MSc Exit Seminar- Zobia Anwar (Rhee Lab)

Investigating the Role of Onecut1 in Establishment of Cell-type-Specific Enhancers during Mouse Motor Neuron Differentiation

Thursday, October 22^nd, 2020 at 2:00pm

Join Zoom Meeting:

https://utoronto.zoom.us/j/81258699707

Meeting ID: 812 5869 9707

Passcode: 148248

Abstract:

Enhancers are short regulatory DNA sequences that act to ‘enhance’ associated gene transcription. The immense complexity and diversity within the central nervous system provides an excellent opportunity to study the role of enhancers during cell fate specification. The identity of spinal motor neurons is established by three programming transcription factors (TFs), also known as lineage-determining TFs: Ngn2, Isl1 and Lhx3. Two LIM homeodomain TFs, Isl1 and Lhx3, bind to enhancers in nascent motor neurons regulating the expression of many effector genes, but Lhx3 is downregulated as nascent motor neurons mature. Then, Isl1 is relocated to the enhancers bound by Onecut1 TF in maturing motor neurons ensuring stable motor neuron gene expression. The objective of this thesis was to characterize the role of Onecut1 in maintenance of gene expression in maturing spinal motor neurons. I employed the CRISPR-Cas9 system to generate embryonic stem (ES) cell line containing Onecut1 knockout mutation, followed by in-vitro differentiation of these ES cells into spinal motor neurons. I performed analyses for gene expression changes and genomic mapping in wildtype motor neurons and differentiated cells from the Onecut1 knockout mutant. My gene expression analysis revealed attenuation of expression of a subset of motor neuron genes in Onecut1 mutant motor neurons. Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) analysis showed a global redistribution of Isl1 binding and active enhancers in the absence of Onecut1. These results suggest that Onecut1 plays a role in the maintenance of Isl1-bound enhancers linked to motor neuron genes. Together, my analyses revealed that Onecut1 is associated with the expression of a subset of neuronal genes by binding with Isl1 at motor neuron enhancers.