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.

The glucocorticoid steroid hormone cortisol is released by the adrenal glands in response to stress and serves as a messenger in circadian rhythms. Transcriptional responses to this hormonal signal are mediated by the glucocorticoid receptor (GR). We determined GR binding throughout the human genome by using chromatin immunoprecipitation followed by next-generation DNA sequencing, and measured related changes in gene expression with mRNA sequencing in response to the glucocorticoid dexamethasone (DEX). We identified 4392 genomic positions occupied by the GR and 234 genes with significant changes in expression in response to DEX. This genomic census revealed striking differences between gene activation and repression by the GR. While genes activated with DEX treatment have GR bound within a median distance of 11 kb from the transcriptional start site (TSS), the nearest GR binding for genes repressed with DEX treatment is a median of 146 kb from the TSS, suggesting that DEX-mediated repression occurs independently of promoter-proximal GR binding. In addition to the dramatic differences in proximity of GR binding, we found differences in the kinetics of gene expression response for induced and repressed genes, with repression occurring substantially after induction. We also found that the GR can respond to different levels of corticosteroids in a gene-specific manner. For example, low doses of DEX selectively induced PER1, a transcription factor involved in regulating circadian rhythms.

Mot1 is an essential Snf2/Swi2-related ATPase and TATAbinding protein (TBP)-associated factor (TAF). In vitro, Mot1 utilizes ATP hydrolysis to disrupt TBP-DNA complexes, but the relationship of this activity to Mot1's in vivo function is unclear. Chromatin immunoprecipitation was used to determine how Mot1 affects the assembly of preinitiation complexes (PICs) at Mot1-controlled promoters in vivo. We find that the Mot1-repressed HSP26 and INO1 promoters are both regulated by TBP recruitment; inactivation of Mot1 leads to increased PIC formation coincident with derepression of transcription. For the Mot1-activated genes BNA1 and URA1, inactivation of Mot1 also leads, remarkably, to increased TBP binding to the promoters, despite the fact that transcription of these genes is obliterated in mot1 cells. In contrast, levels of Taf1, TFIIB, and RNA polymerase II are reduced at Mot1-activated promoters in mot1 cells. These results suggest that Mot1-mediated displacement of TBP underlies its mechanism of repression and activation at these genes. We suggest that at activated promoters, Mot1 disassembles transcriptionally inactive TBP, thereby facilitating the formation of a TBP complex that supports functional PIC assembly.

Although pathways for assembly of RNA polymerase (Pol) II transcription preinitiation complexes (PICs) have been well established in vitro, relatively little is known about the dynamic behavior of Pol II general transcription factors in vivo. In vitro, a subset of Pol II factors facilitates reinitiation by remaining very stably bound to the promoter. This behavior contrasts markedly with the highly dynamic behavior of RNA Pol I transcription complexes in vivo, which undergo cycles of disassembly/reassembly at the promoter for each round of transcription. To determine whether the dynamic behavior of the Pol II machinery in vivo is fundamentally different from that of Pol I and whether the static behavior of Pol II factors in vitro fully recapitulates their behavior in vivo, we used fluorescence recovery after photobleaching (FRAP). Surprisingly, we found that all or nearly all of the TATA-binding protein (TBP) population is highly mobile in vivo, displaying FRAP recovery rates of <15 s. These high rates require the activity of the TBP-associated factor Mot1, suggesting that TBP/chromatin interactions are destabilized by active cellular processes. Furthermore, the distinguishable FRAP behavior of TBP and TBP-associated factor 1 indicates that there are populations of these molecules that are independent of one another. The distinct FRAP behavior of most Pol II factors that we tested suggests that transcription complexes assemble via stochastic multistep pathways. Our data indicate that active Pol II PICs can be much more dynamic than previously considered.fluorescence recovery after photobleaching ͉ Mot1 ͉ TFIID T ranscription preinitiation complex (PIC) formation involves the assembly of general transcription factors (GTFs) at appropriately remodeled chromatin templates. Regulation of RNA polymerase (Pol) II transcription initiation can occur by many different mechanisms involving facilitated recruitment of PIC components or stimulation of their activities at the promoter. Although the diversity of such regulatory mechanisms is widely appreciated, comparatively little is known about the dynamic behavior of GTFs in vivo vis a vis their interactions with each other and with chromatin. Most nuclear proteins studied to date, including a number of gene-specific transcription factors and chromatin-modifying enzymes, display highly dynamic behavior in vivo (1-3). In many cases, this dynamic behavior is energy-independent (4). Even chromatin structural proteins once thought to be stably bound to DNA display dynamic behavior in vivo (3-9). However, the dynamic behavior of GTFs is largely unexplored. In vitro, a PIC scaffold nucleated by TATA binding protein (TBP) is very stable, a property that facilitates multiple rounds of transcription at an activated promoter (10). A stable TBP association with chromatin is supported by analysis in human cells, which yields a slow fluorescence recovery time for TBP of 20 min (11). In striking contrast, in vivo analysis of RNA Pol I PICs yields a much more dynamic picture, with fluores...

Mot1 is a conserved Snf2/Swi2-related transcriptional regulator that uses ATP hydrolysis to displace TATAbinding protein (TBP) from DNA. Several models of the enzymatic mechanism have been proposed, including Mot1-catalyzed distortion of TBP structure, competition between Mot1 and DNA for the TBP DNA-binding surface, and ATP-driven translocation of Mot1 along DNA. Here, DNase I footprinting studies provide strong support for a 'DNA-based' mechanism of Mot1, which we propose involves ATP-driven DNA translocation. Mot1 forms an asymmetric complex with the TBP core domain (TBPc)-DNA complex, contacting DNA both upstream and within the major groove of the TATA Box. Contact with upstream DNA is required for Mot1-mediated displacement of TBPc from DNA. Using the SsoRad54-DNA complex as a model, DNA-binding residues in Mot1 were identified that are critical for Mot1-TBPc-DNA complex formation and catalytic activity, thus placing Mot1 mechanistically within the helicase superfamily. We also report a novel ATP-independent TBPc displacement activity for Mot1 and describe conformational heterogeneity in the Mot1 ATPase, which is likely a general feature of other enzymes in this class.