The role of the cohesin complex in hematopoietic transformation and leukemia maintenance

项目摘要 在这个建议中，我们将定义通过Stag 2缺失的表观遗传失调对三维细胞的影响。 染色质结构和基因表达。我们将定义关键的谱系特异性染色质特征，并验证 与干细胞自我更新相反，它们在分化中的层次结构。在目标1中，我们将执行低输入Hi-ChIP Ctcf（环/TAD-边界）、Rad 21（泛粘蛋白环）、H3 K27 Ac（增强子）和RNA-的ChIP测定 在表型确定的小鼠和人干细胞和祖细胞上进行seq（基因表达）以确定群体- 特定的循环事件。使用我们新的“开-关-开”双重重组酶（Stag 2GL）等位基因，我们将首先确定 Stag 2的恢复可以逆转对自我更新/分化的影响，并定义染色质环1） 在Stag 2损失期间受到干扰，2）在Stag 2恢复后恢复。基因座特异性功能测定将 通过体外研究确定自我更新所需的关键事件，以在“Stag 2-on”中复制表型或防止 “Stag 2-off”中的反转。在目标2中，使用我们的低细胞输入Hi-C和Hi-ChIP技术来评估染色质环 在我们的完全渗透Stag 2/Flt 3共突变AML模型中鉴定的改变与Stag 2GL 等位基因使用多组学方法，我们将定义Flt 3的染色质景观， 先前的Stag 2突变我们已经创建了一个注释良好的MDS和AML患者生物储存库，以及 髋关节置换术患者健康骨髓的生物储存库。使用这些原始患者样本，我们 将绘制干细胞和祖细胞群体中的染色质环和转录输出，并交叉谱系- 在人类和小鼠中的特定相互作用。染色质环的功能验证丢失或获得的共同- 将在体外评估突变模型，优先考虑在原发性AML患者样品中鉴定的基因座。Stag2 该系统中的逆转将最终确定是否需要Stag 2来维持AML， 染色质可逆性的能力，由此代表Stag 2突变型AML中的遗传依赖性。最后， 这些小鼠模型和连续传代的患者来源的异种移植物资源将构成系统， 评估Stag 2缺失对标准化疗方案（阿糖胞苷）治疗反应的影响 和多柔比星）以及新兴的靶向治疗，如FLT 3-ITD突变AML中的FLT 3抑制 （gilteritinib）。综上所述，本建议的结果将阐明 Stag 2，导致更深入地了解1）染色质重塑的表观遗传机制， 转录调控，表观遗传可塑性，2）Stag 2缺失对MDS肿瘤发生的贡献 和继发性AML;和3）鉴定关键的新治疗靶点。

PROJECT SUMMARY In this proposal, we will define the effects of epigenetic dysregulation via Stag2 loss on three-dimensional chromatin structure and gene expression. We will define key lineage-specific chromatin features and validate their hierarchy in differentiation in contrast to stem cell self-renewal. In Aim 1, we will perform low-input Hi-ChIP assay on Ctcf (loop/TAD-boundary), Rad21 (pan-cohesin loop), ChIP assay for H3K27Ac (enhancer) and RNA- seq (gene expression) on phenotypically defined mouse and human stem and progenitors to define population- specific looping events. Using our novel ‘on-off-on’ dual recombinase (Stag2GL) allele, we will first determine if the restoration of Stag2 can reverse the effects on self-renewal/differentiation and define chromatin loops 1) perturbed during Stag2 loss and 2) restored after Stag2 reversion. Locus-specific functional assays will determine the key events required for self-renewal through in vitro studies to phenocopy in ‘Stag2-on' or prevent reversion in ‘Stag2-off’. In Aim 2, using our low cell input Hi-C and Hi-ChIP techniques to assess chromatin loops alterations identified in our fully penetrant Stag2/Flt3 co-mutant AML models in combination with the Stag2GL allele. Using a multi-omics approach, we will define the chromatin landscape of Flt3 with and without the antecedent Stag2 mutation. We have created a well-annotated MDS and AML patient biorepository, as well as a biorepository of healthy bone marrow from hip arthroplasty patients. Using these primary patient samples, we will map chromatin loops and transcriptional output in stem and progenitor populations and intersect lineage- specific interactions in human and mouse. Functional validation of chromatin loops lost or gained in the co- mutant models will be assessed in vitro, prioritizing loci identified in primary AML patient samples. Stag2 reversion in this system will definitively determine whether Stag2 is required for the maintenance of AML and capability for chromatin reversibility, hereby representing a genetic dependency in Stag2-mutant AML. Finally, these mouse models and serially passaging patient derived xenograft resources will constitute the systems to assess the effects of Stag2 loss on therapeutic response to standard chemotherapeutic regimens (cytarabine and doxorubicin) as well as to emerging targeted therapies such as FLT3 inhibition in FLT3-ITD mutant AML (gilteritinib). Taken together, the results of this proposal will elucidate the pathophysiological mechanism of Stag2, leading to a deeper understanding of 1) epigenetic mechanisms of chromatin remodeling on transcriptional regulation, epigenetic plasticity, 2) the contributions of Stag2 loss towards tumorigenesis in MDS and secondary AML and 3) identification of key novel therapeutic targets.