With all phenomena in nature there are two major questions scientists ask: (1) what is the underlying mechanism (how?) and (2) what is its biological significance (why?). All eukaryotic cells replicate their DNA in a specific temporal sequence but both the mechanism of this "replication timing" program and its biological significance remain a mystery. Replication timing is clearly related to the 3-dimensional organization of chromosomes such that early and late replicating DNA are spatially segregated from each other in the nucleus and the timing program is developmentally regulated at the level of megabase-sized units of chromosome. The primary motivation for our research is that these units that we call "replication domains" provide us with a molecular handle into the higher order structural and functional organization of chromosomes. Since this is such a complex problem, we must ask elementary questions, the answers to which help guide us at major crossroads of inquiry. The work in our laboratory can be divided into three major areas:
1. Developmental Regulation of DNA Replication
(click here for a non-technical summary of this topic)
All cells contain the same genetic information (DNA) but package it with proteins into "chromatin" in characteristic ways that define each cell type. Chromatin is dismantled and re-assembled during each round of DNA replication, and we have discovered that the temporal order in which segments of DNA are replicated changes as stem cells turn into different cell types. Understanding how to manipulate this packaging process may help us engineer different cell types, a central goal in stem cell therapy.
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Replication proceeds via the synchronous firing of clusters of replication origins encompassing domains of several hundred kilobases. These "replication domains" coincide with structural and functional domains of chromosomes and replicate in a defined temporal order during S-phase. Since chromatin is assembled at the replication fork, and since different types of chromatin are assembled at different times during S-phase, it makes sense that replication would be an important regulatory event at which to assemble different types of chromatin in different cell types, but testing this hypothesis has been difficult. Many studies have correlated changes in replication timing to changes in gene expression in different cell lineages and in cancer but none have been able to address the intermediate states that accompany these changes. Mechanistic studies require a system in which these changes can be elicited with sufficient synchrony and homogeneity as to permit biochemical and molecular analyses. Using differentiation systems modeling both mouse and human early development, we detect dynamic changes in replication timing that affect at least half of the genome, some occurring within a single cell cycle. Early to late replication changes that coincide with loss of pluripotency and irreversible down-regulation of embryonic stem cell-specific genes occur in epiblast-like cell types, while late to early changes coincide with germ layer commitment and up-regulation of lineage-specific genes. Since these dynamic replication changes, unlike other functional properties of chromosomes, are regulated at the level of large chromosomal domains, our studies have the potential to open a novel chapter in gene regulation. Our working hypothesis is that changes in replication timing during differentiation reinforce the heritability of changes in chromatin structure across large chromosome domains that in turn modulate the responsiveness of genes during stem cell commitment. Together, this body of work has provided a watershed of information that has upheld or refuted longstanding hypotheses about replication timing and generated many new hypotheses that are now testable with the systems that we have developed. We are taking both genomics approaches to evaluate the significance of replication timing to cell fate changes during differentiation, and more targeted approaches to elucidate the mechanisms relating replication timing and gene expression changes at specific gene loci.
2. Regulation of Replication Timing During the Cell Cycle
(click here for a non-technical summary of this topic)
Studies of DNA replication in mammalian cells suffer from a lack of systems with which to approach the problem at the molecular level. Cis-acting sequences that function as replication origins in mammalian cells have not been identified and the mechanisms that regulate where and when origins will fire during S-phase remain a mystery. Over the last 15 years we have taken a variety of approaches to this problem, revealing several discrete steps during early G1-phase that establish a spatial and temporal program for replication:
Pre-Replication Complex (pre-RC) Assembly: First is the assembly of pre-replication complexes (pre-RCs). Knowledge of this process has been greatly aided by the identification of several proteins that bind to replication origins in yeast and the identification of homologues to these proteins in higher eukaryotes. We have shown that the mammalian homologues to these proteins bind to chromatin very tightly during telophase and render newly assembled nuclei fully competent to replicate in cell-free extracts lacking these proteins.
The Replication Timing Decision Point (TDP): Shortly after the assembly of pre-RCs, a replication timing program is established that determines the order in which chromosomal domains will be replicated (Timing Decision Point; TDP). Early cytogenetic studies by our group and others demonstrated that DNA synthesis takes place in discrete punctate foci within the cell nucleus and that foci replicating at different times during S phase are located in distinct compartments of the nucleus. We have demonstrated that the replication-timing program is established coincident with re-positioning and anchorage of these foci within the nucleus shortly after nuclear re-assembly, a time we called the Timing Decision Point (TDP). The TDP precedes the ODP and several manipulations could uncouple proper replication timing from any particular pattern of replication origin usage. We later demonstrated that determinants for replication timing are lost during S phase, despite maintenance of the 3D organization of chromatin.
Taken together, our findings have led us to propose the “Replication Domain Model”. Our early work showed that replication foci (discussed above) are stable units of chromosome structure consisting of multiple coordinately activated replicons that retain the punctate replication pulse label for many cell generations. We showed that replication foci serve as sites of replication protein assembly and disassembly in a temporal sequence that can be uncoupled from DNA synthesis itself. We found that foci in different compartments have different chromatin composition and proposed that, since chromatin is assembled at the replication fork, organization of the genome into coordinately replicated domains could facilitate rapid domain-wide chromatin changes. Using genomics, we discovered that replication timing consistently changes in units of 400-800 kb, defining molecular coordinates for replication domains. We showed that domain boundaries are functionally relevant in that they can confine the effects of rearrangements. We demonstrated that replication domains correspond to topologically associating domains (TADs) measured by chromatin conformation capture (Hi-C) and their higher order folding corresponds to replication timing such that domains that contact each other replicate at similar times. All of this 3D organization is re-established coincident with the TDP (above), when replication timing is established. In summary, our work shows that the way the genome is organized in 3D space has important functional consequences and provides a unifying model for genome organization that some have generously likened to the discovery of the nucleosome. We propose that anchorage of chromatin could create scaffolds that seed the assembly of sub-nuclear compartments of different molecular composition, a model that has become popular to explain many structure-function relationships in the nucleus. We have also identified a candidate protein (Rif1) that has properties consistent with a cell cycle regulated factor important for both replication timing regulation and 3D organization of chromatin.3. Origin and significance of altered replication timing in pediatric leukemia
B-lineage acute lymphocytic leukemia (B-ALL) is the most common childhood malignancy, yet we still do not understand the molecular mechanisms that lead to the genesis of this disease and there are no known strategies for its prevention. We recently demonstrated that pediatric B-ALL cells deviate from non-leukemic human B cells in the temporal order in which segments of their chromosomal DNA are duplicated (replication timing; RT). We also identified specific features in these alterations in DNA RT that are linked to particular sub-types of B-ALL defined by their hallmark mutations. We are now engaged in three areas of research to follow up these exciting new results: 1) In collaboration with the Children’s Oncology Group (COG), we are generating genome-wide RT profiles from a large cohort of banked pediatric B-ALL samples lacking prognostic markers and developing statistical linkages to outcome to potentially identify a whole new genre of biomarkers for pediatric leukemia; 2) We are profiling the changes in RT that accompany the process by which normal human B-cells are generated to determine which RT alterations in leukemia derive from the cell type of origin; 3) We are developing a novel model of leukemic transformation of normal human hematopoietic cells to assess when and in what cell types evidence of “aberrant RT” first appears during the process of leukemogenesis. RT has the potential to provide a new genre of biomarkers for diagnosis. In addition, RT reports on an undeveloped aspect of chromosome biology that is altered in B-ALL. Hence, studies of the cellular origin and mechanistic determinants of these RT alterations will provide novel insights into the underlying mechanisms of B-ALL.