Biology of terminal R-loops in splicing factor mutant cancers

这份重新提交的R03申请试图确定在剪接因子(SF)中重复突变是如何常见的 导致染色质可及性和表观遗传景观的变化，从而促进肿瘤发生。 SF的复发突变在包括急性髓系白血病在内的多种无关癌症类型中普遍存在 白血病、慢性淋巴细胞白血病、肺癌和黑色素瘤。在成百上千的剪接因素中， 只有4个常见的突变：SF3B1、SRSF2、U2AF1和ZRSR2。SF突变是相互排斥的 非同义词，提示它们作为具有新的功能的肿瘤驱动因素。他们已经确立的角色 在剪接催化中，导致假设肿瘤抑制基因或癌基因的交替剪接是 突变细胞克隆进化的主要机制。而备用剪接事件是 在rna-seq数据集中可以看出，这种变化相当温和，仅限于特定的突变。 子类型。这使得选择性剪接在肿瘤发生中的直接作用受到质疑。最近的研究表明 研究了肿瘤发生的其他分子机制，包括响应于 突变的sf表达。R-环是两个DNA和一个RNA分子在 抄写。R-环是染色质状态的关键调节器，如果不加以抑制，可能会导致基因组 不稳定。这些R环也可引起S期停滞，并可通过RNAseH的过度表达而挽救。 新的证据表明剪接体机制和信使rna之间的密切协调。 加工，包括其3‘端的切割和终止。因此，我们假设终端R-环出现 来自有缺陷的信使核糖核酸。在这项建议的第一个目标中，我们将确定转录的动力学 在SF3B1和U2AF1突变中，通过新生RNA的代谢标记(瞬时转录测序 使用Timelapse化学或TT-TL-Seq)。RNA聚合酶II与R-环和失速相关的定位 DNA复制分叉将通过邻近连接试验确定。第二个目标将探索如何 表观遗传修饰物调节这种病理性R-环的形成和分解。我们的初步结果 使用短发夹状RNA文库揭示了组蛋白脱乙酰酶途径在这一调控中的作用；因此我们 在这一目标中，我将探讨HDAC和R环之间的关系。最后，我们将确定染色质 剪接因子突变型急性髓系白血病患者样本的可及性，因为R-环和开放 染色质是高度相关的。鉴于R03机制的范围有限，该建议主要是 侧重于将异常RNA加工与R环形成联系起来的生化研究。如果成功，它将 形成对SF致癌机制的全面研究的基础 利用适当的体内模型进行突变。

This resubmitted R03 application seeks to determine how recurrent mutations common in splicing factors (SF) lead to changes in chromatin accessibility and epigenetic landscape, thereby contributing to oncogenesis. Recurrent mutations in SF are prevalent across multiple unrelated cancer types including acute myeloid leukemia, chronic lymphocytic leukemia, lung cancer and melanoma. Among the hundreds of splicing factors, only 4 are commonly mutated: SF3B1, SRSF2, U2AF1 and ZRSR2. SF mutations are mutually exclusive and non-synonymous, suggesting their role as tumor drivers with neomorphic function. Their well-established roles in splicing catalysis have led to the presumption that alternate splicing of tumor suppressors or oncogenes is the primary mechanism driving clonal evolution of mutant cells. While alternate splicing events are demonstrable in RNA-seq datasets, such changes are quite modest and restricted to specific mutational subtypes. This makes a direct role of alternative splicing in oncogenesis questionable. Recent studies have looked at alternate molecular mechanisms of oncogenesis including excess R-loops that arise in response to mutant SF expression. R-loops are 3 stranded structures of two DNA and one RNA molecules formed during transcription. R-loops are critical regulators of chromatin states, and when unchecked can lead to genome instability. These R-loops also cause S-phase arrest and can be rescued by the over-expression of RNAseH. Emerging evidence points to the close coordination between spliceosomal machinery and messenger RNA processing, including its 3' end cleavage and termination. We hence hypothesize that terminal R-loops arise from defective mRNA processing. In the first aim of this proposal, we will determine dynamics of transcription in SF3B1 and U2AF1 mutations through metabolic labeling of nascent RNA (transient transcript sequencing with Timelapse chemistry or TT-TL-seq). Location of RNA Polymerase II in relation to R-loops and stalled DNA replication forks will be determined through proximity ligation assay. The second aim will explore how epigenetic modifiers regulate the formation and resolution of such pathologic R-loops. Our preliminary results using a short hairpin RNA library reveals the role of histone deacetylase pathway in this regulation; hence we will explore the relation between HDAC and R-loops in this aim. Finally, we will determine chromatin accessibility in splicing factor mutant acute myeloid leukemia patient samples, since R-loops and open chromatin are highly correlated. Given the limited scope of the R03 mechanism, the proposal is primarily focused on biochemical studies linking aberrant RNA processing and R-loop formation. If successful, it will form the basis of comprehensive investigations into the mechanisms of oncogenesis conferred by SF mutations utilizing appropriate in vivo models.