Mapping of physiological DNA double stranded breaks in normal breast cells and breast cancer cells [BLISS]. Mapping of physiological DNA double stranded breaks in normal breast cells and breast cancer cells [BLISS]

DNA double-stranded breaks (DSBs) pose a significant threat to genomic integrity, and their generation during essential cellular processes like transcription remains poorly understood. In this study, we employed the advanced BLISS techniques to map DSBs, with different genetic manipulations to comprehensively investigate the interplay between transcription, DSBs, Topoisomerase 1 (TOP1), and R-loops. Our findings revealed the presence of DSBs at highly expressed genes enriched with TOP1 and R-loops, indicating their crucial involvement in transcription-associated genomic instability. Depletion of R-loops and TOP1 specifically reduced DSBs at highly expressed genes, uncovering their pivotal roles in transcriptional DSB formation. By elucidating the intricate interplay between TOP1cc trapping, R-loops, and DSBs, our study provides novel insights into the mechanisms underlying transcription-associated genomic instability. Moreover, we establish a link between transcriptional DSBs and early molecular changes driving cancer development. Notably, our study highlights the distinct etiology and molecular characteristics of driver mutations compared to passenger mutations, shedding light on the potential for targeted therapeutic strategies. Overall, these findings deepen our understanding of the regulatory mechanisms governing DSBs in hypertranscribed genes associated with carcinogenesis, opening avenues for future research and therapeutic interventions. Overall design: In-suspension break labeling in situ and sequencing (sBLISS) for MCF-7 cells, for control cells transfected with scramble siRNA and invected with empty vector (EV), cells after TOP1 knockdown (KD), cells after RNase H overexpression (OE), cells after both manipulations (TOP1 KD + RNase H OE). sBLISS was also performed on MCF-7 cells after E2 treatment, E2 treatment and RNase H OE, and control cells treated with vehicle and infected with EV. Additionally sBLISS was also performed on MCF-7 cells after E2 treatment, E2 treatment and TOP1 KD, and control cells treated with vehicle and transfected with scrambleRNA. sBLISS was also performed on synchronized MCF-7 cells in the G1 cell cycle stage. sBLISS was also performed on HMLE cells, and MCF-10A WT cells as well as MCF-10A RAS transformed cells. all BLISS experiments were replicated in duplicates, and in quadruplicates in HMLE cells.

DNA双链断裂（DNA double-stranded breaks, DSBs）对基因组完整性构成严重威胁，而其在转录等关键细胞过程中的产生机制至今仍不甚明确。本研究采用先进的BLISS技术对DSBs进行全基因组图谱绘制，并结合多种基因操作手段，全面探究转录、DSBs、拓扑异构酶1（Topoisomerase 1, TOP1）及R环之间的相互作用网络。
研究结果显示，在富集TOP1与R环的高表达基因区域存在大量DSBs，表明这些因素与转录相关的基因组不稳定性密切相关。敲低R环与TOP1可特异性降低高表达基因区域的DSBs水平，揭示了二者在转录相关DSB形成中的关键调控作用。通过阐明TOP1cc捕获、R环与DSBs之间的复杂相互作用，本研究为转录相关基因组不稳定性的潜在分子机制提供了全新见解。
此外，本研究建立了转录相关DSBs与驱动癌症发生的早期分子变化之间的潜在关联。值得注意的是，本研究对比了驱动突变与乘客突变的不同病因学特征与分子特性，为靶向治疗策略的开发提供了新思路。
总体而言，这些发现加深了我们对致癌相关高转录基因中DSBs调控机制的理解，为未来相关研究与治疗干预开辟了全新方向。

整体实验设计：对MCF-7细胞开展悬浮断裂标记原位测序（in-suspension break labeling in situ and sequencing, sBLISS）实验，涵盖以下组别：转染乱序小干扰RNA（scramble siRNA）并感染空载体（empty vector, EV）的对照细胞、TOP1敲低（knockdown, KD）细胞、核糖核酸酶H（RNase H）过表达（overexpression, OE）细胞，以及同时进行两种操作的细胞（TOP1 KD + RNase H OE）。
此外，还对经雌二醇（E2）处理的MCF-7细胞、经E2处理并过表达RNase H的MCF-7细胞，以及经溶剂对照处理并感染EV的对照细胞开展sBLISS实验。同时，对经E2处理的MCF-7细胞、经E2处理并敲低TOP1的MCF-7细胞，以及经溶剂对照处理并转染乱序RNA的对照细胞进行sBLISS检测。
另外，还对处于G1细胞周期时相的同步化MCF-7细胞开展sBLISS实验。此外，对HMLE细胞、MCF-10A野生型（wild type, WT）细胞以及MCF-10A RAS转化细胞进行sBLISS实验。
所有BLISS实验均设置两次生物学重复，其中HMLE细胞的实验设置四次生物学重复。