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Dissecting the sensory hair cell nucleus: Development of a novel method for investigating chromatin interactions (Tn5-Capture) in small cell numbers

Award Number: R21DC020773
ORGANIZATION: NATIONAL INSTITUTE ON DEAFNESS AND OTHER COMMUNICATION DISORDERS
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 12/01/2022
PERIOD OF PERFORMANCE END DATE: 05/31/2025

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Dissecting the sensory hair cell nucleus: Development of a novel method for investigating chromatin interactions (Tn5-Capture) in small cell numbers - 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.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2025 ( Subtotal = -$123 )
2025	2024	CREIGHTON UNIVERSITY	2500 CALIFORNIA PLAZA	OMAHA	NE	68178	DOUGLAS	USA	Research Related to Deafness and Communication Disorders	000	2	9/24/2025	NON-COMPETING CONTINUATION	-$123
														Subtotal = -$123

Issue Date FY: 2024 ( Subtotal = $181,875 )
2024	2024	CREIGHTON UNIVERSITY	2500 CALIFORNIA PLAZA	OMAHA	NE	68178	DOUGLAS	USA	Research Related to Deafness and Communication Disorders	001	2	5/2/2024	NON-COMPETING CONTINUATION	$18,187
2024	2024	CREIGHTON UNIVERSITY	2500 CALIFORNIA PLAZA	OMAHA	NE	68178	DOUGLAS	USA	Research Related to Deafness and Communication Disorders	000	2	11/16/2023	NON-COMPETING CONTINUATION	$163,688
														Subtotal = $181,875

Issue Date FY: 2023 ( Subtotal = $181,875 )
2023	2023	CREIGHTON UNIVERSITY	2500 CALIFORNIA PLAZA	OMAHA	NE	68178	DOUGLAS	USA	Research Related to Deafness and Communication Disorders	000	1	10/21/2022	NEW	$181,875
														Subtotal = $181,875

Grand Total All Awards = $363,627

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Dissecting the sensory hair cell nucleus: Development of a novel method for investigating chromatin interactions (Tn5-Capture) in small cell numbers

Award Number: R21DC020773

ORGANIZATION: NATIONAL INSTITUTE ON DEAFNESS AND OTHER COMMUNICATION DISORDERS

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 12/01/2022

PERIOD OF PERFORMANCE END DATE: 05/31/2025

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