Project Summary:
Chromatins are packed into the tiny nucleus in a way that distal enhancers interact with target gene promoters
in 3D space to facilitate the expression of the target genes in a cell type-specific manner, though enhancers
and promoters are separated by several kilobases or sometimes megabases in the linear forms of chromatins.
Thus, understanding of the chromatin interactions is important for us to interpret the transcription regulatory
networks dictating cell identifies and to reverse or convert cell fate through transcription manipulations.
However, millions of cells are required to profile genome-wide chromatin interactions by conventional proximity
ligation-based chromatin conformation capture methods, making it difficult to interrogate chromatin interactions
in sensory hair cells of the organ of Corti. The lack of enhancer-promoter interaction information in the hair
cells restrains our understanding of the regulatory networks governing hair cell-specific gene expression and
hinders the identification of potential targets for hair cell regeneration through expression manipulation. To
address this need, we are proposing to investigate enhancer-promoter interactions in sensory hair cells
utilizing the “cut-and-paste” transposition activity of Tn5 transposase (Tn5-Capture). The feasibility of Tn5-
Catpure to detect enhancer-promoter interactions has been demonstrated by our preliminary study, we will
start enhancer-promoter interaction analysis for a few essential transcription factor genes using Tn5-Capture
followed by qPCR, and then expand the analysis to genome-wide scale by combining Tn5-Capture and Next
Generation Sequencing. Finally, with the CRISPRi (CRISPR-dCAS9-EZH2) system, we will validate the
regulatory functions of selected enhancer-promoter interactions for already known and newly discovered
enhancer-promoter interactions. Through this project, we will collect datasets of enhancer-promoter
interactions in hair cells, which can be integrated with available transcriptomic and epigenetic data to establish
hair cell regulatory networks governing hair cell-specific gene expression and to identify potential targets to
stimulate hair cell regeneration. Implementation of the CRISPRi technology in the inner ear system also
provide a powerful tool in the context of therapeutical treatment of deafness. Besides, the Tn5-Capture
method, a new chromatin interaction interrogating technique, will have broad applications in research outside
of the inner ear system.
