GSE195809
Identification of a small genomic segment required for the Plpp3 replication timing domain integrity
Early and late replication timing (RT) domains are Mb-sized structural units of mammalian chromosomes that can change RT in the context of development and disease. Identification of cis- and trans-acting factors involved in RT regulation is important for understanding higher-ordered chromosome structure and function in the nucleus. Here we examined the effect of CRISPR/Cas9-mediated genomic deletions within an early RT domain (Plpp3 domain) on mouse chromosome 4. We found that a small deletion (1.7-kb deletion) within the domain caused a domain-wide shift towards later replication. Single-cell DNA replication profiling revealed that the RT defect occurred uniformly across individual cells. Transcription of the Plpp3 gene that is affected by this deletion is unrelated to the integrity of the domain since other types of deletions that affect Plpp3 transcription have little or no effect on its early RT. The identified small segment is, however, not sufficient to form an early RT domain at an ectopic genomic location. Our results demonstrate that a small deletion could cause domain-wide alteration of RT and also underscore the complexity of the mechanism by which RT domains are regulated. Overall design: BrdU-IP Repli-seq and single-cell Repli-seq experiments in wild type and mutant mouse embryonic stem cells (CBMS1 mESCs).