The DAXX/ATRX/H3.3 axis in ERV regulation and tumor suppression

项目摘要 基因组测序已经发现了新的候选肿瘤抑制基因，包括DAXX和ATRX，它们是 在43%的胰腺神经内分泌肿瘤(PanNETs)中发生突变。尽管有这种强烈的疾病联系，我们的 对DAXX和ATRX的生理功能的了解仍然有限。生化与细胞 生物学研究表明，它们共同调节表观基因组，作为伴侣复合体 组蛋白变异体H3.3，使H3.3沉积在异染色质中。附加的非组蛋白伴侣功能有 这两种基因都有报道。我们最近为Daxx开发了一个条件性小鼠模型，并展示了 Daxx的丧失创造了一种允许的状态，与环境应激(炎症)和其他 基因损伤(MEN1缺失)影响转录图谱和影响细胞状态，最终损害 胰腺在体内的恢复和再生。转录水平的改变与转录因子的失调有关。 ERV，从而扩大了我们对DNA和组蛋白甲基化以外的体细胞调控的理解 体内，并暗示ERV失调的胰腺神经内分泌肿瘤的发生。重要的是，我们还 证实ERV附近的内源性基因在人PanNETs中也被观察到 Daxx突变。这项建议对这些研究进行了扩展，以更好地理解DAXX基因突变是如何 有助于肿瘤的发生。我们将首先使用我们健壮的生理小鼠模型来获得全面的 了解Daxx丢失下游ERV失调的表观遗传机制。我们 将对H3.3掺入、DNA甲基化和组蛋白甲基化进行综合分析。我们会 用基因实验来补充这些基因组研究，以直接询问Daxx：H3.3和 Daxx：使用新产生的小鼠等位基因在体内进行ATRX相互作用。最后，我们将把我们的研究扩展到一个 创新的人类多潜能干细胞模型，可分化为内胚层血统和 随后的胰腺和内分泌细胞状态。这将揭示DAXX损失和 阐明导致肿瘤发生的特定下游变化。K22职业转折奖 不仅将推进这些研究目标，而且还将有助于实现我长期的职业目标 指导一个富有成效和影响力的独立研究计划，专注于 肿瘤发生的基础。具体地说，它将支持我在一个新的 主办机构。它还将允许提交我的第一份资深作者手稿，并提供 基金会和初步数据，以迅速申请和具有竞争力的R01资金。总而言之，这是 该提案支持在细胞内稳态的表观遗传调控方面的研究和职业发展 以及调节失调如何促进胰腺神经内分泌肿瘤的发生。

Project Summary Genome sequencing has implicated novel candidate tumor suppressors, including DAXX and ATRX which are mutated in 43% of pancreatic neuroendocrine tumors (PanNETs). Despite this strong disease association, our understanding of the physiological functions of DAXX and ATRX remains limited. Biochemical and cell biological studies have demonstrated that together they regulate the epigenome as a chaperone complex for the histone variant H3.3, depositing H3.3 in heterochromatin. Additional non-histone chaperone functions have been reported for both genes. We recently developed a conditional mouse model for Daxx and demonstrated that Daxx loss creates a permissive state that cooperates with environmental stress (inflammation) and other genetic lesions (Men1 loss) to affect the transcriptional profile and impact cell state, which ultimately impairs pancreas recovery and regeneration in vivo. The transcriptional changes are associated with dysregulation of ERVs, thereby expanding our understanding of the somatic regulation beyond DNA and histone methylation in vivo, and implicating ERV dysregulation of pancreatic neuroendocrine tumorigenesis. Importantly, we also demonstrated that dysregulation of endogenous genes near ERVs is also observed in human PanNETs with DAXX mutations. This proposal expands on these studies to better understand how mutations in DAXX contribute to tumorigenesis. We will first use our robust physiological mouse models to obtain a comprehensive understanding of the epigenetic mechanisms that underlie ERV dysregulation downstream of Daxx loss. We will conduct an integrative analysis of H3.3 incorporation, DNA methylation, and histone methylation. We will complement these genomic studies with genetic experiments to directly interrogate the Daxx:H3.3 and Daxx:Atrx interactions in vivo using newly generated mouse alleles. Finally, we will extend our studies to an innovative human pluripotent stem cell model that can be differentiated to an endoderm lineage and subsequent pancreas and endocrine cell states. This will reveal context-dependent effects of DAXX loss and illuminate the specific downstream changes that contribute to tumorigenesis. The K22 Career Transition award will not only advance these research objectives, but it will also help achieve my long-term career goal of directing a productive and impactful independent research program focused on the molecular mechanisms that underlie tumorigenesis. Specifically, it will support the establishment of my independent laboratory at a new sponsoring institution. It will further allow for the submission of my first senior author manuscript and provide the foundations and preliminary data to rapidly apply and be competitive for R01 funding. In summary, this proposal supports both research and career development in the epigenetic regulation of cellular homeostasis and how dysregulation contributes to pancreatic neuroendocrine tumorigenesis.