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.

Mapping DNase I hypersensitive (HS) sites is an accurate method of identifying the location of genetic regulatory elements, including promoters, enhancers, silencers, insulators, and locus control regions. We employed high-throughput sequencing and whole-genome tiled array strategies to identify DNase I HS sites within human primary CD4+ T cells. Combining these two technologies, we have created a comprehensive and accurate genome-wide open chromatin map. Surprisingly, only 16%-21% of the identified 94,925 DNase I HS sites are found in promoters or first exons of known genes, but nearly half of the most open sites are in these regions. In conjunction with expression, motif, and chromatin immunoprecipitation data, we find evidence of cell-type-specific characteristics, including the ability to identify transcription start sites and locations of different chromatin marks utilized in these cells. In addition, and unexpectedly, our analyses have uncovered detailed features of nucleosome structure.

No abstract

Little is known about the regulation of neuronal and other cell-type specific epigenomes from the brain. Here, we map the genomewide distribution of trimethylated histone H3K4 (H3K4me3), a mark associated with transcriptional regulation, in neuronal and nonneuronal nuclei collected from prefrontal cortex (PFC) of 11 individuals ranging in age from 0.5 to 69 years. Massively parallel sequencing identified 12,704 H3K4me3 enriched regions (peaks), the majority located proximal to (within 2 kb of) the transcription start site (TSS) of annotated genes. These included peaks shared by neurons in comparison with three control (lymphocyte) cell types, as well as peaks specific to individual subjects. We identified 6,213 genes that show highly enriched H3K4me3 in neurons versus control. At least 1,370 loci, including annotated genes and novel transcripts, were selectively tagged with H3K4me3 in neuronal but not in nonneuronal PFC chromatin. Our results reveal agecorrelated neuronal epigenome reorganization, including decreased H3K4me3 at approximately 600 genes (many function in developmental processes) during the first year after birth. In comparison, the epigenome of aging (>60 years) PFC neurons showed less extensive changes, including increased H3K4me3 at 100 genes. These findings demonstrate that H3K4me3 in human PFC is highly regulated in a cell type-and subject-specific manner and highlight the importance of early childhood for developmentally regulated chromatin remodeling in prefrontal neurons.D evelopmentally regulated changes in histone modifications and DNA methylation, shaping gene expression patterns and genome organization, are critical intermediates for numerous genetic and environmental factors affecting neuronal functions in healthy and diseased brains (1). For example, there is increasing evidence that epigenetic alterations in the cerebral cortex and hippocampus play an important role in the etiology of schizophrenia and other neurodevelopmental disease (2, 3). Cortical neurons permanently exit from the cell cycle during the fetal period, before the dramatic changes in functional connectivity, both on a micro-(e.g., synapse) and macroscale (e.g., network activity, cortical gray matter volumes), that extend into early childhood years and continue throughout adolescence and even beyond (4, 5). To date, however, comprehensive and genomewide maps of neuronal epigenomes, and their developmental trajectories, do not exist. This critical deficiency in epigenetic information, as it pertains to the human-and more generally animal-brain, finally can be addressed because recently it became possible to efficiently separate neuronal chromatin from other chromatin in tissue, thereby avoiding potential confounds such as the highly dynamic changes in glia cell densities during cortical ontogenesis and maturation (6). Here, we employ ChIP-Seq (7) to study the genome-wide distribution of histone H3K4 trimethylation (H3K4me3)-an epigenetic mark highly enriched at start sites of actual or potential transcription (8)-in n...

The cJun NH2-terminal kinase (JNK) stress signaling pathway is implicated in the metabolic response to the consumption of a high fat diet, including the development of obesity and insulin resistance. These metabolic adaptations involve altered liver function. Here we demonstrate that hepatic JNK potently represses the nuclear hormone receptor peroxisome proliferator-activated receptor α (PPARα). JNK therefore causes decreased expression of PPARα target genes that increase fatty acid oxidation / ketogenesis and promote the development of insulin resistance. We show that the PPARα target gene fibroblast growth factor 21 (Fgf21) plays a key role in this response because disruption of the hepatic PPARα - FGF21 hormone axis suppresses the metabolic effects of JNK-deficiency. This analysis identifies the hepatokine FGF21 as a critical mediator of JNK signaling in the liver.

For a binary polymer brush layer, we investigated the morphological state, the structure reordering, and the nanomechanical properties as a function of treatment with selective solvents. Two incompatible polymers, poly(methyl acrylate) (PMA) and poly(styrene-co-2,3,4,5,6-pentafluorostyrene) (PSF), were randomly grafted one after another onto a silicon wafer via the "grafting from" method producing thick (20-150 nm) dense mixed brush layers. The resulting layers possessed a nanostructured surface exhibiting either complete vertical or a combination of vertical and lateral microphase segregation of the two components. The lateral and vertical reorganization of the mixed brush layer was observed to be quick (on the order of a few minutes) and reversible for at least 100 "switches" between good and bad solvent states for each component. Atomic force microscopy (AFM) images revealed different surface structure states upon exposure to different solvents. Since PSF and PMA are mechanically dissimilar (glassy and rubbery, respectively) at room temperature, phase imaging was used to roughly verify the resulting structure. However, to determine vertical segregation in addition to truly authenticating the lateral ordering, surface nanomechanical mapping was conducted, which also allowed, for the first time, to directly determine the elastic modulus and adhesion. Results show the bimodal response of the mechanically heterogeneous surface, with elastic modulus and adhesion distributions very different for the "glassy state" and the "rubbery state". Furthermore, depth profiling of the elastic modulus conducted for binary brushes confirmed the vertical segregation in the mixed brush. Results demonstrated the dramatic mechanical contrast of the surface as a function of solvent conditions and decisively revealed the modes of phase segregation in a binary polymer brush.

The identification of regulatory elements from different cell types is necessary for understanding the mechanisms controlling cell type–specific and housekeeping gene expression. Mapping DNaseI hypersensitive (HS) sites is an accurate method for identifying the location of functional regulatory elements. We used a high throughput method called DNase-chip to identify 3,904 DNaseI HS sites from six cell types across 1% of the human genome. A significant number (22%) of DNaseI HS sites from each cell type are ubiquitously present among all cell types studied. Surprisingly, nearly all of these ubiquitous DNaseI HS sites correspond to either promoters or insulator elements: 86% of them are located near annotated transcription start sites and 10% are bound by CTCF, a protein with known enhancer-blocking insulator activity. We also identified a large number of DNaseI HS sites that are cell type specific (only present in one cell type); these regions are enriched for enhancer elements and correlate with cell type–specific gene expression as well as cell type–specific histone modifications. Finally, we found that approximately 8% of the genome overlaps a DNaseI HS site in at least one the six cell lines studied, indicating that a significant percentage of the genome is potentially functional.

Mapping histone methylation landscapes in neurons from human, chimpanzee, and macaque brains reveals coordinated, human-specific epigenetic regulation at hundreds of regulatory sequences.