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

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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