Functions, mechanisms, and therapeutic potential of chromatin looping

描述（由申请人提供）：真核生物基因组在间期细胞核中是非随机组织的。关键的控制元件可以物理地相互接触，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-珠蛋白位点建立环状染色质相互作用至关重要。具体来说，我们发现在未成熟的红细胞中通过人工锌指蛋白将Ldb1系在位点上，足以促进-珠蛋白增强子和启动子之间的环相互作用，并有效地激活转录。这首次表明，染色质环是基因表达的因果基础，并提出了强迫染色质环可能被用来有效地操纵基因表达的可能性。本研究基于这些发现，在Specific Aim 1中研究了Ldb1的功能机制及其在基因组组织中的广泛作用。在特异性目标2中，我们将进一步发展强制染色质环的方法，以增强和扩大其用途。在具体目标3中，我们将研究强制染色质环作为一种方法来重新编程小鼠和人类的-珠蛋白位点。在Specific Aim 4中，概念验证研究将检验通过染色质环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性贫血。据我们所知，为了调节基因表达而操纵高阶染色质结构在其设计上是独特而新颖的。与传统方法相比，复杂调控元件的并放在一起有望在基因激活方面发生更大的变化，并且也可能被用于抑制基因转录的探索或治疗目的。

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.