title: Bubble-seq analysis of the human genome reveals distinct chromatin-mediated mechanisms for regulating early- and late-firing origins.
abstract: We have devised a method for isolating virtually pure and comprehensive libraries of restriction fragments that contained replication initiation sites (bubbles) in vivo. We have now sequenced and mapped the bubble-containing fragments from GM06990, a near-normal EBV-transformed lymphoblastoid cell line, and have compared origin distributions with a comprehensive replication timing study recently published for this cell line. We find that early-firing origins, which represent ∼32% of all origins, overwhelmingly represent zones, associate only marginally with active transcription units, are localized within large domains of open chromatin, and are significantly associated with DNase I hypersensitivity. Origin "density" falls from early- to mid-S-phase, but rises again in late S-phase to levels only 17% lower than in early S-phase. Unexpectedly, late origin density calculated on the 1-Mb scale increases as a function of increasing chromatin compaction. Furthermore, the median efficiency of origins in late-replicating, heterochromatic domains is only 25% lower than in early-replicating euchromatic loci. Thus, the activation of early- and late-firing origins must be regulated by quintessentially different mechanisms. The aggregate data can be unified into a model in which initiation site selection is driven almost entirely by epigenetic factors that fashion both the long-range and local chromatin environments, with underlying DNA sequence and local transcriptional activity playing only minor roles. Importantly, the comprehensive origin map we have prepared for GM06990 overlaps moderately well with origin maps recently reported for the genomes of four different human cell lines based on the distributions of small nascent strands.
authors.affiliation: Department of Biochemistry & Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
authors.forename: Larry D
authors.initials: LD
authors.lastname: Mesner
authors.forename: Veena
authors.initials: V
authors.lastname: Valsakumar
authors.forename: Marcin
authors.initials: M
authors.lastname: Cieslik
authors.forename: Rebecca
authors.initials: R
authors.lastname: Pickin
authors.forename: Joyce L
authors.initials: JL
authors.lastname: Hamlin
authors.forename: Stefan
authors.initials: S
authors.lastname: Bekiranov
mesh_terms: D045744:Cell Line, Tumor; D002843:Chromatin / Q000235:genetics* / Q000378:metabolism*; D042522:DNA Replication Timing; D003850:Deoxyribonuclease I / Q000378:metabolism; D057890:Epigenomics; D056426:Genetic Loci; D015894:Genome, Human; D006367:HeLa Cells; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D018741:Replication Origin / Q000235:genetics*; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D015533:Transcriptional Activation
chemical_list: D002843:Chromatin; D003850:Deoxyribonuclease I