Consequences of chromatin replication on RNAPII activity and transcriptional outcome [HU]

In the nucleus of the eukaryote cell, the genetic material is organized in the form of chromatin where DNA associates with nucleosomes, formed by an octamer of histone proteins, to provide the correct environment to regulate gene expression, maintain cellular identity and avoid disease. Despite the important role of chromatin organization in cellular function, in each cell division chromatin is totally disorganized. Nucleosomes are evicted ahead of the replication fork and will have to be recycled together with newly synthetized histones to maintain nucleosome density in the two daughter strands. This produce changes in chromatin accessibility and a dilution in epigenetic information. To date, it is not clear how these chromatin and epigenetic changes can affect gene expression. Here, we have used the cutting-edge technology ChOR-seq (Chromatin Occupancy after Replication and sequencing) and ChrRNA-seq (Chromatin-associated RNA-seq) to investigate the distribution and abundance of the RNA Polymerase II (RNAPII) in newly replicated chromatin and nascent RNA levels in human cells. Analysis of S2P RNAPII distribution and abundance in newly replicated and mature chromatin by ChOR-seq in HeLa S3 cells. All experiments were done in duplicates. Replicated chromatin was labelled in early S and treated with hydroxyurea (HU) to slow down replication fork speed. Chromatin of Drosophila melanogaster S2 cells were used as spike-in.