Functions, mechanisms, and therapeutic potential of chromatin looping

描述（由申请人提供）：真核基因组在间期核中非随机组织。关键控制元件可以物理地彼此接触以形成染色质环。染色质纤维的环状构型在许多基因位点都有描述，然而，这些结构的建立机制及其与基因表达的功能关系仍不清楚。转录辅因子Ldb 1对于在鼠的ε-珠蛋白基因座上建立环状染色质相互作用是至关重要的。具体地说，我们表明，通过人工锌指蛋白在未成熟红系细胞中将Ldb 1拴系到该位点足以促进<$-珠蛋白增强子和启动子之间的环状相互作用，并有效地激活转录。这第一次表明，染色质成环的因果关系的基因表达的基础，并提出了强制染色质成环可能被用来有效地操纵基因表达的可能性。该提案建立在这些发现的基础上，通过研究特定目标1中Ldb 1功能的机制及其在基因组组织中的广泛作用。在具体目标2中，我们将进一步发展强迫染色质成环的方法，以增强和扩大其实用性。在具体目标3中，我们将研究强迫染色质循环作为一种方法来发育重编程小鼠和人类的<$-珠蛋白基因座。在Specific Aim 4中，概念验证研究将检查通过染色质成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，操纵染色质的高级结构，以调节基因表达的目的是独特的和新颖的设计。与传统方法相比，复杂调控元件的并列有望使基因激活发生更显著的变化，并且也可能用于抑制基因转录以用于探索或治疗目的。

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.