Our goal is to understand how mammalian chromosomes are organized within the nucleus to carry out their functions. DNA replication provides an excellent forum in which to study chromosome structure and function. Structural and functional units of chromosomes replicate coordinately, often through the synchronous firing of clusters of replication origins that encompass domains of approximately 0.5 Mb. Each of these replication domains is programmed to replicate at a specific time during S-phase. In general, transcriptionally active (euchromatin) domains replicate early in S-phase, and transcriptionally silent (heterochromatin) domains replicate late. Programmed changes in replication timing accompany key stages of animal development and are often coupled to changes in gene expression. Our working hypothesis is that structural, functional, and replication domains share topographical boundaries and represent basic units of chromosome organization. We would like to understand what regulates where and when replication begins, how developmental cues communicate with the cell-cycle machinery to elicit changes in the program for replication and how that program is disrupted in cancer. Read more
The temporal order of DNA replication (replication timing [RT]) is highly coupled with genome architecture, but cis-elements regulating either remain elusive. We created a series of CRISPR-mediated deletions and inversions of a pluripotency-associated topologically associating domain (TAD) in mouse ESCs. CTCF-associated domain boundaries were dispensable for RT. CTCF protein depletion weakened most TAD boundaries but had no effect on RT or A/B compartmentalization genome-wide. By contrast, deletion of three intra-TAD CTCF-independent 3D contact sites caused a domain-wide early-to-late RT shift, an A-to-B compartment switch, weakening of TAD architecture, and loss of transcription. The dispensability of TAD boundaries and the necessity of these “early replication control elements” (ERCEs) was validated by deletions and inversions at additional domains. Our results demonstrate that discrete cis-regulatory elements orchestrate domain-wide RT, A/B compartmentalization, TAD architecture, and transcription, revealing fundamental principles linking genome structure and function.