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

人肽精氨酸脱亚胺酶型基因（PADI）编码一种重要的翻译后修饰酶，将蛋白质结合的精氨酸残基转化为瓜氨酸残基。它的表达仅限于几种细胞类型，包括表皮颗粒层的角质形成细胞和毛囊内根鞘的角质形成细胞。在这些细胞中，该酶参与中间丝状结合蛋白如聚丝蛋白和毛透明蛋白的末端加工。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，我们使用转染了PADI3与荧光素酶基因偶联的5'-上游区域的各种缺失片段的人角质形成细胞来表征其启动子区域。我们发现，只要在转录起始位点上游129个bps处，就足以指导报告基因的转录。电泳迁移率和染色质免疫沉淀实验表明，NF-Y和Sp 1 /Sp3在体外和体内均与该区域结合。此外，Sp1或NF-Y结合基序的突变显著降低了PADI3启动子的活性。此外，Sp1或NF-YA （NF-Y亚基）小干扰rna在低钙和高钙培养基培养的角质形成细胞中都能有效地降低PADI3的表达。这些数据表明，PADI3的表达是由Sp1/Sp3和NF-Y结合到启动子区域驱动的。为了研究在转录水平上控制PADI1在角质形成细胞中表达的分子机制，我们克隆并表征了其启动子区域，使用转染了PADI1与荧光素酶基因偶联的5'-上游区域的各种缺失片段的人角质形成细胞。我们发现，转录起始位点上游只要195个就足以直接转录报告基因。MZF1或sp1结合位点的突变显著降低了PADI1启动子活性。染色质免疫沉淀实验显示MZF1和Sp1/Sp3在体内与该区域结合。此外，MZF1或Sp1小干扰rna可有效降低在低钙和高钙培养基中培养的角化细胞中PADI1的表达。这些数据表明PADI1的表达是由MZF1和Sp1/Sp3结合到其启动子区域驱动的。少

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