Drosophila Mi-2 negatively regulates dDREF by inhibiting its DNA-binding activity.
Drosophila melanogaster DNA replication-related element (DRE) factor (dDREF) is a transcriptional regulatory factor required for the expression of genes carrying the 5'-TATCGATA DRE. dDREF has been reported to bind to a sequence in the chromatin boundary element, and thus, dDREF may play a part in regulating insulator activity. To generate further insights into dDREF function, we carried out a Saccharomyces cerevisiae two-hybrid screening with DREF polypeptide as bait and identified Mi-2 as a DREF-interacting protein. Biochemical analyses revealed that the C-terminal region of Drosophila Mi-2 (dMi-2) specifically binds to the DNA-binding domain of dDREF. Electrophoretic mobility shift assays showed that dMi-2 thereby inhibits the DNA-binding activity of dDREF. Ectopic expression of dDREF and dMi-2 in eye imaginal discs resulted in severe and mild rough-eye phenotypes, respectively, whereas flies simultaneously expressing both proteins exhibited almost-normal eye phenotypes. Half-dose reduction of the dMi-2 gene enhanced the DREF-induced rough-eye phenotype. Immunostaining of polytene chromosomes of salivary glands showed that dDREF and dMi-2 bind in mutually exclusive ways. These lines of evidence define a novel function of dMi-2 in the negative regulation of dDREF by its DNA-binding activity. Finally, we postulated that dDREF and dMi-2 may demonstrate reciprocal regulation of their functions.
Hirose
Fumiko
F
Division of Biochemistry, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya 464-8681, Japan. fsegawa@aichi-cc.jp
Ohshima
Nobuko
N
Kwon
Eun-Jeong
EJ
Yoshida
Hideki
H
Yamaguchi
Masamitsu
M
D000251:Adenosine Triphosphatases; D000818:Animals; D001324:Autoantigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism*; D005124:Eye Abnormalities / Q000235:genetics; D017344:Genes, Insect; D010449:Peptide Mapping; D010641:Phenotype; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D014157:Transcription Factors / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D001324:Autoantigens; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C099494:Dref protein, Drosophila; D029721:Drosophila Proteins; C116095:Mi-2 protein, Drosophila; D018809:Proliferating Cell Nuclear Antigen; D014157:Transcription Factors; D000251:Adenosine Triphosphatases