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

转录和疾病的基因组机制：使用 CRISPR/Cas9 基因组工程在 Pitx2 基因座生成偏侧性缺陷小鼠模型

• 批准号: 9244676
• 负责人: Frances L Chen
• 金额: $ 4.05万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-01 至 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9244676
• 关键词: 3-Dimensional; Alleles; Animal Genetics; Animal Model; Architecture; Atrial Fibrillation; Automobile Driving; Axenfeld-Rieger syndrome; Bilateral; Biological Assay; Body cavities; CCCTC-binding factor; CRISPR/Cas technology; Cells; ChIP-seq; Chickens; Chromatin; Chromatin Loop; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Communities; Congenital Abnormality; Congenital exomphalos; DNA; Data; Defect; Development; Disease; Distal; Dorsal; Enhancers; Failure; Fluorescent in Situ Hybridization; Gene Expression; Genes; Genetic; Genetic Enhancer Element; Genetic Transcription; Genome engineering; Genomics; Goals; Handedness; Heart; Heart Abnormalities; Human; Imagery; In Vitro; Individual; Ink; Intestinal Volvulus; Junk DNA; Knockout Mice; Left; Life; Link; Mediating; Mental Retardation; Mesentery; Modeling; Molecular Genetics; Morphogenesis; Mus; Mutation; Names; Newborn Infant; Oranges; Organ; Organogenesis; Patients; Positioning Attribute; Process; Protein Isoforms; Proteins; Regulatory Element; Role; Side; Site; System; Technology; Tissues; Transcription Initiation Site; Untranslated RNA; Vertebrates; Work; body cavity; cellular engineering; craniofacial; egg; embryonic stem cell; gene discovery; genetic manipulation; genome editing; genomic tools; in vivo; innovation; knock-down; mouse model; mutant; organ growth; promoter; public health relevance; screening; transcription factor

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.

描述（通过应用程序提供）：转录因子PITX2是左右（LR）非对称器官形态发生的必不可少的调节因子，无法建立左侧特异性PITX2表达与威胁生命的心脏缺陷以及新生儿中肠道畸形和肠道差异和肠道肠道差异和肠胃不变有关。人pitx2中的突变是轴菲尔德综合征（ARS）的因果关系，其特征是静脉蜂窝状的症状。二进制LR不对称的肠道肠系膜（DM），中胚层组织的桥梁在体腔2中的肠道仅在DM左侧表达，从而通过执行DM左VS。特定的转录曲线，我们发现基因在右侧pitx2（链接的基因）的较大的pitx2附近和远端与右侧IC PITX2上的DM相邻。在PITX2基因座的不对称染色质镜面，并在3维（3D染色质拓扑坐标PITX2）中发生变化。利用DM作为可聊天模型hromatin拓扑仪的LR转录，Pitx2基因座的LR转录。 PITX2基因座的基因沙漠是为了模仿小鼠的ARS缺陷。在DM的右边，CCCTC结合因子的作用CTCF，一种参与染色质循环的结构蛋白，在调节PITX2转录中来自小鼠ES细胞的CHIP-seq数据的Perturbs L对称性表达了一个保守的峰，该峰分隔了多个PITX2同工型的Trantition Transtion T位点，CTCF表明Pitx2-Isoform of Pitx2-Isoform of Pitx2-Isoform ofertial pitx2-Isoform sworge。发育的前所未有的潜力。