Determining the role of Nkx2.2 in the maintenance of pancreatic a cell identity
确定 Nkx2.2 在维持胰腺 a 细胞身份中的作用
基本信息
- 批准号:10386038
- 负责人:
- 金额:$ 3.34万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2021
- 资助国家:美国
- 起止时间:2021-09-15 至 2024-09-14
- 项目状态:已结题
- 来源:
- 关键词:AdultAlpha CellApoptosisAreaBeta CellBindingBiological AssayBlood GlucoseBody WeightCell CountCell LineCell MaintenanceCell physiologyCellsCellular biologyChIP-seqCo-ImmunoprecipitationsComplexCoupledD CellsDNADataDevelopmentDiabetes MellitusDiseaseDown-RegulationEndocrineEnhancersEpigenetic ProcessFastingFibrinogenFunctional disorderGene ExpressionGene Expression ProfilingGenesGenetic TranscriptionGlucagonHealthHistonesHypoglycemiaImmunofluorescence ImmunologicImpairmentIslet CellIslets of LangerhansKnock-outKnockout MiceLightMaintenanceMass Spectrum AnalysisMeasurementMeasuresMolecularMonitorMusPathologyPopulationPromoter RegionsRegulationReporterResearchRoleSiteSpecific qualifier valueStructure of alpha Cell of isletTestingTomatoesTreatment Side Effectsbiological systemsblood glucose regulationcell typechromatin immunoprecipitationexperimental studyglucose monitorin vivoinsulin toleranceisletknock-downnegative affectnext generation sequencingnovelnull mutationprogramspromoterprotein expressiontranscription factortranscriptome sequencing
项目摘要
Similar to many other biological systems, maintenance of pancreatic islet cell types is governed by transcription
factor function. NKX2.2 is one such transcription factor that is critical for all stages of islet cell development,
including α and β cells8-12. Mice carrying Nkx2.2 null mutations have impaired differentiation of α and β cells and
die shortly after birth9. In adult β cells, NKX2.2 promotes and actively maintains β cell identity by regulating
several cell specific genes, including repressing the α cell master regulator Arx13. NKX2.2 is also expressed in
the α cell population, but its role in maintaining α cell identity and function is not yet known9. To determine
whether NKX2.2 is also important for α cell identity and function, I generated a constitutive α cell specific
knockout (KO) of Nkx2.2. The preliminary in vivo data shows there is a reduction in α cell expression area that
appears to be accompanied by a loss of α cell identity genes, with a concomitant gain of non-α islet cell genes.
This suggests that in α cells, NKX2.2 is promoting the α cell transcriptional program, while repressing alternate
endocrine cell genes. In preliminary studies to identify NKX2.2 α cell-specific DNA occupancy, an immortalized
α cell line (αTC)14 was used to perform NKX2.2 chromatin immunoprecipitation coupled with next generation
sequencing (ChIP-seq) and qPCR. Interestingly, this analysis identified a novel α cell-specific occupancy site in
the promoter region of Arx – an α gene that is repressed by NKX2.2 in β cells. When combined with data from a
pilot αTC RNA-seq of a Nkx2.2 knockdown in αTC cells that shows significant Arx downregulation, these data
suggest Arx is a direct transcriptional target of NKX2.2 in α cells. Furthermore, comparisons between expression
profiles of α cells versus β or δ cells shows that within α cells, NKX2.2 is binding to significantly more β or δ cell
specific genes than α cell genes. This suggests that a major part of NKX2.2’s role in α cells is repressing alternate
islet transcriptional programs. Lastly, NKX2.2 binds more frequently overall to promoter regions in α cells;
whereas it predominantly occupies intergenic enhancer regions in β cells, suggesting global differences in
NKX2.2 occupancy in α versus β cells13. Together, previous research and preliminary evidence supports the
overarching hypothesis that in α cells, NKX2.2 is important for maintaining α cell identity by promoting α
cell gene transcription and repressing β and δ cell genes. Aim 1 of this proposal assesses the role of NKX2.2
in the maintenance of α cell identity and function using morphometrics, gene expression analysis, and functional
assays in α cell specific Nkx2.2 KO mice. Aim 2 determines the molecular mechanism of NKX2.2 identity
regulation in α cells using Nkx2.2 ChIP-correlated RNA-seq to find direct targets, ChIP-seq to explore the
functional epigenetics of NKX2.2 occupancy, and co-immunoprecipitation coupled with mass spectrometry (Co-
IP-MS) to identify interacting factors. These experiments will further define the role of NKX2.2 in α cell
maintenance and function, and NKX2.2’s molecular mechanism in α cells.
与许多其他生物系统类似,胰岛细胞类型的维持受转录控制
因子函数。 NKX2.2 就是这样一种转录因子,对于胰岛细胞发育的所有阶段都至关重要,
包括α和β细胞8-12。携带 Nkx2.2 无效突变的小鼠的 α 和 β 细胞分化受损,并且
9. 出生后不久就去世了。在成体 β 细胞中,NKX2.2 通过调节
几个细胞特异性基因,包括抑制 α 细胞主调节因子 Arx13。 NKX2.2也表达于
α 细胞群,但其在维持 α 细胞身份和功能中的作用尚不清楚9。确定
NKX2.2对于α细胞身份和功能是否也很重要,我生成了一个组成型α细胞特异性
Nkx2.2 的敲除(KO)。初步体内数据显示 α 细胞表达面积减少,
似乎伴随着α细胞识别基因的丧失,同时伴随着非α胰岛细胞基因的增加。
这表明在 α 细胞中,NKX2.2 促进 α 细胞转录程序,同时抑制交替
内分泌细胞基因。在鉴定 NKX2.2 α 细胞特异性 DNA 占据的初步研究中,一种永生化的
使用α细胞系(αTC)14进行NKX2.2染色质免疫沉淀与下一代结合
测序 (ChIP-seq) 和 qPCR。有趣的是,该分析发现了一个新的 α 细胞特异性占据位点
Arx 的启动子区域——一种 α 基因,在 β 细胞中被 NKX2.2 抑制。当与来自的数据结合时
αTC 细胞中 Nkx2.2 敲低的试点 αTC RNA-seq 显示 Arx 显着下调,这些数据
表明 Arx 是 α 细胞中 NKX2.2 的直接转录靶标。此外,表达之间的比较
α 细胞与 β 或 δ 细胞的概况显示,在 α 细胞内,NKX2.2 与明显更多的 β 或 δ 细胞结合
比α细胞基因更特殊的基因。这表明 NKX2.2 在 α 细胞中的作用的主要部分是抑制交替
胰岛转录程序。最后,NKX2.2 与 α 细胞中的启动子区域的结合更加频繁;
而它主要占据β细胞中的基因间增强子区域,这表明
NKX2.2 在 α 细胞和 β 细胞中的占据情况13。先前的研究和初步证据共同支持了
总体假设是,在 α 细胞中,NKX2.2 通过促进 α 来维持 α 细胞身份非常重要
细胞基因转录并抑制 β 和 δ 细胞基因。该提案的目标 1 评估 NKX2.2 的作用
使用形态计量学、基因表达分析和功能来维持 α 细胞的身份和功能
在 α 细胞特异性 Nkx2.2 KO 小鼠中进行测定。目标 2 确定 NKX2.2 身份的分子机制
使用 Nkx2.2 ChIP 相关的 RNA-seq 寻找直接靶标,ChIP-seq 探索 α 细胞的调控
NKX2.2 占据的功能表观遗传学,以及免疫共沉淀与质谱联用(Co-
IP-MS)来识别相互作用的因素。这些实验将进一步明确NKX2.2在α细胞中的作用
NKX2.2在α细胞中的维护和功能,以及NKX2.2的分子机制。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Elliott Brooks其他文献
Elliott Brooks的其他文献
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{{ truncateString('Elliott Brooks', 18)}}的其他基金
Determining the role of Nkx2.2 in the maintenance of pancreatic a cell identity
确定 Nkx2.2 在维持胰腺 a 细胞身份中的作用
- 批准号:
10683229 - 财政年份:2021
- 资助金额:
$ 3.34万 - 项目类别:
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