Estradiol down regulates the binding activity of an avian vitellogenin gene repressor (MDBP-2) and triggers a gradual demethylation of the mCpG pair of its DNA binding site.
A negative regulating protein (MDBP-2) from rooster liver nuclear extracts binds preferentially to a methylated promoter region 5'TTCACCTTmCGCTATGAGGGGGATCATACTGG3' of the avian vitellogenin II gene (Nucleic Acids Res. 19, 1029-1034, 1991). Treatment of adult and immature roosters with estradiol results in a 90% decrease in the binding activity of MDBP-2 within three days. This corresponds to the level found in egg laying hens. The decrease in the binding activity of MDBP-2 precedes the onset of vitellogenin gene transcription. At the same time, there is a two-fold increase in the binding activity of NHP-1 (tested with the same oligonucleotide as for MDBP-2), a protein thought to be involved in the active demethylation of DNA. The methylated oligonucleotide binds either MDBP-2 or NHP-1 and there is no complex formation between the two proteins and DNA. Estradiol treatment does not change the equilibrium binding constant of MDBP-2 which is about 10(-9)M for the methylated oligonucleotide. The early kinetics of demethylation of the mCpG pair in the binding site of MDBP-2 was studied by means of genomic sequencing. A low level of demethylation of mCpG starts gradually on both DNA strands already 4 hours after estradiol treatment during the lag phase of vitellogenin mRNA synthesis. It is concluded that the lowering of the binding activity of MDBP-2 may have a stronger effect on the derepression of the gene than the slow demethylation of MDBP-2 DNA binding site. The role of the methylated CpG is to assure a high binding affinity of the repressor to DNA.
Jost
J P
JP
Friedrich Miescher-Institut, Basel, Switzerland.
Saluz
H P
HP
Pawlak
A
A
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002645:Chickens; D004247:DNA / Q000378:metabolism*; D015226:Dinucleoside Phosphates / Q000378:metabolism; D015536:Down-Regulation; D004958:Estradiol / Q000494:pharmacology*; D005260:Female; D007700:Kinetics; D008297:Male; D008745:Methylation; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*; D014158:Transcription, Genetic; D014819:Vitellogenins / Q000235:genetics*
D004247:DNA; D015226:Dinucleoside Phosphates; D004958:Estradiol; D009841:Oligonucleotides; D012097:Repressor Proteins; D014819:Vitellogenins; C015772:CpG dinucleotide