Sequenciando o DNA recém-replicado revela a ampla plasticidade no tempo de replicação humana

Sequencing newly replicated DNA reveals widespread plasticity in human replication timing

R. Scott Hansen a,1, Sean Thomas b, Richard Sandstrom b, Theresa K. Canfield b, Robert E. Thurman b, Molly Weaver b, Michael O. Dorschner b, Stanley M. Gartler a,b and John A. Stamatoyannopoulos b,1

- Author Affiliations

aDepartment of Medicine, Division of Medical Genetics, and

bDepartment of Genome Sciences, University of Washington School of Medicine, Seattle, WA 98195

Contributed by Stanley M. Gartler

Abstract

Faithful transmission of genetic material to daughter cells involves a characteristic temporal order of DNA replication, which may play a significant role in the inheritance of epigenetic states. We developed a genome-scale approach—Repli Seq—to map temporally ordered replicating DNA using massively parallel sequencing and applied it to study regional variation in human DNA replication time across multiple human cell types. The method requires as few as 8,000 cytometry-fractionated cells for a single analysis, and provides high-resolution DNA replication patterns with respect to both cell-cycle time and genomic position. We find that different cell types exhibit characteristic replication signatures that reveal striking plasticity in regional replication time patterns covering at least 50% of the human genome. We also identified autosomal regions with marked biphasic replication timing that include known regions of monoallelic expression as well as many previously uncharacterized domains. Comparison with high-resolution genome-wide profiles of DNaseI sensitivity revealed that DNA replication typically initiates within foci of accessible chromatin comprising clustered DNaseI hypersensitive sites, and that replication time is better correlated with chromatin accessibility than with gene expression. The data collectively provide a unique, genome-wide picture of the epigenetic compartmentalization of the human genome and suggest that cell-lineage specification involves extensive reprogramming of replication timing patterns.

chromatin structure gene expression tissue specificity
Footnotes

1To whom correspondence may be addressed. E-mail: supreme@u.washington.edu or jstam@u.washington.edu.

Author contributions: R.S.H., T.K.C., M.O.D., S.M.G., and J.A.S. designed research; R.S.H., T.K.C., and M.W. performed research; R.S.H., S.T., R.S., R.E.T., and J.A.S. contributed new reagents/analytic tools; R.S.H., S.T., R.S., R.E.T., S.M.G., and J.A.S. analyzed data; and R.S.H., S.M.G., and J.A.S. wrote the paper.

The authors declare no conflict of interest.

Data deposition: The RNA levels determined by Affymetrix Exon array analysis are available from the NCBI GEO database under accession numbers GSM472898, GSM472903, GSM472910, GSM472944, and GSM472945. The digital DNaseI chromatin accessibility data are available as released from the ENCODE Project through the UCSC Genome Browser at http://genome.ucsc.edu (subId = 295 and subId = 106, narrow peak data). Newly replicated DNA sequencing data are deposited to the NCBI Short Read Archive under the Study accession number SRP001393.1.

This article contains supporting information online at www.pnas.org/cgi/content/full/0912402107/DCSupplemental.

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