PROJECT SUMMARY/ABSTRACT
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.