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Regulatory Mechanism of Peptidylarginine Deiminase Genes Expressed in the Human Epidermal Cells

人表皮细胞表达的肽基精氨酸脱亚胺酶基因的调控机制

基本信息

批准号：
18591265
负责人：
KAWADA Akira
金额：
$ 2.57万
依托单位：
Kinki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18591265/
关键词：
peptidylarginine deiminase human keratinocytes gene regulation PADI3 PADI1 Sp1 Sp3 NF-Y MZF1 peptidylarginine deiminas TATA-box Peptidylarginine deiminase 転写制御基本転写因子

项目摘要

Human peptidylarginine deiminase type gene (PADI) encodes a crucial post-translational modification enzyme that converts protein-bound arginine residues to citrulline residues. Its expression is restricted to a few cell types, including keratinocytes in the granular layer of the epidermis and in the inner root sheath of hair follicles. In these cells, the enzyme is involved in terminal processing of intermediate filament-binding proteins such as filaggrin and trichohyalin. To study the molecular mechanisms that control the expression of PADI3 in human keratinocytes at the transcriptional level, we characterized its promoter region using human keratinocytes transfected with variously deleted fragments of the 5'-upstream region of PADI3 coupled to the luciferase gene. We found that as few as 129 bps upstream from the transcription initiation site were sufficient to direct transcription of the reporter-gene. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed … More that NF-Y and Sp l/Sp3 bind to this region in vitro and in vivo.Moreover, mutation of the Sp1 or NF-Y binding motif markedly reduced PADI3 promoter activity. Furthermore, Sp1 or NF-YA (NF-Y subunit) small interfering RNAs effectively diminished PADI3 expression both in low and high calcium medium cultured keratinocytes. These data indicate that PADI3 expression is driven by Sp1/Sp3 and NF-Y binding to the promoter region. To study the molecular mechanisms that control the expression of PADI1 in keratinocytes at the transcriptional level, we cloned and characterized its promoter region using human keratinocytes transfected with variously deleted fragments of the 5'-upstream region of PADI1 coupled to the luciferase gene. We found that as few as 195 by upstream from the transcription initiation site was sufficient to direct transcription of the reporter gene.Mutations of MZF1 or Sp1-binding sites markedly reduced PADI1 promoter activity. Chromatin immunoprecipitation assays revealed that MZF1 and Sp1/Sp3 bind to this region in vivo. Furthermore, MZF1 or Sp1 small interfering RNAs effectively diminished PADI1 expression in keratinocytes cultured in both low- and high-calcium containing medium. These data indicate that expression of PADI1 is driven by MZF1 and Sp1/Sp3 binding to its promoter region. Less

人肽基精氨酸脱亚胺酶基因（PADI）编码一种重要的翻译后修饰酶，可将蛋白质结合的精氨酸残基转化为瓜氨酸残基。其表达仅限于少数细胞类型，包括表皮颗粒层和毛囊内根鞘中的角质形成细胞。在这些细胞中，该酶参与中间体结合蛋白如聚丝蛋白和透明质酸的末端加工。为了研究在转录水平上控制PADI3在人角质形成细胞中表达的分子机制，我们使用与荧光素酶基因偶联的PADI3的5 '上游区的各种缺失片段转染的人角质形成细胞来表征其启动子区。我们发现，从转录起始位点上游129个碱基对就足以指导转录因子基因的转录。电泳迁移率变化和染色质免疫沉淀试验显示， ...更多信息在体外和体内实验中，NF-Y和Sp1/Sp3与该区域结合，并且Sp1或NF-Y结合基序的突变显著降低了PADI3启动子活性。此外，Sp1或NF-YA（NF-Y亚基）小干扰RNA有效地降低了低钙和高钙培养基培养的角质形成细胞中的PADI3表达。这些数据表明，PADI3表达是由Sp1/Sp3和NF-Y结合到启动子区驱动的。为了研究在转录水平上控制PADI 1在角质形成细胞中表达的分子机制，我们使用与荧光素酶基因偶联的PADI 1的5 '上游区的各种缺失片段转染的人角质形成细胞克隆并表征其启动子区。我们发现，只要从转录起始位点上游195个碱基就足以指导报告基因的转录。MZF 1或Sp1结合位点的突变显著降低了PADI 1启动子的活性。染色质免疫沉淀分析表明，MZF1和Sp1/Sp3结合到这个区域在体内。此外，MZF1或Sp1小干扰RNA有效地降低了在低钙和高钙培养基中培养的角质形成细胞中的PADI1表达。这些数据表明，PADI1的表达是由MZF 1和Sp1/Sp3结合其启动子区驱动的。少