The genomic mechanisms of transcription and disease: generating mouse models for laterality defects using CRISPR/Cas9 genome engineering at the Pitx2 locus

描述（由申请人提供）：转录因子Pitx 2是左右（LR）不对称器官形态发生的重要调节因子，未能建立左特异性Pitx 2表达与新生儿中危及生命的心脏缺陷和肠旋转不良和肠扭转有关。人类PITX 2的突变是导致Axenovir-Rieger综合征（ARS）的原因，其特征在于智力迟钝、颅面出生缺陷和脐疝。ARS患者的筛选已经确定了在Pitx 2编码序列中没有突变但具有大的缺失的个体，所述缺失包括缺乏编码基因的相邻基因沙漠，这表明在驱动Pitx 2表达中的顺式调节作用。尽管Pitx 2在发育中起着关键作用，但驱动左特异性Pitx 2表达的基因组顺式调节机制仍不清楚。Kurpios实验室已经建立了二元LR不对称肠背肠系膜（DM），这是一种将肠道悬挂在体腔内的中胚层组织桥，作为一种强大的体内系统，其中仅在DM左侧的Pitx 2表达驱动了保守的肠循环过程。通过进行DM左室与右室特异性转录谱分析，我们发现，与Pitx 2紧邻的基因以及位于Pitx 2侧翼的大保守基因沙漠（连锁基因）的近端和远端的基因在DM中仅在右侧表达，与左室特异性Pitx 2相反。使用DNA荧光原位杂交（FISH），我们进行了染色质可视化的L与R DM，并了解到，二元不对称DM组织反映了不对称染色质结构在Pitx 2基因座，这表明在3维（3D）染色质拓扑结构的变化协调Pitx 2转录。利用DM作为一个易处理的模型，本建议的目标是阐明如何差分染色质拓扑结构协调LR转录的Pitx 2基因座。在我的第一个目标，我的特点的调控作用的基因沙漠在Pitx 2位点，以模仿ARS缺陷小鼠。我们最近发现了一个增强子元件e926位于基因沙漠，它在DM左侧的活动，但它也重叠的转录起始位点的长非编码RNA（lncRNA）的右侧DM上表达。因此，在我的第二个目标，我将功能解剖的LR不对称器官发生过程中e926的作用。我的第三个目标是确定CCCTC结合因子CTCF在调控Pitx 2转录中的作用，CTCF是一种参与染色质循环的结构蛋白。我们发现，在小鼠ES细胞中敲低CTCF会破坏Pitx 2基因座的染色质组织，并干扰相关基因的LR不对称表达。此外，来自小鼠ES细胞的ChIP-seq数据鉴定了分离多种Pitx 2同种型的关键转录起始位点的保守峰，表明CTCF调节Pitx 2同种型转换。总的来说，这项工作为科学界提供了第一项研究，即在脊椎动物中驱动LR形态发生的差异LR染色质拓扑学，并为开发Pitx 2相关疾病的小鼠模型提供了前所未有的潜力。

DESCRIPTION (provided by applicant): The transcription factor Pitx2 is an essential regulator of left-­right (LR) asymmetric organ morphogenesis and failure to establish left-specific Pitx2 expression is linked to life-threatening heart defects and gut malrotation and volvulus in newborns. Mutations in human PITX2 are causal for Axenfeld-Rieger Syndrome (ARS), characterized by mental retardation, craniofacial birth defects, and umbilical hernias. Screening of ARS patients has identified individuals who possess no mutations in Pitx2 coding sequences but harbor large deletions that encompass an adjacent gene desert devoid of coding genes, suggesting a cis-regulatory role in driving Pitx2 expression. Despite key roles of Pitx2 in development, the genomic cis-regulatory mechanisms driving left-specific Pitx2 expression remain unclear. The Kurpios lab has established the binary LR asymmetric gut dorsal mesentery (DM), a bridge of mesodermal tissue that suspends the gut within the body cavity, as a powerful in vivo system where Pitx2 expression exclusively on the DM left side drives the conserved process of leftward gut looping. By performing DM left vs. right compartment-specific transcriptional profiling, we discovered that genes immediately neighboring Pitx2 and positioned either proximally and distally to a large conserved gene desert flanking Pitx2 (linked genes) are expressed in the DM exclusively on the right side, opposite to left-specific Pitx2. Using DNA fluorescent in situ hybridization (FISH), we performed chromatin visualization of the L vs. R DM and learned that the binary asymmetric DM organization is mirrored by asymmetric chromatin architecture at the Pitx2 locus, suggesting that changes in 3 dimensional (3D) chromatin topology coordinate Pitx2 transcription. Utilizing the DM as a tractable model, the goal of this proposal is to elucidate how differential chromatin topology coordinates LR transcription of the Pitx2 locus. In my first aim, I characterize the regulatory role of the gene desert at the Pitx2 locus in order to mimic ARS defects in mice. We recently identified an enhancer element e926 located within the gene desert, which has left-sided activity in the DM but it also overlaps the transcriptional start site of a long noncoding RNA (lncRNA) expressed on the right side of the DM. Hence in my second aim I will functionally dissect the role of e926 during LR asymmetric organogenesis. My third aim will determine the role of CCCTC-binding factor, CTCF, an architectural protein involved in chromatin looping, in regulating Pitx2 transcription. We showed that knockdown of CTCF in mouse ES cells disrupts chromatin organization of the Pitx2 locus and perturbs LR asymmetric expression of linked genes. Moreover, ChIP-seq data from mouse ES cells identified a conserved peak separating the critical transcriptional start site of the multiple Pitx2 isoforms suggesting that CTCF regulates Pitx2-isoform switching. Collectively, this work provides to the scientific community the first study of differential LR chromatin topolog driving LR morphogenesis in vertebrates and offers unprecedented potential for developing mouse models for Pitx2-inked disease.