UC San Diego

The identity of each cell in the human body is established and maintained through distinct gene expression program, which is regulated in part by the chromatin accessibility. Until recently, our understanding of chromatin accessibility has depended largely upon bulk measurements in populations of cells. Recent advances in the sequencing techniques have allowed for the identification of open chromatin regions in single cells. During my Ph.D., I have developed and used single cell sequencing techniques to study the diverse gene regulatory programs underlie the different cell types in mammalian complex tissues. In chapter 1, colleague and I developed Single Nucleus Assay of Transpose Accessible Chromatin using Sequencing (snATAC-seq), a combinatorial barcoding-assisted single-cell assay for probing accessible chromatin in single cells. We then used snATAC-seq to generate an epigenomic atlas of early developing mouse brain. The high-level noise of each single cell chromatin accessibility profile and the large volume of the datasets pose unique computational challenges. In chapter 2, I developed a comprehensive bioinformatics software package called SnapATAC for analyzing large-scale single cell ATAC-seq dataset. SnapATAC resolves the heterogeneity in complex tissues and maps the trajectories of cellular states. As a demonstration of its utility, SnapATAC was applied to 55,592 single-nucleus ATAC-seq profiles from the mouse secondary motor cortex. To further determine the target genes of the distal regulatory elements identified using snATAC-seq in different cell types, in chapter 3, colleague and I developed PLAC-seq, a cost-efficient method that identifies the long-range chromatin interaction at kilobase resolution. PLAC-seq improves the efficiency of detecting chromatin conformation by over 10-fold and reduces the input requirement by nearly 100-fold compared to the prior techniques. Finally, to probe the in vivo function of the regulatory sequences, I present a high-throughput CRISPR screening method (CREST-seq) for the unbiased discovery and functional assessment of enhancer sequences in the human genome. We used it to interrogate the 2-Mb POU5F1 locus in human embryonic stem cells and discovered that sequences previously annotated as promoters of functionally unrelated genes can regulate the expression of POU5F1 from a long distance. We anticipate that these studies will help us understand the gene regulatory programs across diverse biological systems ranging from human disease to the evolution of species.