Defining the Chromatin Landscape and Transcriptional Drivers Of Proliferation and Migration In Human Glioblastoma.

最近的分子研究揭示了胶质母细胞瘤（GBM）中的整体染色质紊乱，但这如何影响 个体基因位点在体内驱动肿瘤生长和迁移仍然知之甚少。此前，我们有 显示出显着的相似性，在染色质重塑之间的germ matrix干/祖细胞（NSPCs） 和胶质瘤的EGFR启动子，并涉及几个表观遗传修饰的转录 该致癌基因座的激活（Erfani等人，2015）。我们现在扩大我们的研究重点， 系统地，在功能不同的人群中，发育调控的转录网络 在神经胶质瘤中变得病理性地增选，以维持细胞迁移和细胞增殖的致瘤特性。 增殖为此，我们开发了一种简单的EGF配体结合策略， 来自新鲜人样品和患者来源的神经胶质瘤异种移植物（PDX）的具有干细胞特性的群体， 其中迁移的人GBM细胞可以与肿瘤中心的那些细胞区域性地分离（托姆- Garcia等人，Stem Cell Rep，在最终修订版中接受）。全转录组和染色质可及性 在这些新鲜分选的人GBM干细胞群体中的分析揭示了不同的调控区域， 细胞迁移与自我更新，后者也存在于NSPCs中。因此我们假设肿瘤的特性 GBM中的细胞生长和迁移是通过开放区域的独特转录因子（TF）调节的。 染色质，其中一些是从正常的神经发育状态中选出的。为了验证这一假设， 我们打算分析转录组（通过RNA-seq）和相关的开放染色质景观（通过ATAC-seq） 在从PDX神经胶质瘤分离的表型定义的迁移性与核心增殖性人GBM群体中， 并将它们与我们以前在开发NSPC时生成的数据进行比较。RNAseq的差异分析和 浸润性与肿瘤核心GBM细胞中的ATACseq数据将使我们能够定义转录途径， 染色质可及性调节区驱动体内迁移GBM细胞命运的适应， 与NSPCs中类似数据的比较分析将阐明发育途径是如何异常地协同的， 在GBM中选择并重新激活。ATACseq/RNAseq组合数据分析将为特定的 在浸润性肿瘤细胞中激活的基因位点处的TF结合占据，产生TF的候选列表 GBM中细胞迁移的调节剂，我们将通过生物化学方法证实。 在肿瘤性和非肿瘤性中定义特定的表观遗传景观和相关的转录表型 来自新鲜人类和PDX样本的祖细胞群体将深入推进我们对 维持GBM干细胞可塑性的机制以及GBM干细胞亚群中特定的分子适应性， 这些细胞走向迁移细胞的命运。通过将我们的数据存储在开放访问平台中，我们将最大限度地 数据传播和促进进一步调查细胞迁移在GBM中的作用，希望能揭示 针对目前逃避手术切除的浸润性肿瘤细胞的治疗新途径。

Recent molecular studies reveal global chromatin disorganization in glioblastoma (GBM), but how that affects individual gene loci to drive tumor growth and migration in vivo is still poorly understood. Previously, we have shown remarkable similarities in chromatin remodeling between germinal matrix stem/progenitors (NSPCs) and gliomas at the EGFR promoter, and implicated several epigenetic modifiers for the transcriptional activation of this oncogenic locus (Erfani et al, 2015). We now expand our study focus to investigate more systematically and in functionally distinct populations how developmentally regulated transcriptional networks become pathologically co-opted in gliomas to maintain tumorigenic properties of cell migration and cell proliferation. To this end, we have developed a simple EGF ligand-binding strategy to prospectively isolate populations with stem cell properties from fresh human samples and patient-derived glioma xenografts (PDX), in which the migratory human GBM cells can be regionally separated from those at the tumor core (Tome- Garcia et al, Stem Cell Rep, accepted in final revisions). Full transcriptome and chromatin accessibility analyses in these freshly sorted human GBM stem cell populations revealed distinct regulatory regions related to cell migration vs. self-renewal, the latter also present in NSPCs. We thus hypothesized that tumor properties of cell growth and migration in GBM are regulated through unique transcription factors (TFs) at regions of open chromatin, some of which are co-opted from a normal neural developmental state. To test this hypothesis, here we intend to profile the transcriptome (by RNA-seq) and associated open chromatin landscape (by ATAC-seq) in phenotypically defined migratory vs. core-proliferative human GBM populations, isolated from PDX gliomas, and compare them to our previously generated data in developing NSPCs. Differential analysis of RNAseq and ATACseq data in infiltrative vs. tumor-core GBM cells will allow us to define the transcriptional pathways and chromatin accessibility regulatory regions driving adaptations for a migratory GBM cell fate in vivo, while comparative analysis with similar data in NSPCs will elucidate how developmental pathways are aberrantly co- opted and re-activated in GBM. Combined ATACseq/RNAseq data analysis will provide a blueprint for specific TF binding occupancy at gene loci activated in infiltrative tumor cells, generating a candidate list of TF regulators for cell migration in GBM, which we will confirm biochemically. Defining the specific epigenetic landscape and associated transcriptional phenotype in neoplastic and progenitor populations derived from fresh human and PDX samples will advance deeply our understanding of the mechanisms maintaining plasticity in GBM stem cells and the specific molecular adaptations in a subset of these cells towards a migratory cell fate. By depositing our data in an open access platform, we will maximize data dissemination and facilitate further investigation into the role of cell migration in GBM, hoping to uncover new avenues for therapy aimed at targeting infiltrative tumor cells that currently evade surgical resection.