We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.

Traditional culture-based methods have incompletely defined the etiology of common recalcitrant human fungal skin diseases including athlete’s foot and toenail infections. Skin protects humans from invasion by pathogenic microorganisms, while providing a home for diverse commensal microbiota1. Bacterial genomic sequence data have generated novel hypotheses about species and community structures underlying human disorders2,3,4. However, microbial diversity is not limited to bacteria; microorganisms such as fungi also play major roles in microbial community stability, human health and disease5. Genomic methodologies to identify fungal species and communities have been limited compared with tools available for bacteria6. Fungal evolution can be reconstructed with phylogenetic markers, including ribosomal RNA gene regions and other highly conserved genes7. Here, we sequenced and analyzed fungal communities of 14 skin sites in 10 healthy adults. Eleven core body and arm sites were dominated by Malassezia fungi, with species-level classifications revealing greater topographical resolution between sites. By contrast, three foot sites, plantar heel, toenail, and toeweb, exhibited tremendous fungal diversity. Concurrent analysis of bacterial and fungal communities demonstrated that skin physiologic attributes and topography differentially shape these two microbial communities. These results provide a framework for future investigation of interactions between pathogenic and commensal fungal and bacterial communities in maintaining human health and contributing to disease pathogenesis.

Summary Biogeography and individuality shape the structural and functional composition of the human skin microbiome. To explore these factors’ contribution to skin microbial community stability, we generated metagenomic sequence data from longitudinal samples collected over months and years. Analyzing these samples using a multi-kingdom, reference-based approach, we found that despite the skin’s exposure to the external environment, its bacterial, fungal, and viral communities were largely stable over time. Site, individuality, and phylogeny were all determinants of this stability; foot sites exhibited the most variability, individuals differed in stability, and transience was a particular characteristic of eukaryotic viruses, which showed little site-specificity in colonization. Strain and single nucleotide variant level analysis showed that individuals maintain, rather than reacquire prevalent microbes from the environment. Longitudinal stability of skin microbial communities generates hypotheses about colonization resistance and empowers clinical studies exploring alterations observed in disease states.

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.

Public health officials have raised concerns that plasmid transfer between Enterobacteriaceae species may spread resistance to carbapenems, an antibiotic class of last resort, thereby rendering common healthcare-associated infections nearly impossible to treat. We performed comprehensive surveillance and genomic sequencing to identify carbapenem-resistant Enterobacteriaceae in the NIH Clinical Center patient population and hospital environment in order to to articulate the diversity of carbapenemase-encoding plasmids and survey the mobility of and assess the mobility of these plasmids between bacterial species. We isolated a repertoire of carbapenemase-encoding Enterobacteriaceae, including multiple strains of Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli, Enterobacter cloacae, Citrobacter freundii, and Pantoea species. Long-read genome sequencing with full end-to-end assembly revealed that these organisms carry the carbapenem-resistance genes on a wide array of plasmids. Klebsiella pneumoniae and Enterobacter cloacae isolated simultaneously from a single patient harbored two different carbapenemase-encoding plasmids, overriding the epidemiological scenario of plasmid transfer between organisms within this patient. We did, however, find evidence supporting horizontal transfer of carbapenemase-encoding plasmids between Klebsiella pneumoniae, Enterobacter cloacae and Citrobacter freundii in the hospital environment. Our comprehensive sequence data, with full plasmid identification, challenges assumptions about horizontal gene transfer events within patients and identified wider possible connections between patients and the hospital environment. In addition, we identified a new carbapenemase-encoding plasmid of potentially high clinical impact carried by Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae and Pantoea species, from unrelated patients and the hospital environment.

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.

While landmark studies have shown that microbiota activate and educate host immunity, how immune systems shape microbiomes and contribute to disease is incompletely characterized. Primary immunodeficiency (PID) patients suffer recurrent microbial infections, providing a unique opportunity to address this issue. To investigate the potential influence of host immunity on the skin microbiome, we examined skin microbiomes in patients with rare monogenic PIDs: hyper-IgE (STAT3-deficient), Wiskott-Aldrich, and dedicator of cytokinesis 8 syndromes. While specific immunologic defects differ, a shared hallmark is atopic dermatitis (AD)-like eczema. We compared bacterial and fungal skin microbiomes (41 PID, 13 AD, 49 healthy controls) at four clinically relevant sites representing the major skin microenvironments. PID skin displayed increased ecological permissiveness with altered population structures, decreased site specificity and temporal stability, and colonization with microbial species not observed in controls, including Clostridium species and Serratia marcescens. Elevated fungal diversity and increased representation of opportunistic fungi (Candida, Aspergillus) supported increased PID skin permissiveness, suggesting that skin may serve as a reservoir for the recurrent fungal infections observed in these patients. The overarching theme of increased ecological permissiveness in PID skin was counterbalanced by the maintenance of a phylum barrier in which colonization remained restricted to typical human-associated phyla. Clinical parameters, including markers of disease severity, were positively correlated with prevalence of Staphylococcus, Corynebacterium, and other less abundant taxa. This study examines differences in microbial colonization and community stability in PID skin and informs our understanding of host-microbiome interactions, suggesting a bidirectional dialogue between skin commensals and the host organism.

A key component of the ongoing ENCODE project involves rigorous comparative sequence analyses for the initially targeted 1% of the human genome. Here, we present orthologous sequence generation, alignment, and evolutionary constraint analyses of 23 mammalian species for all ENCODE targets. Alignments were generated using four different methods; comparisons of these methods reveal large-scale consistency but substantial differences in terms of small genomic rearrangements, sensitivity (sequence coverage), and specificity (alignment accuracy). We describe the quantitative and qualitative trade-offs concomitant with alignment method choice and the levels of technical error that need to be accounted for in applications that require multisequence alignments. Using the generated alignments, we identified constrained regions using three different methods. While the different constraint-detecting methods are in general agreement, there are important discrepancies relating to both the underlying alignments and the specific algorithms. However, by integrating the results across the alignments and constraint-detecting methods, we produced constraint annotations that were found to be robust based on multiple independent measures. Analyses of these annotations illustrate that most classes of experimentally annotated functional elements are enriched for constrained sequences; however, large portions of each class (with the exception of protein-coding sequences) do not overlap constrained regions. The latter elements might not be under primary sequence constraint, might not be constrained across all mammals, or might have expendable molecular functions. Conversely, 40% of the constrained sequences do not overlap any of the functional elements that have been experimentally identified. Together, these findings demonstrate and quantify how many genomic functional elements await basic molecular characterization.