Role of Sp1 in Transcription of Human ATP2A2 Gene in Keratinocytes

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Role of Sp1 in Transcription of Human ATP2A2 Gene in Keratinocytes Atsushi Takagi, Chiharu Nishiyama, Keiko Maeda, Tomoko Tokura, Hiroshi Kawada, Shunsuke Kanada, Yusuke Niwa, Nobuhiro Nakano, Nobuyasu Mayuzumi, Makoto Nishiyama, Shigaku Ikeda, Ko Okumura, Hideoki Ogawa Journal of Investigative Dermatology Volume 128, Issue 1, Pages 96-103 (January 2008) DOI: 10.1038/sj.jid.5700937 Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Identification of transcription factor binding to cis-enhancing elements in human ATP2A2 promoter by EMSA. EMSA was performed with FITC-labeled probe corresponding to (a) −550/−528 or (b) –488/−465 with nuclear extract from NHK. Journal of Investigative Dermatology 2008 128, 96-103DOI: (10.1038/sj.jid.5700937) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Sp1 binds to human ATP2A2 promoter region in vivo. Binding between the human ATP2A2 promoter (a) or cis control region of ATP2A2 gene (b) and Sp1 or Sp3 was analyzed by ChIP assay using anti-Sp1, anti-Sp3, or isotype control Ab (mouse IgG1 and mouse IgG2a as controls for anti-Sp1 and anti-Sp3, respectively). Binding was quantitatively analyzed by real-time PCR. Relative input units are calculated from cycle threshold values as described in “Materials and Methods”. Results are expressed as means±SD of triplicate samples. Representative results of three independent experiments are shown. Journal of Investigative Dermatology 2008 128, 96-103DOI: (10.1038/sj.jid.5700937) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Knockdown of Sp1 expression by siRNA results in downregulation of ATP2A2 promoter activity. (a) Fluorescence microscopy of FITC-labeled control oligonucleotide-introduced keratinocytes. After 24hours culture from transfection of oligonucleotides, cells were washed and visualized by phase microscopy to visualize cells (left) and by fluorescence microscopy using a green fluorescent protein filter (right). Bar=100μm. (b) Sp1 mRNA level in Sp1 siRNA-treated keratinocytes analyzed by real-time PCR. (c) Sp1 protein level in Sp1 siRNA-treated keratinocytes analyzed by Western blotting. (d) Transcription activity of human. ATP2A2 promoter in Sp1 siRNA-introduced keratinocytes. The ratio of luciferase activity in each transfectant to that in cells transfected with pGL3-Basic and control siRNA is given as relative activity. (e) ATP2A2 mRNA level in Sp1 siRNA-introduced keratinocytes analyzed by real-time PCR. Journal of Investigative Dermatology 2008 128, 96-103DOI: (10.1038/sj.jid.5700937) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Nucleotide sequence of the 5′-flanking region of the human ATP2A2 gene. The transcription initiation site is indicated as +1, based on the 5′-end of the human ATP2A2 mRNA sequence (accession number NM_170665). Possible transcription factor binding sites found in cis-enhancing elements identified by the publicly available MOTIF search services are underlined, and Sp1-binding sites confirmed by EMSA are boxed. This genomic region corresponds to 109203135/109203873 of chromosome 12 published in NCBI human genomic DNA sequence site. Three regions underlined with dotted black line are identified to be highly conserved sequences by UCSC (chromosome 12: 109203326–109203493, lod=178; chromosome 12: 109203225–109203285, lod=41; chromosome 12: 109203813–109203827, lod=28). Journal of Investigative Dermatology 2008 128, 96-103DOI: (10.1038/sj.jid.5700937) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions