From Genome to Regulatory Networks: For biologists, having a genome in hand is only the beginning—much more investigation is still needed to characterize how the genome is used to help to produce a functional organism (see the Perspective by Blaxter ). In this vein, Gerstein et al. (p. 1775 ) summarize for the Caenorhabditis elegans genome, and The modENCODE Consortium (p. 1787 ) summarize for the Drosophila melanogaster genome, full transcriptome analyses over developmental stages, genome-wide identification of transcription factor binding sites, and high-resolution maps of chromatin organization. Both studies identified regions of the nematode and fly genomes that show highly occupied targets (or HOT) regions where DNA was bound by more than 15 of the transcription factors analyzed and the expression of related genes were characterized. Overall, the studies provide insights into the organization, structure, and function of the two genomes and provide basic information needed to guide and correlate both focused and genome-wide studies.

A large collection of new modENCODE and ENCODE genome-wide chromatin data sets from cell lines and developmental stages in worm, fly and human are analysed; this reveals many conserved features of chromatin organization among the three organisms, as well as notable differences in the composition and locations of repressive chromatin. This study describes numerous new genome-wide chromatin data sets from cell lines and developmental stages of Homo sapiens, Drosophila melanogaster and Caenorhabditis elegans generated by the ENCODE and modENCODE consortia. The results point to many conserved features of chromatin organization among the three organisms, while identifying differences in the composition and locations of repressive chromatin. Genome function is dynamically regulated in part by chromatin, which consists of the histones, non-histone proteins and RNA molecules that package DNA. Studies in Caenorhabditis elegans and Drosophila melanogaster have contributed substantially to our understanding of molecular mechanisms of genome function in humans, and have revealed conservation of chromatin components and mechanisms1,2,3. Nevertheless, the three organisms have markedly different genome sizes, chromosome architecture and gene organization. On human and fly chromosomes, for example, pericentric heterochromatin flanks single centromeres, whereas worm chromosomes have dispersed heterochromatin-like regions enriched in the distal chromosomal ‘arms’, and centromeres distributed along their lengths4,5. To systematically investigate chromatin organization and associated gene regulation across species, we generated and analysed a large collection of genome-wide chromatin data sets from cell lines and developmental stages in worm, fly and human. Here we present over 800 new data sets from our ENCODE and modENCODE consortia, bringing the total to over 1,400. Comparison of combinatorial patterns of histone modifications, nuclear lamina-associated domains, organization of large-scale topological domains, chromatin environment at promoters and enhancers, nucleosome positioning, and DNA replication patterns reveals many conserved features of chromatin organization among the three organisms. We also find notable differences in the composition and locations of repressive chromatin. These data sets and analyses provide a rich resource for comparative and species-specific investigations of chromatin composition, organization and function.

The origin recognition complex (ORC) specifies replication origin location. The Saccharomyces cerevisiae ORC recognizes the ARS (autonomously replicating sequence) consensus sequence (ACS), but only a subset of potential genomic sites are bound, suggesting other chromosomal features influence ORC binding. Using high-throughput sequencing to map ORC binding and nucleosome positioning, we show that yeast origins are characterized by an asymmetric pattern of positioned nucleosomes flanking the ACS. The origin sequences are sufficient to maintain a nucleosome-free origin; however, ORC is required for the precise positioning of nucleosomes flanking the origin. These findings identify local nucleosomes as an important determinant for origin selection and function.

The origin recognition complex (ORC) is an essential DNA replication initiation factor conserved in all eukaryotes. In Saccharomyces cerevisiae, ORC binds to specific DNA elements; however, in higher eukaryotes, ORC exhibits little sequence specificity in vitro or in vivo. We investigated the genome-wide distribution of ORC in Drosophila and found that ORC localizes to specific chromosomal locations in the absence of any discernible simple motif. Although no clear sequence motif emerged, we were able to use machine learning approaches to accurately discriminate between ORC-associated sequences and ORC-free sequences based solely on primary sequence. The complex sequence features that define ORC binding sites are highly correlated with nucleosome positioning signals and likely represent a preferred nucleosomal landscape for ORC association. Open chromatin appears to be the underlying feature that is deterministic for ORC binding. ORC-associated sequences are enriched for the histone variant, H3.3, often at transcription start sites, and depleted for bulk nucleosomes. The density of ORC binding along the chromosome is reflected in the time at which a sequence replicates, with early replicating sequences having a high density of ORC binding. Finally, we found a high concordance between sites of ORC binding and cohesin loading, suggesting that, in addition to DNA replication, ORC may be required for the loading of cohesin on DNA in Drosophila.

The size of a eukaryotic genome presents a unique challenge to the cell: package and organize the DNA to fit within the confines of the nucleus while at the same time ensuring sufficient dynamics to allow access to specific sequences and features such as genes and regulatory elements. This is achieved via the dynamic nucleoprotein organization of eukaryotic DNA into chromatin. The basic unit of chromatin, the nucleosome, comprises a core particle with 147 bp of DNA wrapped 1.7 times around an octamer of histones. The nucleosome is a highly versatile and modular structure, both in its composition, with the existence of various histone variants, and through the addition of a series of posttranslational modifications on the histones. This versatility allows for both short-term regulatory responses to external signaling, as well as the long-term and multigenerational definition of large functional chromosomal domains within the nucleus, such as the centromere. Chromatin organization and its dynamics participate in essentially all DNA-templated processes, including transcription, replication, recombination, and repair. Here we will focus mainly on nucleosomal organization and describe the pathways and mechanisms that contribute to assembly of this organization and the role of chromatin in regulating the DNA replication program.

For more than three decades, investigators have sought to identify the precise locations where DNA replication initiates in mammalian genomes. The development of molecular and biochemical approaches to identify start sites of DNA replication (origins) based on the presence of defining and characteristic replication intermediates at specific loci led to the identification of only a handful of mammalian replication origins. The limited number of identified origins prevented a comprehensive and exhaustive search for conserved genomic features that were capable of specifying origins of DNA replication. More recently, the adaptation of origin-mapping assays to genome-wide approaches has led to the identification of tens of thousands of replication origins throughout mammalian genomes, providing an unprecedented opportunity to identify both genetic and epigenetic features that define and regulate their distribution and utilization. Here we summarize recent advances in our understanding of how primary sequence, chromatin environment, and nuclear architecture contribute to the dynamic selection and activation of replication origins across diverse cell types and developmental stages.