Project description:E/L Repli-seq is a powerful tool for detecting cell type-specific replication landscapes in mammalian cells, but its potential to monitor DNA replication under replication stress awaits better understanding. Here, we used E/L Repli-seq to examine the temporal order of DNA replication in human retinal pigment epithelium cells treated with the topoisomerase I inhibitor camptothecin. We found that the replication profiles by E/L Repli-seq exhibits characteristic patterns after replication-stress induction, including the loss of specific initiation zones within individual early replicating timing domains. We also observed global disappearance of the replication timing domain structures in the profiles, which can be explained by checkpoint-dependent suppression of replication initiation. Thus, our results demonstrate the effectiveness of E/L Repli-seq at identifying cells with replication-stress-induced altered DNA replication programs.
Project description:Replication timing is cell type specific, is tightly linked to the 3D nuclear organisation of the genome and is considered an epigenetic fingerprint. In spite of its importance in maintaining the epigenome the developmental regulation of replication timing in mammals in vivo has not been explored. Here, using single cell Repli-seq, we generated the genome-wide replication timing maps of mouse embryos from the zygote until the blastocyst stage.
Project description:The replication timing program, or the order in which DNA is duplicated during S-phase, is associated with various features of chromosome structure and function, including gene expression, histone modifications, and 3-D compartmentalization of chromatin.
Project description:The replication timing program, or the order in which DNA is duplicated during S-phase, is associated with various features of chromosome structure and function, including gene expression, histone modifications, and 3-D compartmentalization of chromatin. 3 cell types, with a total of 6 individual replicates
Project description:Cycling cells duplicate their DNA content during S phase, following a defined program called replication timing (RT). Early and late replicating regions differ in terms of mutation rates, transcriptional activity, chromatin marks and sub-nuclear position. Moreover, RT is regulated during development and is altered in disease . Exploring mechanisms linking RT to other cellular processes in normal and diseased cells will be facilitated by rapid and robust methods with which to measure RT genome wide. Here, we describe a rapid, robust and relatively inexpensive protocol to analyse genome-wide RT by next-generation sequencing (NGS). This protocol yields highly reproducible results across laboratories and platforms. We also provide computational pipelines for analysis, parsing phased genomes using single nucleotide polymorphisms (SNP) for analyzing imprinted RT, and for direct comparison to Repli-chip data obtained by analyzing nascent DNA by microarrays.
Project description:We replaced the endogenous histones of Drosophila melanogaster with either histones containing an H3K9R mutation or histones containing an H4K16R mutation to interrogate established genome-wide correlations between chromatin state, transcription, and DNA replication timing. We performed total RNA-seq in H4K16R males and females to investigate the role of H4K16 in dosage compensation of the male X chromosome. We found that H4K16 directly promotes hyper-expression of the male X chromosome in Drosophila. To generate replication timing profiles, we performed Repli-seq in HWT males and females, H4K16R males and females, and H3K9R females. We found that H3K9 promotes late replication of the pericentromeric heterochromatin and H4K16 promotes early replication of the male X chromosome.
