the propensity to mutate is modulated by the context of surrounding nucleotides and that SHM machinery generates biased substitutions. To investigate the intrinsic mutation frequency and substitution bias of SHMs at the amino acid level, we analyzed functional human antibody repertoires and developed mGSSP (method for gene-specific substitution profile), a method to construct amino acid substitution profiles from next-generation sequencing-determined B cell transcripts. We demonstrated that these gene-specific substitution profiles (GSSPs) are unique to each V gene and highly consistent between donors. We also showed that the GSSPs constructed from functional antibody repertoires are highly similar to those constructed from antibody sequences amplified from non-productively rearranged passenger alleles, which do not undergo functional selection. This suggests the types and frequencies, or mutational space, of a majority of amino acid changes sampled by the SHM machinery to be well captured by GSSPs. We further observed the rates of mutational exchange between some amino acids to be both asymmetric and context dependent and to correlate weakly with their biochemical properties. GSSPs provide an improved, position-dependent alternative to standard substitution matrices, and can be utilized to developing software for accurately modeling the SHM process. GSSPs can also be used for predicting the amino acid mutational space available for antigen-driven selection and for understanding factors modulating the maturation pathways of antibody lineages in a gene-specific context. The mGSSP method can be used to build, compare, and plot GSSPs1; we report the GSSPs constructed for 69 common human V genes (DOI: ) and provide high-resolution logo plots for each (DOI: ).