The formation of improper 3-dimensional (3D) chromatin structures and states can lead to many types of human disease. 3D epigenomic datasets were generated in many different cell-types, using genome-wide chromosome conformation capture-derivative techniques (e.g. Hi-C) and chromatin immunoprecipitation assays with sequencing (ChIP-seq). However, there are many unanswered questions about the role of chromatin interactions in cell-type specific gene regulation. Genomic regions that physically interact with each other with high frequency are called topologically associating domains (TADs). Our preliminary results found common TADs that share boundaries among cell types (aka invariant TADs). Interestingly, a subset of the TADs is heavily enriched with histone modifications, suggesting that the size of the TADs may be tightly associated with epigenetic states. We identified hundreds of H3K27me3-enriched (repressed), H3K9me3- enriched (heterochromatic) and H3K36me3-enriched (active) TADs in multiple human cell lines and primary cells, and we also found common TADs that changed epigenetic states among cell types. To elucidate the epigenomic mechanisms by which TADs are cell-type specific or invariant, and to develop tools that can alter TADs, we propose to use this two-pronged approach.
In Aim 1, we will identify common TADs that have different chromatin states among cell types, using 3D epigenome and transcriptome data generated in >50 cell types by large consortia (e.g. Roadmap of Epigenomics, Encyclopedia of DNA Elements, PsychENCODE, 4DNucleome) and other epigenomic studies. We will classify large-scale structural features (TADs) into invariant or cell type-specific TADs, comparing the size of TADs. We will also identify epigenetic states of TADs in different cell and tissue types, integrating Hi-C, ChIP-seq, and RNA-seq datasets. In the process of carrying out this aim, we will develop databases and bioinformatics tools that facilitate researchers to identify epigenetic states of common TADs.
In Aim 2, we will develop technologies to alter epigenetic states of the TADs using targeted epigenome editing. As preliminary data, we have selected candidate common TADs that are enriched with histone marks and have changed chromatin states between normal and prostate cancer. We also demonstrated that targeted epigenetic editing using the CRISPRi system enabled long-term repression of target genes. Using CRISPRi and gRNAs targeting boundaries of the H3K36me3-enriched TADs and active regulatory elements within the TADs, we will edit the epigenetic states of the selected TADs. Moreover, we will test if changing epigenetic states can alter chromatin structures or expression levels of multiple genes that are located in the TADs, using ChIP-seq, capture Hi-C and RNA-seq. We designed each of the aims can be performed, independently of the others. Our proposed studies will not only provide new insights into transcriptional regulation in 3D human epigenome but also further the development of therapeutic tools for targeted epigenome editing.
Advances in genomics research allow us to map 3-dimensional epigenome and to identify chromatin structures and epigenetic features that lead to human diseases. This proposal addresses an unmet medical need by characterizing variation of large-scale structural and epigenetic features, integrating next generation sequencing datasets in different cell types and developing new tools for targeted epigenome editing. The findings of this study will elucidate novel mechanisms of cell-type specificity and provide new directions to improve human health.