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 origin recognition complex (ORC) binds sites from which DNA replication is initiated. We address ORC binding selectivity in vivo by mapping ∼52,000 ORC2 binding sites throughout the human genome. The ORC binding profile is broader than those of sequence-specific transcription factors, suggesting that ORC is not bound or recruited to specific DNA sequences. Instead, ORC binds nonspecifically to open (DNase I-hypersensitive) regions containing active chromatin marks such as H3 acetylation and H3K4 methylation. ORC sites in early and late replicating regions have similar properties, but there are far more ORC sites in early replicating regions. This suggests that replication timing is due primarily to ORC density and stochastic firing of origins. Computational simulation of stochastic firing from identified ORC sites is in accord with replication timing data. Large genomic regions with a paucity of ORC sites are strongly associated with common fragile sites and recurrent deletions in cancers. We suggest that replication origins, replication timing, and replication-dependent chromosome breaks are determined primarily by the genomic distribution of activator proteins at enhancers and promoters. These activators recruit nucleosome-modifying complexes to create the appropriate chromatin structure that allows ORC binding and subsequent origin firing.DNA replication | replication origins | chromatin | replication timing | ORC R eplication origins are established by the assembly of the prereplication complex at discrete sites of the genome. The first step of this process involves binding of the highly conserved six-subunit origin recognition complex (ORC), which serves as a loading platform for the subsequent assembly of helicases, DNA polymerases, and cofactors required for DNA synthesis (1, 2). In the yeast Saccharomyces cerevisiae, ORC binds DNA in an ATP-dependent manner and recognizes a specific DNA sequence (3). In Drosophila, ORC localizes to regions of open chromatin with contributions from activating histone modifications, DNA sequence, DNA binding proteins, and nucleosome remodelers (4-6). In mammals, the mechanism(s) through which ORC is localized and establishes a functional origin remains unclear.A great deal of effort and a variety of experimental approaches have been devoted to describing the nature and position of replication origins in mammalian genomes. DNA combing technology, replication timing analysis, short nascent strand (SNS) enrichment, and bubble trapping approaches suggest that DNA replication initiation sites are enriched in CpG-rich regions, open chromatin domains, and transcriptional regulatory elements (7-17). However, these methods lack the necessary resolution to investigate important relationships of ORC binding with other features of the genome. In addition, the divergence in protocols and bioinformatic pipelines between laboratories has led to some controversial and nonreproducible observations. Finally, these studies assume that the identified replication initiation sites are compa...

HBO1, an H4-specific histone acetylase, is a co-activator of the DNA replication licensing factor Cdt1. HBO1 acetylase activity is required for licensing, because a HAT-defective mutant of HBO1 bound at origins is unable to load the MCM complex. H4 acetylation at origins is cell-cycle regulated, with maximal activity at the G1/S transition, and co-expression of HBO1 and Jade1 increases histone acetylation and MCM complex loading. Overexpression of the Set8 histone H4 tail-binding domain specifically inhibits MCM loading, suggesting that histones are a physiologically relevant target for licensing. Lastly, Geminin, inhibits HBO1 acetylase activity in the context of a Cdt1-HBO1 complex, and it associates with origins and inhibits H4 acetylation and licensing in vivo. Thus, H4 acetylation at origins by HBO1 is critical for replication licensing by Cdt1, and negative regulation of licensing by Geminin is likely to involve inhibition of HBO1 histone acetylase activity.

HBO1 histone acetylase is important for DNA replication licensing. In human cells, HBO1 associates with replication origins specifically during the G1 phase of the cell cycle in a manner that depends on the replication licensing factor Cdt1, but is independent of the Cdt1 repressor Geminin. HBO1 directly interacts with Cdt1, and it enhances Cdt1-dependent rereplication. Thus, HBO1 plays a direct role at replication origins as a coactivator of the Cdt1 licensing factor. As HBO1 is also a transcriptional coactivator, it has the potential to integrate internal and external stimuli to coordinate transcriptional responses with initiation of DNA replication. The initial step of DNA replication involves the formation of the prereplication complex (pre-RC) on origins of replication distributed throughout the genome. Sequential assembly of two multiprotein complexes, ORC (origin recognition complex) and MCM (minichromosome maintenance), result in a pre-RC that is "licensed" for replication that will occur in the subsequent S phase (Bell and Dutta 2002). ORC associates with replication origins throughout the entire cell cycle, whereas the MCM complex is specifically loaded during late mitosis through G1 phase under the control of ORC and ORCassociated licensing factors Cdt1 and Cdc6 (Thommes and Blow 1997;Bell and Dutta 2002). As cells enter S phase, origins are activated to initiate DNA replication, whereupon the pre-RC disassembles. Relicensing of replication origins does not occur in S phase, thereby restricting DNA synthesis to once per cell cycle.Regulation of Cdt1 plays a critical role in the licensing process. Inhibition of Cdt1 activity occurs by multiple mechanisms, including ubiquitin-dependent proteolysis and binding to its potent inhibitor Geminin (Wohlschlegel et al. 2000;Tada et al. 2001;Arias and Walter 2007). Misregulation of the licensing process and defects in the ordered assembly and disassembly of the pre-RC alter the integrity of the genome (Vaziri et al. 2003;Saxena and Dutta 2005;Niida and Nakanishi 2006;Tatsumi et al. 2006;Hook et al. 2007). Thus, cell cycle regulation of Cdt1 expression and function is responsible for licensing replication origins in G1 and preventing relicensing and hence rereplication in S phase.As expected for any molecular process involving DNA in eukaryotic cells, DNA replication initiation is influenced by chromatin structure. In particular, histone acetylation is linked to pre-RC assembly and the control of initiation of DNA replication. In yeast and mammalian cells, early-firing origins are typically localized in genomic regions that are transcribed and contain hyperacetylated chromatin, whereas late-firing origins lie in silenced heterochromatic domains (Kemp et al. 2005;Zhou et al. 2005;Karnani et al. 2007;Lucas et al. 2007;Goren et al. 2008). In addition, histone acetylation is involved in origin activation during early development in Xenopus (Danis et al. 2004) and at the chorion gene loci in Drosophila follicle cells (Aggarwal and Calvi 2004;Hartl et al. 2007). Howeve...

Faithful DNA replication is essential for the maintenance of genome integrity. Incomplete genome replication leads to DNA breaks and chromosomal rearrangements, which are causal factors in cancer and other human diseases. Despite their importance, the molecular mechanisms that control human genome stability are incompletely understood. Here, we report a pathway that is required for human genome replication and stability. This pathway has three components: an E3 ubiquitin ligase, a transcriptional repressor, and a replication protein. The E3 ubiquitin ligase RBBP6 ubiquitinates and destabilizes the transcriptional repressor ZBTB38. This repressor negatively regulates transcription and levels of the MCM10 replication factor on chromatin. Cells lacking RBBP6 experience reduced replication fork progression and increased damage at common fragile sites due to ZBTB38 accumulation and MCM10 downregulation. Our results uncover a pathway that ensures genome-wide DNA replication and chromosomal stability.