Genome-Wide Mapping of Transcription-Replication Interactions Using Transcription-Replication Immunoprecipitation on Nascent DNA Followed by Next Generation Sequencing (TRIPn-Seq)

Purpose: Transcription and replication are both essential processes that require the processive translocation of large protein complexes through a complex chromatin environment. Transcription-replication interactions (TRIs) arise when transcription and replication occur simultaneously on the same tract of DNA and have been hypothesized to lead to detrimental effects on genome stability. Much of the work analyzing TRIs in mammals has been performed using artificial DNA constructs, chemical agents, and in immortalized cell lines that have abnormal cellular processes that allows for indefinite cellular division. Methods: To understand where TRIs naturally arise in primary B cells, we performed a sequential immunoprecipitation (IP) of RNA polymerase II phosphorylated at serine 5 followed by an IP of nascent DNA in a process we call Transcription-Replication IP on Nascent DNA coupled with high-throughput sequencing (TRIPn-Seq). Results: TRIPn-Seq defined the locations of 1009 independent TRI loci in rapidly proliferating mouse primary B cells. TRIs are associated with flanking RNA polymerase peaks mapping primarily to transcription start sites, where they often colocalize with R-loop structures and have a high propensity for G quadruplex formation. TRIs are likely difficult to replicate areas at the interface between early replicating regions and nascent transcription. TRIs are also enriched for DNA double-strand breaks that appear during S phase, stalled replication forks, and accumulate mutations in mouse tumors. Genes associated with TRIs are enriched for essential genes involved in early development and post-translational modifications, aging, and cancer. Conclusion: We propose that replication stress at TRIs induces mutations at transcription start sites that change expression levels which may contribute to age-related phenotypes and tumor formation in mice and humans. Overall design: TRIPn-Seq was performed on stimulated WT mouse primary B cells to locate transcription-replication interactions (TRIs) genome-wide. Experimental samples were treated with BrdU for 30 minutes to label nascent DNA. RNAP2 ChIP-Seq and DRIP-Seq were also performed to charaterize RNAP2pSer5 and R-loop profiles at TRIs.