22. maj 2019

Transcription Re-Orders the Epigenome after DNA Replication

Published in Molecular Cell, a study from the Groth laboratory at the Biotech Research and Innovation Centre, University of Copenhagen, shows that transcription, the first step in the process by which genes are expressed, pauses during DNA replication, and that restart of transcription is important in organizing the proteins that package the genome. However, this transcription restart does not occur with the same speed throughout the genome; some regions are faster than others.

Instead of being naked within the nucleus, DNA is wrapped around histones, proteins that package and organize DNA into nucleosomes. These nucleosomes are the fundamental unit of chromatin. Separate proteins, specifically transcription factors and RNA polymerase, bind DNA and initiate transcription, but can only do this when DNA is not wrapped into a nucleosome. Promoters, DNA sequences just upstream of gene coding regions, and enhancers, separate DNA sequences that act to modulate expression of gene coding regions, are normally accessible to the transcription machinery. These are therefore called “accessible” regions of chromatin. When these regions are inaccessible, transcription is shut down.

It is known that DNA replication entails eviction of all proteins, including the transcription machinery and histone proteins, from DNA. To understand how the act of DNA replication impacts transcription, members of the Groth lab developed a method called repli-ATAC-seq, for replication-coupled Assay for Transposase-Accessible Chromatin followed by next-generation sequencing. This method can measure accessibility just after DNA replication throughout the genome, and can also be used to track how accessibility changes as chromatin is re-assembled following replication fork passage.

DNA replication shuts down transcription

Using repli-ATAC-seq, the researchers showed that immediately following DNA replication, chromatin was inaccessible, meaning it had little to no transcriptional activity. They additionally used another recently developed method in the lab, Chromatin Occupancy after Replication (ChOR-seq), to show that RNA polymerase, the central protein in transcription, was not bound to newly replicated DNA. Based on these results, the researchers concluded that immediately following DNA replication, chromatin is inaccessible and transcriptionally silenced.

Transcription resumes and re-orders chromatin after DNA replication

The researchers next investigated how transcription resumes on DNA after it has been replicated. By both repli-ATAC-seq and ChOR-seq, they saw transcription restarting within 30 minutes of the replication fork passing at some loci, but genome-wide pre-replicative accessibility was not observed until 2 hours after replication. Different regions restored with different rates, and super enhancers, or highly active enhancers, restored especially fast. To further explore the relationship between the return of accessibility and transcription restarting, researchers used drugs to block transcription and performed repli-ATAC-seq. They saw that accessibility is not restored when transcription is blocked, indicating that it is transcription restart that re-orders nucleosomes to establish accessibility after DNA replication.