Enhancer Driven Gene Regulation During Lymphocyte Development

描述：淋巴样肿瘤（白血病和淋巴瘤）是人类最常见的恶性肿瘤之一，尤其是儿童。尽管它们的病因多种多样，但大多数与癌基因、肿瘤抑制因子和/或转录因子的表达改变有关，这些对谱系描述很重要。为了确定这些癌症的各种形式的潜在原因，了解在正常细胞中调节基因表达的不同细胞过程的机制是很重要的。我们已经把我们的努力集中在基因调控的一个特定方面，仍然知之甚少：增强子介导的基因调控网络。确定支撑分化的增强子介导的基因调控网络仍然是一个挑战，因为很难确定增强子的靶标。事实上，在某些情况下，单个增强子可以控制多个基因，而这些基因不一定位于顺式基因中物理距离很近的位置。一些研究试图利用染色体构象捕获来识别增强子和启动子之间的相互作用来解决这个问题。然而，目前尚不清楚相互作用是否能预测功能调控，因此这些方法并不能明确确定单个增强子的调控目标以及它们对基因表达的影响程度。为了检验单个增强子的作用，有必要对这些元件进行突变，并表征与基因活性和转录因子结合耦合的相互作用的变化。在这个应用中，我们的目标是执行一系列全基因组分析，以检查这些方面的控制，使用小鼠携带与免疫球蛋白和T细胞受体位点相关的增强子缺失。迄今为止，我们所进行的实验数据支持这样一个模型，即每个增强子通过物理地将每个基因座重新定位到特定的位点来参与转录，对其进行扰动（例如通过删除特定的增强子）可以对在同一位点表达或抑制的许多基因座（包括顺式和反式）产生广泛的影响。基于这些发现和我们实验室的数据以及其他显示共同调节基因在细胞核中聚集的数据，我们假设转录因子或调节因子的结合诱导了一种独特的细胞类型模式，特定阶段的相互作用，形成了一个有助于控制基因表达的网络。在本应用中，我们的目标是进一步探索这一点，并通过解决以下问题来测试我们模型的局限性：(1)增强子介导的基因调控网络的潜在机制是什么？(2) RAG在控制抗原受体增强子介导的网络中的作用是什么？我们能从基因上操纵E吗？观察增强子介导的调控网络的变化？总的来说，我们在这里提出的工作将在我们理解细胞类型特异性基因网络和协调位点表达在淋巴细胞发育过程中经历正常和异常DNA重组事件的意义方面开辟新的领域。

DESCRIPTION: Lymphoid neoplasms (leukemias and lymphomas) are among the most common malignancies in humans, particularly children. Although they arise from diverse etiologies, the majority are linked to alterations in expression of oncogenes, tumor suppressors and / or transcription factors that are important for lineage specification. In order to identify te underlying cause of the various forms of these cancers it is important to understand the mechanisms underlying the different cellular processes that regulate gene expression in normal cells. We have focused our efforts on one particular aspect of gene regulation that remains poorly understood: enhancer mediated gene regulatory networks. Identifying the enhancer-mediated gene regulatory networks that underpin differentiation remains a challenge because it has been difficult to define the targets of enhancers. Indeed, individual enhancers can in some instances control multiple genes that are not necessarily located in close physical proximity in cis. Some studies have tried to address this using chromosome conformation capture to identify interactions between enhancers and promoters. However, it is not clear whether interactions are predictive of functional regulation so these approaches do not definitively determine the regulatory targets of individual enhancers and the extent to which they contribute to gene expression. To examine the effect of individual enhancers it is necessary to mutate these elements and characterize changes in interactions coupled with gene activity and binding of transcription factors. In this application we aim to perform a series of genome wide analyses to examine these aspects of control using mice that harbor deletions of enhancers associated with immunoglobulin and T cell receptor loci. The data from experiments we have performed to date support a model in which each enhancer participates in transcription by physically relocating each locus into specific sites, perturbation of which (e.g. by deleting a specific enhancer) can have extensive consequences on many loci (both in cis and in trans) that are expressed or repressed at the same site. Based on these findings and data from our lab and others showing that co-regulated genes come together in the nucleus, we hypothesize that the binding of transcription factors or regulators induces a distinct pattern of cell type, stage specific interactions, which form a network that contributes to the control of gene expression. In this application we aim to explore this further and test the limitations of our model by addressing the following questions: (1) What are the mechanisms underlying enhancer-mediated gene regulatory networks? (2) What is the contribution of RAG in controlling antigen receptor enhancer mediated networks? Can we genetically manipulate E? and observe changes in the enhancer mediated regulatory network? Overall the work we propose here will break new ground in our understanding of cell type-specific gene networks and the implications of coordinate locus expression during lymphoid development in cells undergoing normal and aberrant DNA recombination events.