Effect of CTCF-binding motif on regulation of PAX6 transcription.
OBJECTIVE
Previous studies indicate that the CCCTC binding transcription factor (CTCF) regulates homeobox PAX6 gene transcription in corneal epithelial cells. In the present study, the effect was investigated of CTCF activity on PAX6 transcription through interaction with five essential motifs located in an 80-bp region upstream from the PAX6 P0 promoter.


METHODS
An electrophoretic mobility shift assay (EMSA) was used to determine the interaction between CTCF and DNA binding motifs. DNA mutagenesis was applied in identification of DNA motif functions. Immunohistochemistry and Western blot analyses were performed to detect the stress-induced effect on CTCF activity.


RESULTS
The five identified CTCF-binding motifs were mutated one by one or in different combinations. Interactions of CTCF with these mutated motifs were determined by EMSA and DNA-binding competitions. All five CCCTC motifs were functional for the CTCF binding and DNA-binding activity of CTCF was proportionally decreased after increases in mutations of motif numbers. In addition, ultraviolet (UV) irradiation and epidermal growth factor (EGF) induced suppression and activation of CTCF expression, respectively. Effects of UV and EGF induction were due to alterations in CTCF expression and activity resulting in changes in CTCF DNA binding activity to the PAX6 promoter region detected by EMSA.


CONCLUSIONS
These findings indicate that CTCF regulates PAX6 expression in response to stress-induced conditions and that the molecular base of CTCF controlling PAX6 expression is through five functional and specific motifs in the region upstream from the PAX6 P0 promoter in corneal epithelial cells.
Wu
Dan
D
Health Science Center, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, People's Republic of China.
Li
Tie
T
Lu
Zhenyu
Z
Dai
Wei
W
Xu
Ming
M
Lu
Luo
L
D000818:Animals; D015153:Blotting, Western; D000076246:CCCTC-Binding Factor; D002417:Cattle; D018929:Cell Culture Techniques; D004268:DNA-Binding Proteins / Q000502:physiology*; D024202:Electrophoretic Mobility Shift Assay; D004728:Endothelium, Corneal / Q000378:metabolism* / Q000528:radiation effects; D019573:Epithelium, Corneal / Q000378:metabolism* / Q000528:radiation effects; D005136:Eye Proteins / Q000235:genetics*; D005786:Gene Expression Regulation; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D016297:Mutagenesis, Site-Directed; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D012097:Repressor Proteins / Q000235:genetics* / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014162:Transfection; D014466:Ultraviolet Rays; D016335:Zinc Fingers / Q000502:physiology*
D000076246:CCCTC-Binding Factor; C000619697:CTCF protein, human; D004268:DNA-Binding Proteins; D005136:Eye Proteins; D018398:Homeodomain Proteins; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D051761:Paired Box Transcription Factors; D012333:RNA, Messenger; D012097:Repressor Proteins