Single Cell Analysis of Chromatin Accessibility

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

Functional diversity of CTCFs is encoded in their binding motifs

CTCF ChIP-seq data. Cell lines and statistics for the ChIP-seq data used in the study. (DOCX 55 kb

Transcriptionally active HERV-H retrotransposons demarcate topologically associating domains in human pluripotent stem cells.

Chromatin architecture has been implicated in cell type-specific gene regulatory programs, yet how chromatin remodels during development remains to be fully elucidated. Here, by interrogating chromatin reorganization during human pluripotent stem cell (hPSC) differentiation, we discover a role for the primate-specific endogenous retrotransposon human endogenous retrovirus subfamily H (HERV-H) in creating topologically associating domains (TADs) in hPSCs. Deleting these HERV-H elements eliminates their corresponding TAD boundaries and reduces the transcription of upstream genes, while de novo insertion of HERV-H elements can introduce new TAD boundaries. The ability of HERV-H to create TAD boundaries depends on high transcription, as transcriptional repression of HERV-H elements prevents the formation of boundaries. This ability is not limited to hPSCs, as these actively transcribed HERV-H elements and their corresponding TAD boundaries also appear in pluripotent stem cells from other hominids but not in more distantly related species lacking HERV-H elements. Overall, our results provide direct evidence for retrotransposons in actively shaping cell type- and species-specific chromatin architecture

Joint profiling of DNA methylation and chromatin architecture in single cells.

We report a molecular assay, Methyl-HiC, that can simultaneously capture the chromosome conformation and DNA methylome in a cell. Methyl-HiC reveals coordinated DNA methylation status between distal genomic segments that are in spatial proximity in the nucleus, and delineates heterogeneity of both the chromatin architecture and DNA methylome in a mixed population. It enables simultaneous characterization of cell-type-specific chromatin organization and epigenome in complex tissues

Spatiotemporal DNA methylome dynamics of the developing mouse fetus

Cytosine DNA methylation is essential for mammalian development but understanding of its spatiotemporal distribution in the developing embryo remains limited. Here, as part of the mouse Encyclopedia of DNA Elements (ENCODE) project, we profiled 168 methylomes from 12 mouse tissues or organs at 9 developmental stages from embryogenesis to adulthood. We identified 1,808,810 genomic regions that showed variations in CG methylation by comparing the methylomes of different tissues or organs from different developmental stages. These DNA elements predominantly lose CG methylation during fetal development, whereas the trend is reversed after birth. During late stages of fetal development, non-CG methylation accumulated within the bodies of key developmental transcription factor genes, coinciding with their transcriptional repression. Integration of genome-wide DNA methylation, histone modification and chromatin accessibility data enabled us to predict 461,141 putative developmental tissue-specific enhancers, the human orthologues of which were enriched for disease-associated genetic variants. These spatiotemporal epigenome maps provide a resource for studies of gene regulation during tissue or organ progression, and a starting point for investigating regulatory elements that are involved in human developmental disorders

Spatiotemporal DNA methylome dynamics of the developing mouse fetus

Cytosine DNA methylation is essential for mammalian development but understanding of its spatiotemporal distribution in the developing embryo remains limited. Here, as part of the mouse Encyclopedia of DNA Elements (ENCODE) project, we profiled 168 methylomes from 12 mouse tissues or organs at 9 developmental stages from embryogenesis to adulthood. We identified 1,808,810 genomic regions that showed variations in CG methylation by comparing the methylomes of different tissues or organs from different developmental stages. These DNA elements predominantly lose CG methylation during fetal development, whereas the trend is reversed after birth. During late stages of fetal development, non-CG methylation accumulated within the bodies of key developmental transcription factor genes, coinciding with their transcriptional repression. Integration of genome-wide DNA methylation, histone modification and chromatin accessibility data enabled us to predict 461,141 putative developmental tissue-specific enhancers, the human orthologues of which were enriched for disease-associated genetic variants. These spatiotemporal epigenome maps provide a resource for studies of gene regulation during tissue or organ progression, and a starting point for investigating regulatory elements that are involved in human developmental disorders

A transcriptomic and epigenomic cell atlas of the mouse primary motor cortex.

Single-cell transcriptomics can provide quantitative molecular signatures for large, unbiased samples of the diverse cell types in the brain1-3. With the proliferation of multi-omics datasets, a major challenge is to validate and integrate results into a biological understanding of cell-type organization. Here we generated transcriptomes and epigenomes from more than 500,000 individual cells in the mouse primary motor cortex, a structure that has an evolutionarily conserved role in locomotion. We developed computational and statistical methods to integrate multimodal data and quantitatively validate cell-type reproducibility. The resulting reference atlas-containing over 56 neuronal cell types that are highly replicable across analysis methods, sequencing technologies and modalities-is a comprehensive molecular and genomic account of the diverse neuronal and non-neuronal cell types in the mouse primary motor cortex. The atlas includes a population of excitatory neurons that resemble pyramidal cells in layer 4 in other cortical regions4. We further discovered thousands of concordant marker genes and gene regulatory elements for these cell types. Our results highlight the complex molecular regulation of cell types in the brain and will directly enable the design of reagents to target specific cell types in the mouse primary motor cortex for functional analysis