NF-Y and Sp1/Sp3 are involved in the transcriptional regulation of the peptidylarginine deiminase type III gene (PADI3) in human keratinocytes
NF-Y and Sp1/Sp3 are involved in the transcriptional regulation of the peptidylarginine deiminase type III gene (PADI3) in human keratinocytes
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DOI：
10.1042/bj20051939
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发表时间：
2006-08-01
影响因子：
4.1
通讯作者：
Takahara, Hidenari
中科院分区：
文献类型：
--
作者：
Dong, Sijun;Kanno, Takuya;Takahara, Hidenari
人肽精氨酸脱亚胺酶III型基因（PADI3）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129bp就足以直接转录报告基因。电泳迁移位移和染色质免疫沉淀实验表明，NF-Y（核因子Y）和Sp1/Sp3（特异性蛋白1/3）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可有效降低在低钙和高钙培养基中培养的角质形成细胞中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。
Human peptidylarginine deiminase type III gene (PADI3) encodes a crucial post-translational modification enzyme that converts protein-bound arginine residues into 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 bp upstream from the transcription initiation site were sufficient to direct transcription of the reporter gene. Electrophoretic mobility-shift and chromatin immunoprecipitation assays revealed that NF-Y (nuclear factor Y) and Sp1/Sp3 (specificity protein 1/3) 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 in keratinocytes cultured in both low- and high-calcium medium. These data indicate that PADI3 expression is driven by Sp1/Sp3 and NF-Y binding to the promoter region.