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.

Localized accessibility of critical DNA sequences to the regulatory machinery is a key requirement for regulation of human genes. Here we describe a high-resolution, genome-scale approach for quantifying chromatin accessibility by measuring DNase I sensitivity as a continuous function of genome position using tiling DNA microarrays (DNase-array). We demonstrate this approach across 1% ( approximately 30 Mb) of the human genome, wherein we localized 2,690 classical DNase I hypersensitive sites with high sensitivity and specificity, and also mapped larger-scale patterns of chromatin architecture. DNase I hypersensitive sites exhibit marked aggregation around transcriptional start sites (TSSs), though the majority mark nonpromoter functional elements. We also developed a computational approach for visualizing higher-order features of chromatin structure. This revealed that human chromatin organization is dominated by large (100-500 kb) 'superclusters' of DNase I hypersensitive sites, which encompass both gene-rich and gene-poor regions. DNase-array is a powerful and straightforward approach for systematic exposition of the cis-regulatory architecture of complex genomes.

SUMMARY Some Ts in nuclear DNA of trypanosomes and Leishmania are hydroxylated and glucosylated to yield base J (β-D-glucosyl-hydroxymethyluracil). In Leishmania, about 99% of J is located in telomeric repeats. We show here that most of the remaining J is located at chromosome-internal RNA polymerase II termination sites. This internal J and telomeric J can be reduced by a knockout of J-binding protein 2 (JBP2), an enzyme involved in the first step of J biosynthesis. J levels are further reduced by growing Leishmania JBP2 knockout cells in BrdU-containing medium, resulting in cell death. The loss of internal J in JBP2 knockout cells is accompanied by massive readthrough at RNA polymerase II termination sites. The readthrough varies between transcription units but may extend over 100 kb. We conclude that J is required for proper transcription termination and infer that the absence of internal J kills Leishmania by massive readthrough of transcriptional stops.

The genome sequence of the genetically tractable, mesophilic, hydrogenotrophic methanogen Methanococcus maripaludis contains 1,722 protein-coding genes in a single circular chromosome of 1,661,137 bp. Of the protein-coding genes (open reading frames [ORFs]), 44% were assigned a function, 48% were conserved but had unknown or uncertain functions, and 7.5% (129 ORFs) were unique to M. maripaludis. Of the unique ORFs, 27 were confirmed to encode proteins by the mass spectrometric identification of unique peptides. Genes for most known functions and pathways were identified. For example, a full complement of hydrogenases and methanogenesis enzymes was identified, including eight selenocysteine-containing proteins, with each being paralogous to a cysteine-containing counterpart. At least 59 proteins were predicted to contain iron-sulfur centers, including ferredoxins, polyferredoxins, and subunits of enzymes with various redox functions. The methanogenic Archaea (methanogens) occupy a unique metabolic niche, as they produce methane, which is a useful energy source and a powerful greenhouse gas. These organisms are found in diverse anaerobic habitats, ranging from aquatic and marine sediments to sewage digesters and the rumens and large intestines of herbivores and other mammals (127). In these habitats, the degradation of organic matter results in the production of H 2 and other intermediates by fermentative organisms. By maintaining an extremely low partial pressure of H 2 , the methanogens keep fermentative pathways energetically favorable. In addition, some methanogens may occupy niches where hydrogen is produced predominately by geothermal reactions.Metabolically, methanogens are divided into those that specialize in CO 2 reduction and those that also use acetate and/or methyl compounds. The former group, the hydrogenotrophs, use H 2 as an electron donor to reduce CO 2 to methane. Many hydrogenotrophic species can substitute formate or certain low-molecular-weight alcohols and ketones for H 2 . Complete genome sequences have been published for three hydrogenotrophic methanogens, Methanocaldococcus jannaschii (13), Methanothermobacter thermautotrophicus (105), and Methanopyrus kandleri (104), all of which are thermophiles or hyperthermophiles. Of the methanogens that utilize acetate and methyl compounds, complete genome sequences have been published for two species, Methanosarcina acetivorans (26) and Methanosarcina mazei (19), both of which are mesophiles. In addition, partial sequences have been published for two psychrophiles, the hydrogenotroph Methanogenium frigidum and the methylotroph Methanolobus burtonii (97).Genome sequences of methanogens have answered many questions, but they have inspired many others. More than half of the genes in Methanocaldococcus jannaschii lack a predicted function (13), and this proportion has not declined significantly as other methanogen sequences have been determined. The proportions of genes of unknown functions, which are either homologous to other genes of unknown function...