Premium
Characterization of Ets‐binding sequence of human transglutaminase 3 gene promoter
Author(s) -
Kim Chang Deok,
Seo EunYoung,
Sung YeulHoon,
Kim HyunWoong,
Seo YoungJoon,
Park JangKyu,
Lee JeungHoon
Publication year - 2004
Publication title -
experimental dermatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.108
H-Index - 96
eISSN - 1600-0625
pISSN - 0906-6705
DOI - 10.1111/j.0906-6705.2004.00188.x
Subject(s) - electrophoretic mobility shift assay , reporter gene , microbiology and biotechnology , sequence motif , gene , transfection , promoter , biology , gene expression , regulatory sequence , consensus sequence , binding site , oligonucleotide , transcription (linguistics) , genetics , peptide sequence , linguistics , philosophy
The Ets‐ and Sp1‐transcription factors have been implicated in the epithelial specific expression of transglutaminase 3 gene (TGM3) in vitro . By electrophoretic mobility shift assay (EMSA), the core motif of Ets‐binding sequence of TGM3 was determined as ACAGGAAT (−118 to −111 bp from transcriptional start site). However, a sequence extending from −120 to +10 of TGM3 proximal promoter region failed to induce the expression of CAT reporter in transfected normal human epidermal keratinocytes (NHEKs). In contrast, a construct extending from −126 to +10 bp showed the highest expression of CAT gene, indicating the presence of an important element(s) between −126 and −120 bp that affects TGM3 expression in NHEKs. To find the critical sequences in this promoter region, we performed EMSA analysis with competitor oligonucleotides, in which the upstream nucleotides of Ets core motif were mutated. The results showed that the mutation of A at −125 dramatically reduced the binding of Ets to its recognition sequence. Transient transfection assay revealed that the mutation in this nucleotide greatly diminished the expression of CAT reporter gene in NHEKs. Together, these results suggest that the upstream sequence of Ets core motif is critical for the expression of TGM3 in NHEKs cultured in vitro .