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Pitx2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Pitx2表达与新生儿危及生命的心脏缺陷、肠道旋转不良和肠扭转有关。人类PITX2突变是导致Axenfeld-Rieger综合征（ARS）的原因，其特征是智力迟钝，颅面出生缺陷和脐疝。通过对ARS患者的筛选，发现了Pitx2编码序列没有突变，但包含大量缺失的个体，这些缺失包含了缺乏编码基因的邻近基因沙漠，这表明在驱动Pitx2表达中具有顺式调控作用。尽管Pitx2在发育中起关键作用，但驱动左特异性Pitx2表达的基因组顺式调控机制尚不清楚。Kurpios实验室已经建立了二元LR非对称肠背肠系膜（DM），这是一个将肠道悬浮在体腔内的中表皮组织的桥梁，是一个强大的体内系统，其中仅在DM左侧的Pitx2表达驱动左肠环的保守过程。通过进行DM左右区室特异性转录分析，我们发现紧邻Pitx2的基因，以及位于Pitx2两侧一个大的保守基因沙漠的近端和远端（连锁基因），在DM中只在右侧表达，与左特异性Pitx2相反。利用DNA荧光原位杂交（FISH），我们对L和R DM进行了染色质可视化，并了解到二元不对称DM组织是由Pitx2位点的不对称染色质结构反映的，这表明三维（3D）染色质拓扑结构的变化协调了Pitx2转录。利用DM作为一个可处理的模型，本提案的目标是阐明差异染色质拓扑如何协调Pitx2位点的LR转录。在我的第一个目标中，我描述了Pitx2位点的基因沙漠的调节作用，以模拟小鼠的ARS缺陷。我们最近在基因沙漠中发现了一个增强子元件e926，它在DM中具有左侧活性，但它也与DM右侧表达的长链非编码RNA （lncRNA）的转录起始位点重叠。因此，在我的第二个目标中，我将从功能上剖析e926在LR不对称器官发生中的作用。我的第三个目标是确定ccctc结合因子CTCF在调节Pitx2转录中的作用，CTCF是一种参与染色质环的建筑蛋白。我们发现，小鼠胚胎干细胞中CTCF的敲低会破坏Pitx2位点的染色质组织，并扰乱相关基因的LR不对称表达。此外，来自小鼠胚胎干细胞的ChIP-seq数据发现了一个分离多个Pitx2亚型的关键转录起始位点的保守峰，这表明CTCF调节Pitx2亚型的转换。总的来说，这项工作为科学界首次研究了脊椎动物LR形态发生的差异染色质拓扑结构，并为开发pitx2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.