Exploring stem cell pluripotency through long range chromosome interactions

2018-10-16Exploration of the genome and functional elements contained within the DNA sequence continue to bring forth surprising discoveries about nature and dynamics of the nuclear architecture. As many new methods for examining chromatin interactions have emerged in the past decade, there now is a better understanding of the overall spatial configuration of the chromosomes in the interphase cell nucleus and overarching principles of nuclear compartmentalization. In particular, biochemical approaches like chromosome conformation capture (3C) techniques and its variations have been developed further by various research groups to continue deciphering the matrix of interactions in different types of cells. These advances have helped bolster the idea that spatial organization of the chromatin provides an additional layer of regulation within the cell for controlling transcriptional programs. However, limitations inherent to the 3C method have made study of long range interacting chromatin loci difficult. To address the shortcomings encountered in 3C methods, I have developed a novel assay combining the specific locus targeting by CRISPR-Cas9 and the DNA labeling function of the DNA adenine methyltransferase protein utilized in DamID assays. Drawing upon the strengths of both techniques and performing the assay in live cells, CRISPR-Dam is able to better characterize the proximity mediated interaction network at a specified locus of interest. Using the well studied pluripotency gene network in mouse embryonic stem cells as a model system to validate the new assay, we focused on the interactions mediated by the Oct4 distal enhancer (DE). The Oct4 DE is required for Oct4 gene expression and maintenance of stem cell pluripotency. Previously, 4C-seq performed to characterize the Oct4 DE interactome in mouse embryonic stem cells (ESC) was unsuccessful in identifying distally located functional target genes of the DE. Performing the CRISPR-Dam assay at the Oct4 DE in mouse ESCs helped identify two candidate genes, lethal giant larvae 2 (Llgl2) and growth factor receptor-bound protein 7 (Grb7), both located on chromosome 11. Deletion of the Oct4 DE via Cre-loxP recombination showed that Llgl2 and Grb7 are directly regulated by the Oct4 DE and their expression cannot be rescued by ectopic OCT4. We further characterized Llgl2 and Grb7 roles in supporting ESC pluripotency and found that both genes are necessary to maintain ESC pluripotency and overexpression of either gene enhances reprogramming efficiency, suggesting that Llgl2 and Grb7 play functional roles in ESC biology