The Genomic Mechanisms of Transcription and Disease: Generating Mouse Models for Laterality Defects Using CRISPR/Cas9 Genome Engineering at the Pitx2 Locus
Fellow: Frances Chen
Mentor: Natasza Kurpios, John Schimenti
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 therole 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 topology driving LR morphogenesis in vertebrates and offers unprecedented potential for developing mouse models for Pitx2-‐‑linked disease.