Mechanisms of gene expression regulation in pancreatic cancer

项目总结 胰腺导管腺癌(PDAC)预计将成为世界上第二大致死性癌症 接下来的10年。由于NO的存在，迫切需要研究PDAC转化的新机制 目前存在有效的PDAC治疗方法。转化过程中的转录调控是多方面的 基因突变和表观遗传改变发挥着至关重要的相互联系的作用。一个关键的未阐明的方面 转化背景下的基因调控是调节细胞周期变化的分子机制。 染色质重组。CHIP-SEQ和CHIP-PCR在致癌KRAS的诱导模型中的研究 PDAC中的启动因子，表明异染色质重新定位到核周。这些薄板 相关结构域(LAD)富含H3K9me2，位于核层。芯片序列和 Rna-seq检测到LAD结合的活性增强子区域的丢失和LAD的下调 分别是相关基因。为了研究调节LAD下游组装的机制 致癌的KRAS我们进行了几项分子和生化研究。我们进行了一次生物识别筛选 利用已知可与LAD相互作用的核膜核心成分Lamin A作为诱饵。拉明A生物ID 鉴定了一种新的肌球蛋白，肌球蛋白18a(MYO18a)，在致癌KRAS下富含在核层 发信号。免疫细胞化学显示MYO18a的核定位增加，并在 KRAS激活后的片层。生化分析证实了MYO18a-Lamin A的相互作用 MYO18a与染色质的相互作用。SiRNA敲除MYO18a挽救了相关基因的表达 致癌的KRAS介导的LAD。这些数据将我们引向我们的核心假设，即MYO18a作为一个 KRAS下游效应器调节核膜染色质定位以抑制致癌作用 基因表达。为了验证这一假设，我们将同时进行CHIP-SEQ和RNA-SEQ实验，以 在体外的PDAC模型中检测MYO18a相关的LADS。LAD组件随后将在 MYO18A的存在/不存在。我们还将开发MYO18a基因工程小鼠模型，以探索 MYO18a和核MYO18a在PDAC发生发展中的作用对此作进一步的研究 机制可以为转化过程中的基因调控提供有价值的见解，并将识别 潜在的可下药目标。

PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDAC) is predicted to become the second deadliest cancer within the next 10 years. Investigations into novel mechanisms of PDAC transformation are urgently needed as no effective PDAC treatment currently exists. Transcriptional regulation during transformation is multifaceted with genetic mutations and epigenetic alterations playing vital interconnected roles. A key unelucidated facet of gene regulation in the context of transformation is the molecular mechanism which mediates changes in chromatin reorganization. ChIP-seq and ChIP-PCR studies in an inducible model of oncogenic KRAS, a key initiating factor in PDAC, demonstrate a relocation of heterochromatin to the nuclear periphery. These lamina associated domains (LADs) are enriched in H3K9me2 and positioned at the nuclear lamina. ChIP-seq and RNA-seq identified loss of active enhancer regions incorporated into LADs and downregulation of LAD associated genes, respectively. To investigate the mechanism regulating the assembly of LADs downstream of oncogenic KRAS we performed several molecular and biochemical studies. We conducted a screening BioID utilizing Lamin A, a core component of the nuclear lamina known to interact with LADs, as bait. Lamin A BioID identified a novel myosin, Myosin 18a (MYO18a), enriched at the nuclear lamina under oncogenic KRAS signaling. Immunocytochemistry revealed increased nuclear localization of MYO18a and enrichment at the lamina upon KRAS activation. Biochemical analysis validated MYO18a-Lamin A interaction and confirmed MYO18a interaction with chromatin. siRNA knockdown for MYO18a rescued expression of genes associated with oncogenic KRAS-mediated LADs. These data lead us to our central hypothesis that MYO18a acts as a downstream effector of KRAS to modulate chromatin positioning at the nuclear lamina to silence oncogenic gene expression. To test this hypothesis, we will conduct ChIP-seq and RNA-seq experiments in parallel to detect MYO18a associated LADs in in vitro models of PDAC. LAD assembly will then be investigated in the presence/absence of MYO18a. We will also develop MYO18a genetic engineered mouse models to explore the role of MYO18a and nuclear MYO18a in PDAC development and progression. Further study into this mechanism can provide valuable insight into gene regulation in the transformation process and will identify potentially druggable targets.