SUMMARY Replication timing profiles are cell type-specific and reflect genome organization changes upon differentiation. In this protocol we describe how to analyze replication timing genome-wide in mammalian cells. Asynchronously cycling cells are pulse labeled with the nucleotide analog 5-bromo-2-deoxyuridine (BrdU) and sorted into S-phase fractions based on DNA content using flow cytometry. BrdU-labeled DNA from each fraction is immunoprecipitated, amplified, differentially labeled, and co-hybridized to a whole-genome CGH microarray, which is currently more cost effective than high-throughput sequencing and equally capable of resolving features at the biologically relevant level of tens to hundreds of kilobases. We also present a guide to analyzing the resulting datasets, based on methods we use routinely. Subjects include normalization, scaling, and data quality measures, loess (local polynomial) smoothing of replication timing values, segmentation of data into domains, and assignment of timing values to gene promoters. Finally, we cover clustering methods and means to relate changes in the replication program to gene expression and other genetic and epigenetic datasets. Some experience with R or similar programming languages is assumed. Altogether, the protocol takes approximately 3 weeks to complete.

Abnormal replication timing has been observed in cancer but no study has comprehensively evaluated this misregulation. We generated genome-wide replication-timing profiles for pediatric leukemias from 17 patients and three cell lines, as well as normal B and T cells. Nonleukemic EBV-transformed lymphoblastoid cell lines displayed highly stable replicationtiming profiles that were more similar to normal T cells than to leukemias. Leukemias were more similar to each other than to B and T cells but were considerably more heterogeneous than nonleukemic controls. Some differences were patient specific, while others were found in all leukemic samples, potentially representing early epigenetic events. Differences encompassed large segments of chromosomes and included genes implicated in other types of cancer. Remarkably, differences that distinguished leukemias aligned in register to the boundaries of developmentally regulated replicationtiming domains that distinguish normal cell types. Most changes did not coincide with copy-number variation or translocations. However, many of the changes that were associated with translocations in some leukemias were also shared between all leukemic samples independent of the genetic lesion, suggesting that they precede and possibly predispose chromosomes to the translocation. Altogether, our results identify sites of abnormal developmental control of DNA replication in cancer that reveal the significance of replication-timing boundaries to chromosome structure and function and support the replication domain model of replication-timing regulation. They also open new avenues of investigation into the chromosomal basis of cancer and provide a potential novel source of epigenetic cancer biomarkers.

Many types of epigenetic profiling have been used to classify stem cells, stages of cellular differentiation, and cancer subtypes. Existing methods focus on local chromatin features such as DNA methylation and histone modifications that require extensive analysis for genome-wide coverage. Replication timing has emerged as a highly stable cell type-specific epigenetic feature that is regulated at the megabase-level and is easily and comprehensively analyzed genome-wide. Here, we describe a cell classification method using 67 individual replication profiles from 34 mouse and human cell lines and stem cell-derived tissues, including new data for mesendoderm, definitive endoderm, mesoderm and smooth muscle. Using a Monte-Carlo approach for selecting features of replication profiles conserved in each cell type, we identify “replication timing fingerprints” unique to each cell type and apply a k nearest neighbor approach to predict known and unknown cell types. Our method correctly classifies 67/67 independent replication-timing profiles, including those derived from closely related intermediate stages. We also apply this method to derive fingerprints for pluripotency in human and mouse cells. Interestingly, the mouse pluripotency fingerprint overlaps almost completely with previously identified genomic segments that switch from early to late replication as pluripotency is lost. Thereafter, replication timing and transcription within these regions become difficult to reprogram back to pluripotency, suggesting these regions highlight an epigenetic barrier to reprogramming. In addition, the major histone cluster Hist1 consistently becomes later replicating in committed cell types, and several histone H1 genes in this cluster are downregulated during differentiation, suggesting a possible instrument for the chromatin compaction observed during differentiation. Finally, we demonstrate that unknown samples can be classified independently using site-specific PCR against fingerprint regions. In sum, replication fingerprints provide a comprehensive means for cell characterization and are a promising tool for identifying regions with cell type-specific organization.

BackgroundCellular differentiation and reprogramming are accompanied by changes in replication timing and 3D organization of large-scale (400 to 800 Kb) chromosomal domains (‘replication domains’), but few gene products have been identified whose disruption affects these properties.ResultsHere we show that deletion of esBAF chromatin-remodeling complex components BAF250a and Brg1, but not BAF53a, disrupts replication timing at specific replication domains. Also, BAF250a-deficient fibroblasts reprogrammed to a pluripotency-like state failed to reprogram replication timing in many of these same domains. About half of the replication domains affected by Brg1 loss were also affected by BAF250a loss, but a much larger set of domains was affected by BAF250a loss. esBAF binding in the affected replication domains was dependent upon BAF250a but, most affected domains did not contain genes whose transcription was affected by loss of esBAF.ConclusionsLoss of specific esBAF complex subunits alters replication timing of select replication domains in pluripotent cells.

Facioscapulohumeral muscular dystrophy (FSHD) is linked to contraction of an array of tandem 3.3-kb repeats (D4Z4) at 4q35.2 from 11-100 copies to 1-10 copies. The extent to which D4Z4 contraction at 4q35.2 affects overall 4q35.2 chromatin organization remains unclear. Because DNA replication timing is highly predictive of long-range chromatin interactions, we generated genome-wide replication-timing profiles for FSHD and control myogenic precursor cells. We compared non-immortalized myoblasts from four FSHD patients and three control individuals to each other and to a variety of other human cell types. This study also represents the first genome-wide comparison of replication timing profiles in non-immortalized human cell cultures. Myoblasts from both control and FSHD individuals all shared a myoblast-specific replication profile. In contrast, male and female individuals were readily distinguished by monoallelic differences in replication timing at DXZ4 and other regions across the X chromosome affected by X inactivation. We conclude that replication timing is a robust cell-type specific feature that is unaffected by FSHD-related D4Z4 contraction.