Project 2: Replication Domain Organization During hESC Differentiation Dalton, StephenA.
Specific Aims. Many cytological, genetic and biochemical definitions of higher-order chromosomal domains have been putforth, but none provides a property that delineates boundaries of domains with precision and in a manner thatcan be applied genome-wide. Hence, while the word 'domain' is frequently used to describe properties oflarge, often multi-genie, units of chromosomes, there is no comprehensive definition of a chromosome domain.We demonstrate that the 'replication domain' is a definable chromosomal unit, revealing replication aspotentially the only chromosomal property that affords a comprehensive segmentation of the entire genome atthe megabase level. Importantly, we have discovered that the boundaries of these domains are dramaticallyre-organized during mESC and hESC differentiation to create larger temporally consolidated domains in whichreplication timing correlates more closely with sequence properties of chromosomes. Hence, replicationstructure of chromosomes in ESCs is relatively free from constraints imposed by DNA sequences, defining anovel property of pluripotent cells. We propose to further investigate the biological significance of domainconsolidation, its relationship to the 3D organization of chromatin in the nucleus and the mechanisms by whichconsolidation occurs using hESCs as a model system.
The specific aims are as follows:
Aim 1 : Choreography of Replication Domain Boundaries During Lineage Commitment.In this aim, we will test the hypothesis that replication domain boundaries are characteristic of particular celltypes. More specifically, we propose that smaller temporally distinct replication domains is a characteristic ofstem cells, and that differentiation will be accompanied by a progressively more rigid relationship betweenisochore sequence composition and replication timing. To determine whether boundaries are lineage specific,we will examine changes in replication domain boundaries during differentiation of hESCs to independent germlayers, ectoderm and mesendoderm. To distinguish whether changes in replication domain boundaries occur ina single-step, for example during loss of pluripotence, or whether there is a continuum of re-organization ascell lineage choices become more restricted, we will examine the downstream lineages definitive endodermand mesoderm, as well as mesoderm differentiated to smooth muscle. Finally, to understand the functionalsignificance of domain consolidation, we will determine which genes are affected by consolidation and howthey relate to cell lineage choices.
Aim 2 : Replication Profiling as a Novel Means to Characterize hESCs.Replication profiling provides a convenient comprehensive genome-wide identification method that may shedlight on important relationships between cell types and pluripotent cellular states that are currently beingdebated in the literature. In this Aim, we will identify the domains that distinguish hESCs from theirdifferentiated counterparts. We will examine regions of conserved synteny between mouse and human forevolutionary conservation of replication domain structure. Finally, we will use replication profiling to address thedebate as to whether hESCs are more similar to mESCs or mouse epiblast-like cells.
Aim 3 : Spatial consolidation of chromatin during differentiation.Here we will determine how the consolidation of replication domains defined molecularly in Aim1 correspondsto the three-dimensional re-organization of these domains in the cell nucleus. Using in situ hybridization atdifferent stages of differentiation, we will trace the sub-nuclear localization of replication domains relative tospecific sub-nuclear compartments to ask whether domains that consolidate temporally also consolidatespatially and whether they acquire a more uniform overall level of compaction.
Aim 4 : Coordination of replication forks during domain consolidation. In this Aim, we will analyze thepolarity of replication forks, replicon sizes and regions of replication origin activity on individual stretched DNAfibers from the consolidating domains to identify replicon clusters and the locations of their boundaries relativeto the boundaries of replication domains defined molecularly. These experiments will begin to define themolecular mechanisms by which domain consolidation occurs.
Showing the most recent 10 out of 118 publications
