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Azra Memon,Yuliya Pyao,Yerin Jung,Hwa-Sik Choi,송기덕,이운규 한국유전학회 2021 Genes & Genomics Vol.43 No.4
Background Krüppel–like factor 10 (KLF10) belongs to the Sp1-like transcription factor family, which plays an important role in many directions, e.g., cell proliferation, apoptosis, and diferentiation. Its 5′ upstream regions are conserved across mammalian species. However, the regulatory mechanism has not been elucidated yet. Objective Nonetheless the basal transcriptional regulation mechanisms of these regions are unknown. Here, we characterized it which is indispensable for the basal transcription of the Klf10 gene. Methods Seven deletions of 5′ upstream DNA fragments from the 10 kb mKlf10 genomic DNA were produced by PCR and cloned into the upstream of the luciferase (Luc) reporter gene in the pGL3 basic plasmid. Result The luciferase reporter assay showed that the DNA sequence at positions from −101 to +68 was required for a principle activity in the promoter of mKlf10 gene, in which transcriptional factor binding motifs, one JunB and two Sp1 sites, are included. Mutations at the sequence of JunB motif, but not at the two Sp1, abrogated the promoter activity completely, suggesting the indispensable role of JunB site for basal transcription of mKlf10 gene. Moreover, electrophoretic mobility and supershift assays (EMSA) uncovered that JunB protein bound to this region specifcally. Conclusion Taken together, our study revealed that the JunB but not Sp1 at mKlf10 promoter functions as a positive basic factor for the transcriptional activity of the gene.
Characterization of the Nanog 5'-flanking Region in Bovine
Choi, Don-Ho,Kim, Duk-Jung,Song, Ki-Duk,Park, Hwan-Hee,Ko, Tae Hyun,Pyao, Yuliya,Chung, Ku-Min,Cha, Seok Ho,Sin, Young-Su,Kim, Nam-Hyung,Lee, Woon-Kyu Asian Australasian Association of Animal Productio 2016 Animal Bioscience Vol.29 No.10
Bovine embryonic stem cells have potential for use in research, such as transgenic cattle generation and the study of developmental gene regulation. The Nanog may play a critical role in maintenance of the undifferentiated state of embryonic stem cells in the bovine, as in murine and human. Nevertheless, efforts to study the bovine Nanog for pluripotency-maintaining factors have been insufficient. In this study, in order to understand the mechanisms of transcriptional regulation of the bovine Nanog, the 5'-flanking region of the Nanog was isolated from ear cells of Hanwoo. Results of transient transfection using a luciferase reporter gene under the control of serially deleted 5'-flanking sequences revealed that the -134 to -19 region contained the positive regulatory sequences for the transcription of the bovine Nanog. Results from mutagenesis studies demonstrated that the Sp1-binding site that is located in the proximal promoter region plays an important role in transcriptional activity of the bovine Nanog promoter. The electrophoretic mobility shift assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. In addition, significant inhibition of Nanog promoter activity by the Sp1 mutant was observed in murine embryonic stem cells. Furthermore, chromatin-immunoprecipitation assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. These results suggest that Sp1 is an essential regulatory factor for bovine Nanog transcriptional activity.