The mission of the Encyclopedia of DNA Elements (ENCODE) Project is to enable the scientific and medical communities to interpret the human genome sequence and apply it to understand human biology and improve health. The ENCODE Consortium is integrating multiple technologies and approaches in a collective effort to discover and define the functional elements encoded in the human genome, including genes, transcripts, and transcriptional regulatory regions, together with their attendant chromatin states and DNA methylation patterns. In the process, standards to ensure high-quality data have been implemented, and novel algorithms have been developed to facilitate analysis. Data and derived results are made available through a freely accessible database. Here we provide an overview of the project and the resources it is generating and illustrate the application of ENCODE data to interpret the human genome.

We report a new chemoenzymatic strategy for the rapid and sensitive detection of O-GlcNAc posttranslational modifications. The approach exploits the ability of an engineered mutant of beta-1,4-galactosyltransferase to selectively transfer an unnatural ketone functionality onto O-GlcNAc glycosylated proteins. Once transferred, the ketone moiety serves as a versatile handle for the attachment of biotin, thereby enabling chemiluminescent detection of the modified protein. Importantly, this approach permits the rapid visualization of proteins that are at the limits of detection using traditional methods. Moreover, it bypasses the need for radioactive precursors and captures the glycosylated species without perturbing metabolic pathways. We anticipate that this general chemoenzymatic strategy will have broad application to the study of posttranslational modifications.

Disruptions in local chromatin structure often indicate features of biological interest such as regulatory regions. We find that sonication of cross-linked chromatin, when combined with a sizeselection step and massively parallel short-read sequencing, can be used as a method (Sono-Seq) to map locations of high chromatin accessibility in promoter regions. Sono-Seq sites frequently correspond to actively transcribed promoter regions, as evidenced by their co-association with RNA Polymerase II ChIP regions, transcription start sites, histone H3 lysine 4 trimethylation (H3K4me3) marks, and CpG islands; signals over other sites, such as those bound by the CTCF insulator, are also observed. The pattern of breakage by Sono-Seq overlaps with, but is distinct from, that observed for FAIRE and DNase I hypersensitive sites. Our results demonstrate that Sono-Seq can be a useful and simple method by which to map many local alterations in chromatin structure. Furthermore, our results provide insights into the mapping of binding sites by using ChIP-Seq experiments and the value of reference samples that should be used in such experiments.ChIP-Seq ͉ ENCODE ͉ formaldehyde cross-linking ͉ sonication ͉ DNA sequencing T he accessibility of regulatory elements in chromatin is critical for many aspects of gene regulation. Nucleosomes positioned over regulatory elements inhibit access of transcription factors to DNA; deprotection of the DNA arises from local changes in chromatin conformation. Previous methods for mapping chromatin accessibility include mapping DNase I hypersensitivity sites or formaldehyde-assisted isolation of regulatory elements (FAIRE) regions and analyzing the DNA using microarrays or DNA sequencing (1-3). These methods have mapped many open chromatin sites to promoters of actively transcribed genes as well as to enhancers.The in vivo mapping of regulatory elements is often performed by chromatin immunoprecipitating of a factor of interest followed by analyzing the associated DNA (4-6). Chromatin complexes are preserved through cell fixation with formaldehyde, the chromatin is fragmented, and protein-bound DNA regions are isolated by using antibodies to a specific DNA-associated protein. DNA fragments are purified and used to probe DNA microarrays (ChIPchip) or, more recently, identified by high-throughput DNA sequencing (ChIP-Seq), thereby locating transcription factor binding sites (TFBSs) on a genome-wide scale (4-7). In ChIP experiments, significant targets representing binding regions are found by analyzing signal levels produced by an experimental sample relative to a reference sample. Although several automated scoring algorithms exist for ChIP-Seq data (6,(8)(9)(10)(11), an appreciation of the characteristics and biases inherent to different reference DNA samples and preparation methods is important for understanding the significance of the results obtained.In the work presented here, we examine the signal distributions of commonly used reference samples including sonicated chromatin and investigate the a...

Experimental Section. General. Unless otherwise noted, reagents were purchased from the commercial suppliers Fisher (Fairlawn, NJ) and Sigma-Aldrich (St. Louis, MO) and were used without further purification. Protease and phosphatase inhibitors were purchased from Sigma-Aldrich or Alexis Biochemicals (San Diego, CA). Bovine GalT and ovalbumin were obtained from Sigma-Aldrich. Preparation of rat brain nuclear extracts. Nuclear extracts were prepared as previously reported 1 with minor modifications. The forebrains of Sprague Dawley rats (Charles River Laboratories, Kingston, MA) were dissected on ice and homogenized in 10 volumes of ice-cold buffer A (10 mM HEPES pH 7.9, 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM DTT) containing protease inhibitors (5 µg/ml pepstatin, 5 µg/ml chymostatin, 20 µg/ml leupeptin, 20 µg/ml aprotinin, 20 µg/ml antipain, 0.2 mM PMSF), phosphatase inhibitors (20 mM NaF, 1 mM Na 3 VO 4 , 50 µM Na 2 MoO 4 ), and hexosaminidase inhibitors (50 mM GlcNAc and 10 µM streptozocin). The resulting lysate was centrifuged at 1,000xg at 4 o C for 10 min, and the crude nuclear pellet was washed with buffer A. The pellet was resuspended at 2 mg/ml in buffer B (20 mM HEPES pH 7.9, 0.42 M NaCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 50 mM GlcNAc, 0.2 mM PMSF, 25% (v/v) glycerol) by stirring at 4 o C for 40 min. Following centrifugation at 10,000xg for 30 min, the supernatant was defined as the nuclear extract. The nuclear extract was dialyzed against buffer D (20 mM HEPES pH 7.9, 0.1 M KCl, 0.5 mM DTT, 0.2 mM EDTA, 50 mM GlcNAc, 20% (v/v) glycerol) for 6 h at 4 o C, and proteins were precipitated using (NH 4 ) 2 SO 4 (38% final concentration). The protein pellet obtained after centrifugation at 21,500xg for 10 min was solubilized in buffer C (0.3 volumes; 25 mM Tris pH7.5, 1 mM EDTA, 1 mM DTT containing protease inhibitors) by gentle mixing for 1 h at 4 o C. Following clarification at 21,500xg for 5 min, the supernatant was dialyzed into buffer G (20 mM HEPES pH 7.3, 0.1 M KCl, 0.5 mM DTT, 0.2 mM EDTA, 5 mM MnCl 2 , 0.2 mM PMSF) overnight at 4 o C. The protein concentration was determined using the BCA assay (Pierce/Endogen Biotech, Rockford, IL).

Extensive sonication of formaldehyde-crosslinked chromatin can generate DNA fragments averaging 200 bp in length (range 75–300 bp). Fragmentation is largely random with respect to genomic region and nucleosome position. ChIP experiments employing such extensively fragmented samples show 2- to 4-fold increased enrichment of protein binding sites over control genomic regions, when compared to samples sonicated to a more conventional size range (300–500 bp). The basis of improved fold enrichments is that immunoprecipitation of protein-bound regions is unaffected by fragment size, whereas immunoprecipitation of control genomic regions decreases progressively along with reduced fragment size due to fewer nonspecific binding sites. The use of extensively sonicated samples improves mapping of protein binding sites, and it extends the dynamic range for quantitative measurements of histone density. We show that many yeast promoter regions are virtually devoid of histones.