Duplication of the genome in mammalian cells occurs in a defined temporal order referred to as its replication-timing (RT) program. RT changes dynamically during development, regulated in units of 400-800 kb referred to as replication domains (RDs). Changes in RT are generally coordinated with transcriptional competence and changes in subnuclear position. We generated genome-wide RT profiles for 26 distinct human cell types, including embryonic stem cell (hESC)-derived, primary cells and established cell lines representing intermediate stages of endoderm, mesoderm, ectoderm, and neural crest (NC) development. We identified clusters of RDs that replicate at unique times in each stage (RT signatures) and confirmed global consolidation of the genome into larger synchronously replicating segments during differentiation. Surprisingly, transcriptome data revealed that the well-accepted correlation between early replication and transcriptional activity was restricted to RT-constitutive genes, whereas two-thirds of the genes that switched RT during differentiation were strongly expressed when late replicating in one or more cell types. Closer inspection revealed that transcription of this class of genes was frequently restricted to the lineage in which the RT switch occurred, but was induced prior to a late-to-early RT switch and/or down-regulated after an early-to-late RT switch. Analysis of transcriptional regulatory networks showed that this class of genes contains strong regulators of genes that were only expressed when early replicating. These results provide intriguing new insight into the complex relationship between transcription and RT regulation during human development.

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.

In multicellular organisms, developmental changes to replication timing occur in 400-800 kb domains across half the genome. While examples of epigenetic control of replication timing have been described, a role for DNA sequence in mammalian replication-timing regulation has not been substantiated. To assess the role of DNA sequences in directing developmental changes to replication timing, we profiled replication timing in mice carrying a genetically rearranged Human Chromosome 21 (Hsa21). In two distinct mouse cell types, Hsa21 sequences maintained human-specific replication timing, except at points of Hsa21 rearrangement. Changes in replication timing at rearrangements extended up to 900 kb and consistently reconciled with the wild-type replication pattern at developmental boundaries of replication-timing domains. Our results are consistent with DNA sequence-driven regulation of Hsa21 replication timing during development and provide evidence that mammalian chromosomes consist of multiple independent units of replication-timing regulation.

4609 Abnormal temporal control of replication (“replication timing”) is a universal hallmark of cancer, but the significance of this relationship is not understood. During normal development, changes in replication timing are regulated in defined chromosomal units (“replication domains”) of 400–800 kb, with late replication generally associated with stable gene silencing. We have performed genome-wide replication timing mapping on pediatric acute leukemias and identified patterns of replication-timing mis-regulation (“leukemia fingerprints”) in common across multiple types of pediatric leukemia (“multi-lineage fingerprints”), as well as those linked to specific subtypes of acute leukemia and patient-specific fingerprints. Most of the mis-regulated regions are not associated with copy number variation or translocations and correspond to many of the same domains whose replication timing is regulated during normal development. The existence of multi-lineage fingerprints suggests an early origin of their mis-regulation while type-specific and patient-specific fingerprints suggest that mis-regulation continues to occur downstream of the initiating event. Our hypothesis is that there exists an hierarchy of replication-timing mis-regulation, initiating with frozen vestiges of normal hematopoiesis that are inappropriately maintained, followed by additional changes that evolve as the tumor differentiates. These changes may then affect the ability of downstream lineages to activate or silence genes. Since chromatin is assembled at the replication fork, and different types of chromatin are assembled at different times during S phase, changes in replication timing likely contribute to domain-wide changes in chromatin structure, similar to those observed in long range epigenetic silencing (LRES). In fact, we find that genes within leukemia fingerprint domains are significantly enriched for those transcribed during hematopoiesis and/or mis-regulated in cancers. Replication timing is an exceptionally stable epigenetic property that is not easily disturbed by loss or mis-expression of single master regulators of cell fate and is not directly related to transcription, but rather to the stability of gene silencing. Hence, events occurring early in hematopoiesis could have profound effects on the activation of transcriptional programs in downstream lineages. Elucidating the genesis and genealogy of leukemia-specific replication timing fingerprints and their relationship to transcriptional control will be fundamental to understanding their link to the development of acute leukemias. Disclosures: No relevant conflicts of interest to declare.