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

描述(申请人提供)：转录因子Pitx2是左右(LR)器官形态不对称的重要调节因子，未能建立左-右不对称的Pitx2表达与危及生命的心脏缺陷、肠道旋转不良和新生儿扭转有关。人类PITX2基因突变是导致阿森菲尔德-里格综合征(ARS)的原因，该综合征的特征是智力低下、头面部出生缺陷和脐疝。对ARS患者的筛查发现，在Pitx2编码序列中没有突变，但存在大量缺失的个体，其周围的邻近基因沙漠中没有编码基因，这表明在驱动Pitx2表达的过程中发挥了顺式调节作用。尽管Pitx2在发育过程中发挥了关键作用，但驱动左侧特异性Pitx2表达的基因组顺式调控机制仍不清楚。Kurpios实验室已经建立了二元LR不对称肠道背系膜(DM)，这是一种将肠道悬挂在体腔内的中胚层组织的桥梁，作为一个强大的体内系统，在DM左侧仅表达Pitx2，驱动向左的肠道循环的保守过程。通过执行DM左侧和右侧隔室特异性转录图谱，我们发现紧邻Pitx2的基因位于大型保守基因沙漠侧翼Pitx2的近端和远端(连锁基因)在DM中仅在右侧表达，与左侧特异性Pitx2相反。我们用荧光原位杂交技术对L和R DM进行了染色质显示，发现二元不对称DM的组织结构反映了Pitx2基因座的不对称染色质结构，这表明三维(3D)染色质拓扑结构的变化协调了Pitx2的转录。利用DM作为一个易于处理的模型，本建议的目的是阐明差异染色质拓扑结构如何协调Pitx2基因座的LR转录。在我的第一个目标中，我描述了基因沙漠在Pitx2基因座的调节作用，以便在小鼠中模拟ARS缺陷。我们最近发现了一个位于基因沙漠中的增强子元件e926，它在DM中具有左侧活性，但它也与DM右侧表达的长非编码RNA(LncRNA)的转录起始点重叠。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。我的第三个目标将确定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路的建筑蛋白。我们发现，在小鼠ES细胞中，CTCF的敲除破坏了Pitx2基因位点的染色质组织，并扰乱了相关基因的LR不对称表达。此外，来自小鼠ES细胞的ChIP-SEQ数据发现了一个保守的峰，分离了多个Pitx2亚型的关键转录起点，表明CTCF调控着Pitx2-亚型的转换。总而言之，这项工作为科学界提供了第一个关于驱动脊椎动物LR形态发生的不同LR染色质拓扑结构的研究，并为开发Pitx2连锁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.