Transcription-Replication conflicts are linked to histone H3K79 methylation and R-loop dependent nucleosome eviction [BrdUseq]

Transcription-replication conflicts (TRCs) can provoke genome instability. Progressing transcription and replication machineries profoundly impact their underlying chromatin template and thus, conflict sites are vulnerable to chromatin and epigenome alterations. Here, we engineered an inducible TRC reporter system using a genome-integrated R-loop-prone sequence. Using this system, we characterized the dynamic changes of the local chromatin structure inflicted by TRCs, leading to reduced nucleosome occupancy and replication fork blockage. Consequently, inducing a handful TRCs on the genome results in a measurable global replication stress response. We also find a TRC-specific increase in H3K79 methylation at the R-loop forming TRC-site. Accordingly, inhibition of the H3K79 methyltransferase DOT1L leads to increased cellular TRCs and exacerbated DNA damage response, suggesting that deposition of this mark is required for effective detection and resolution of TRCs. Thus, our work highlights the molecular dynamics and reveals specific epigenetic modifiers bookmarking TRC sites, relevant to cancer and other diseases. Overall design: BrdU Immunprecipitation sequencing (BrdU-seq) time course experiment for analysis of nascent DNA replication in synchronized U-2OS Clone#12 cells. G1 cells as well as S-phase released cells for 2h, 4h, 6h, and 8h were analyzed. BrdU was added to the cell culture medium 30 min before harvesting. Cells were treated with 0 or 1 µg/mL Doxycycline. Immunprecipitations were conducted in biological dublicate.

转录-复制冲突（Transcription-replication conflicts, TRCs）可引发基因组不稳定性。进行中的转录与复制机器会对其下游的染色质模板产生显著影响，因此冲突位点极易发生染色质与表观基因组改变。本研究通过基因组整合型易形成R环的序列，构建了可诱导的TRC报告系统。利用该系统，我们解析了TRC介导的局部染色质结构动态变化，该变化可导致核小体占据率降低与复制叉阻滞。进一步研究发现，在基因组中诱导少量TRC即可触发可检测到的全局复制应激反应。我们还观察到，在形成R环的TRC位点上，H3K79甲基化水平呈现TRC特异性升高。相应地，抑制H3K79甲基转移酶DOT1L会使细胞内TRC水平升高，并加剧DNA损伤应答，提示该修饰的沉积对于有效检测和清除TRC是必需的。综上，本研究阐明了TRC的分子动态机制，并鉴定了标记TRC位点的特异性表观遗传调控因子，该发现与癌症及其他疾病密切相关。

实验设计：采用溴脱氧尿苷免疫沉淀测序（BrdU Immunprecipitation sequencing, BrdU-seq）时间序列实验，用于分析同步化U-2OS Clone#12细胞中的新生DNA复制情况。分别对G1期细胞，以及S期释放后2h、4h、6h、8h的细胞进行检测。在收获细胞前30min，向细胞培养基中添加BrdU。细胞分别经0或1 µg/mL多西环素（Doxycycline）处理。免疫沉淀实验设置生物学重复两次。