Chromatin Dynamics during DNA Replication

Plant cells undergo two types of cell cycles – the mitotic cycle in which DNA replication is coupled to mitosis and the endocycle in which DNA replication occurs in the absence of cell division. We used pulse labeling with the thymidine analog EdU in combination with flow cytometry to examine the DNA replication programs of maize root tip cells as they transition from a mitotic cell cycle to an endocycle during development. Maize root tips were pulse-labeled with EdU, fixed and used for nuclei isolation. Replicating nuclei were fluorescently tagged by conjugating EdU-labeled DNA with Alexa-488. The nuclei were then sorted based on DNA content (DAPI) and EdU incorporation into G1 (2C), early, mid and late S, and G2/M (4C) fractions of the mitotic cycle and into early, mid and late S and fully replicated (8C) fractions of the endocycle. DNA from S phase fractions was immunopreciptated using an Alexa-488 antibody to isolate newly replicated DNA followed by high throughput sequencing to generate genome-wide replication timing profiles. The sorted nuclei were also used to generate full genome maps of selected histone modifications and DNA methylation patterns, while RNA-seq profiles were generated for the overlapping mitotic and endocycling zones of the maize root tip. These datasets are currently being used to examine the relationship(s) between DNA replication timing, transcription and chromatin features in mitotic and endocycling cells during maize root tip development. These studies are the first characterization of replication timing programs in a naturally developing, eukaryotic organ.