Functions, mechanisms, and therapeutic potential of chromatin looping

描述(申请人提供)：真核基因组在间期核中非随机组织。关键的控制元件可以在物理上相互接触，形成染色质环。染色质纤维的环状结构已经在许多基因位点上被描述，然而，这些结构建立的机制以及它们与基因表达的功能关系仍然不清楚。转录辅助因子LDB1对于在小鼠�-珠蛋白基因座上建立染色质环状相互作用至关重要。具体地说，我们证明了在未成熟的红系细胞中，通过人工锌指蛋白将LDB1连接到该基因座上，足以促进�-珠蛋白增强子和启动子之间的环状相互作用，并有效地激活转录。这首次表明染色质循环是基因表达的因果基础，并提出了强制染色质循环可能被用来有效地操纵基因表达的可能性。这项建议建立在这些发现的基础上，通过在特定目标1中研究LDB1的功能及其在基因组组织中的广泛作用来实现。在具体目标2中，我们将进一步发展强制染色质循环的方法，以增强和扩大其有效性。在具体目标3中，我们将研究强制染色质环作为一种对小鼠和人类�-珠蛋白基因座进行发育重新编程的方法。在特定的目标中，4项概念验证研究将检验通过染色质环重新激活胎儿血红蛋白是否可以改善人源化小鼠模型中的镰状细胞贫血。据我们所知，操纵高阶染色质结构以调节基因表达在其设计上是独特和新颖的。与传统方法相比，复杂调控元件的并列有望在基因激活方面发生更戏剧性的变化，也可能被用于探索或治疗目的来抑制基因转录。

DESCRIPTION (provided by applicant): The eukaryotic genome is non-randomly organized in the interphase nucleus. Critical control elements can physically contact each other to form chromatin loops. Looped configurations of the chromatin fiber have been described at numerous gene loci, however, the mechanism by which these structures are established and their functional relationship with gene expression have remained unclear. The transcription co-factor Ldb1 is critical for establishing looped chromatin interactions at the murine �-globin locus. Specifically, we showed that tethering Ldb1 to the locus via artificial zinc finger proteins in immature erythroid cells is sufficient to promote a looped interaction between the �-globin enhancer and promoter and potently activate transcription. This suggested for the first time that chromatin looping causally underlies gene expression and raised the possibility that forced chromatin looping might be employed to effectively manipulate gene expression. This proposal builds on these findings by investigating in Specific Aim 1 the mechanisms of Ldb1 function and its broad role in genome organization. In Specific Aim 2 we will further develop the approach of forced chromatin looping to enhance and broaden its usefulness. In Specific Aim 3 we will examine forced chromatin looping as an approach to developmentally reprogram the murine and human �-globin locus. In Specific Aim 4 proof-of-concept studies will examine whether reactivation of fetal hemoglobin via chromatin looping can ameliorate sickle cell anemia in a humanized mouse model. To our knowledge, the manipulation of higher order chromatin structure for the purpose of regulating gene expression is unique and novel in its design. The juxtaposition of complex regulatory elements promises more dramatic changes in gene activation when compared to conventional approaches, and might also be exploited to repress gene transcription for exploratory or therapeutic purposes.