Molecular interactions between protein complexes and DNA carry out essential gene regulatory functions. Uncovering such interactions by means of chromatin-immunoprecipitation coupled with massively parallel sequencing (ChIP-Seq) has recently become the focus of intense interest. We here introduce QuEST (Quantitative Enrichment of Sequence Tags), a powerful statistical framework based on the Kernel Density Estimation approach, which utilizes ChIP-Seq data to determine positions where protein complexes come into contact with DNA. Using QuEST, we discovered several thousand binding sites for the human transcription factors SRF, GABP and NRSF at an average resolution of about 20 base-pairs. MEME-based motif analyses on the QuEST-identified sequences revealed DNA binding by cofactors of SRF, providing evidence that cofactor binding specificity can be obtained from ChIP-Seq data. By combining QuEST analyses with gene ontology (GO) annotations and expression data, we illustrate how general functions of transcription factors can be inferred.

Transcriptional promoters comprise one of many classes of eukaryotic transcriptional regulatory elements. Identification and characterization of these elements are vital to understanding the complex network of human gene regulation. Using full-length cDNA sequences to identify transcription start sites (TSS), we predicted more than 900 putative human transcriptional promoters in the ENCODE regions, representing a comprehensive sampling of promoters in 1% of the genome. We identified 387 fragments that function as promoters in at least one of 16 cell lines by measuring promoter activity in high-throughput transient transfection reporter assays. These positive functional results demonstrate widespread use of alternative promoters. We show a strong correlation between promoter activity and the corresponding endogenous RNA transcript levels, providing the first experimental quantitative estimate of promoter contribution to gene regulation. Finally, we identified functional regions within a randomly selected subset of 45 promoters using deletion analyses. These experiments showed that, on average, the sequence −300 to −50 bp of the TSS positively contributes to core promoter activity. Interestingly, putative negative elements were identified −1000 to −500 bp upstream of the TSS for 55% of genes tested. These data provide the largest and most comprehensive view of promoter function in the human genome.[Supplemental material is available online at www.genome.org.]The regulation of human gene expression is a critical, highly coordinated, and complex process. Gene regulation plays a crucial role in virtually every biological process from coordinating cell division to responding to extracellular stimuli and directing transcription during development (Pirkkala et al. 2001;Ahituv et al. 2004;Blais and Dynlacht 2004). While knowledge of regulation at the level of individual genes is progressing, global characterization of gene regulation currently represents one of the major challenges and fundamental goals for biomedical research. An initial step in achieving this goal is the comprehensive identification of transcriptional regulatory elements in the human genome. Towards this end, the ENCODE (Encyclopedia of DNA Elements) project began in 2004 as a collective effort of many laboratories to identify the functional elements in 1% of the human genome (The ENCODE Project Consortium 2004). In this paper, we describe our efforts to identify and study the transcriptional promoters in the ENCODE regions.Promoters are the best-characterized transcriptional regulatory sequences in complex genomes because of their predictable location immediately upstream of transcription start sites (TSS). They are often described as having two separate segments: core and extended promoter regions. The core promoter is generally within 50 bp of the TSS, where the preinitiation complex forms and the general transcription machinery assembles. The extended promoter can contain specific regulatory sequences that control spatial and temporal expression of...

To investigate the role of DNA methylation during human development, we developed Methyl-seq, a method that assays DNA methylation at more than 90,000 regions throughout the genome. Performing Methyl-seq on human embryonic stem cells (hESCs), their derivatives, and human tissues allowed us to identify several trends during hESC and in vivo liver differentiation. First, differentiation results in DNA methylation changes at a minimal number of assayed regions, both in vitro and in vivo (2%-11%). Second, in vitro hESC differentiation is characterized by both de novo methylation and demethylation, whereas in vivo fetal liver development is characterized predominantly by demethylation. Third, hESC differentiation is uniquely characterized by methylation changes specifically at H3K27me3-occupied regions, bivalent domains, and low density CpG promoters (LCPs), suggesting that these regions are more likely to be involved in transcriptional regulation during hESC differentiation. Although both H3K27me3-occupied domains and LCPs are also regions of high variability in DNA methylation state during human liver development, these regions become highly unmethylated, which is a distinct trend from that observed in hESCs. Taken together, our results indicate that hESC differentiation has a unique DNA methylation signature that may not be indicative of in vivo differentiation.

Efavirenz is a potent and selective nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Nucleotide sequence analyses of the protease and RT genes (coding region for amino acids 1 to 229) of multiple cloned HIV-1 genomes from virus found in the plasma of patients in phase II clinical studies of efavirenz combination therapy were undertaken in order to identify the spectrum of mutations in plasma-borne HIV-1 associated with virological treatment failure. A K103N substitution was the HIV-1 RT gene mutation most frequently observed among plasma samples from patients for whom combination therapy including efavirenz failed, occurring in at least 90% of cases of efavirenz-indinavir or efavirenzzidovudine (ZDV)-lamivudine (3TC) treatment failure. V108I and P225H mutations were observed frequently, predominantly in viral genomes that also contained other nonnucleoside RT inhibitor (NNRTI) resistance mutations. L100I, K101E, K101Q, Y188H, Y188L, G190S, G190A, and G190E mutations were also observed. V106A, Y181C, and Y188C mutations, which have been associated with high levels of resistance to other NNRTIs, were rare in the patient samples in this study, both before and after exposure to efavirenz. The spectrum of mutations observed in cases of virological treatment failure was similar for patients initially dosed with efavirenz at 200, 400, or 600 mg once a day and for patients treated with efavirenz in combination with indinavir, stavudine, or ZDV-3TC. The proportion of patients carrying NNRTI resistance mutations, usually K103N, increased dramatically at the time of initial viral load rebound in cases of treatment failure after exposure to efavirenz. Viruses with multiple, linked NNRTI mutations, especially K103N-V108I and K103N-P225H double mutants, accumulated more slowly following the emergence of K103N mutant viruses.The reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is critical to the life cycle of HIV and is without a homologue in eukaryotic organisms. As such, it is an attractive target for selective antiviral therapy. Among inhibitors of RT, a large class of chemically diverse, generally HIV-1-specific, nonnucleoside reverse transcriptase inhibitors (NNRTIs) has been identified. These inhibitors generally act by binding to a site on the RT that is distinct from the polymerase catalytic site. While NNRTIs can be potent inhibitors of HIV-1 replication, with favorable safety and pharmacokinetic parameters, rapid emergence of resistant viruses both in vitro (20, 24) and in vivo (11,14,15,25,30), often as the result of single nucleotide changes, has limited the therapeutic utility of these compounds as monotherapy. However, recent clinical trials of the use of NNRTIs in combination with other antiretroviral agents have demonstrated an added benefit from inclusion of