Characterization of Ets-binding sequence of human transglutaminase 3 gene promoter.
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.
Kim
Chang Deok
CD
Department of Dermatology, School of Medicine, Chungnam National University, 640 Daesa-dong, Daejeon, 301-040, South Korea.
Seo
Eun-Young
EY
Sung
Yeul-Hoon
YH
Kim
Hyun-Woong
HW
Seo
Young-Joon
YJ
Park
Jang-Kyu
JK
Lee
Jeung-Hoon
JH
D001665:Binding Sites / Q000235:genetics; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D007668:Kidney / Q000166:cytology; D016296:Mutagenesis; D011401:Promoter Regions, Genetic / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D011503:Transglutaminases / Q000235:genetics* / Q000378:metabolism
D002135:Calcium-Binding Proteins; D014157:Transcription Factors; C101003:ELF2 protein, human; C498712:TGM3 protein, human; D011503:Transglutaminases