Dissecting the sensory hair cell nucleus: Development of a novel method for investigating chromatin interactions (Tn5-Capture) in small cell numbers
Dissecting the sensory hair cell nucleus: Development of a novel method for investigating chromatin interactions (Tn5-Capture) in small cell numbers
批准号:
10571130
负责人:
Litao Tao
金额:
$18.19万
依托单位:
依托单位国家:
美国
项目类别:
财政年份:
2022
资助国家:
美国
项目状态:
已结题
起止时间:
2022-12-01 至 2024-11-30
关键词:
3-DimensionalAddressCRISPR interferenceCell CountCell Differentiation processCell MaturationCell NucleusCellsChromatinClustered Regularly Interspaced Short Palindromic RepeatsCochleaCoinDataData SetDevelopmentDiseaseDistalEZH2 geneEnhancersEpigenetic ProcessGFI1 geneGene ExpressionGenesGenetic TranscriptionGuide RNAHair CellsInvestigationK562 CellsKnowledgeLabyrinthLigationMediatingMethodsModificationOrganOrgan of CortiPaste substancePersonsProtocols documentationQuality ControlRegenerative MedicineResearchResolutionSensory HairSystemTechniquesTechnologyTestingTherapeuticTissuesTn5 transposaseTranscriptional RegulationUnited StatesValidationcell typechromosome conformation capturedeafnessgene regulatory networkgenetic manipulationgenome-widegenome-wide analysishair cell regenerationhearing loss treatmentnext generation sequencingnovelpermanent hearing losspromoterrestrainttooltranscription factortranscription regulatory networktranscriptometranscriptomics
中文摘要
项目概要:
染色质以远端增强子与靶基因启动子相互作用的方式被包装到微小的细胞核中
以促进靶基因以细胞类型特异性方式表达,尽管增强子
并且启动子被几种酶或有时是染色质线性形式的大酶分开。
因此,了解染色质相互作用对于我们解释转录调控机制是非常重要的。
控制细胞身份的网络,并通过转录操作逆转或转换细胞命运。
然而,需要数百万个细胞才能通过常规的邻近分析来分析全基因组染色质相互作用。
基于连接的染色质构象捕获方法,使得难以询问染色质相互作用
在Corti器的感觉毛细胞中。毛发中缺乏增强子-启动子相互作用信息
细胞限制了我们对毛细胞特异性基因表达调控网络的理解,
阻碍了通过表达操作鉴定毛细胞再生的潜在靶标。到
为了满足这一需求,我们建议研究感觉毛细胞中增强子-启动子的相互作用
利用Tn 5转座酶(Tn 5-Capture)的“剪切-粘贴”转座活性。Tn 5的可行性
Catpure检测增强子-启动子相互作用已被我们的初步研究所证明,我们将
应用Tn 5-Capture分析几种重要转录因子基因启动子-增强子相互作用
随后进行qPCR,然后通过结合Tn 5-Capture和Next将分析扩展到全基因组范围
世代排序。最后,利用CRISPRi(CRISPR-dCAS 9-EZH 2)系统,我们将验证
选择的增强子-启动子相互作用对已知和新发现的
增强子-启动子相互作用。通过这个项目,我们将收集增强子-启动子的数据集,
毛细胞中的相互作用,这可以与可用的转录组学和表观遗传学数据相结合,以建立
毛细胞调控网络管理毛细胞特异性基因表达,并确定潜在的目标,
刺激毛细胞再生。在内耳系统中实施CRISPRi技术还
为耳聋的治疗提供了有力的工具。此外,Tn 5-Capture
方法作为一种新的染色质相互作用的检测技术,在国外的研究中有着广泛的应用前景
内耳系统。
英文摘要
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.
期刊论文(0)
专著(0)
科研奖励(0)
会议论文
Pou4f3 enhancers in hearing loss treatment
-
批准号:10662584
-
项目类别:
-
资助金额:$30.03万
-
财政年份:2022
-
负责人:Litao Tao
-
依托单位:
Pou4f3 enhancers in hearing loss treatment
-
批准号:10663166
-
项目类别:
-
资助金额:$25.58万
-
财政年份:2021
-
负责人:Litao Tao
-
依托单位:
海外基金