In vivo estradiol-dependent dephosphorylation of the repressor MDBP-2-H1 correlates with the loss of in vitro preferential binding to methylated DNA.
We have previously shown that estradiol treatment of roosters resulted in a rapid loss of binding activity of the repressor MDBP-2-H1 (a member of the histone H1 family) to methylated DNA that was not due to a decrease in MDBP-2-H1 concentration. Here we demonstrate that MDBP-2-H1 from rooster liver nuclear extracts is a phosphoprotein. Phosphoamino acid analysis reveals that the phosphorylation occurs exclusively on serine residues. Two-dimensional gel electrophoresis and tryptic phosphopeptide analysis show that MDBP-2-H1 is phosphorylated at several sites. Treatment of roosters with estradiol triggers a dephosphorylation of at least two sites in the protein. Phosphatase treatment of purified rooster MDBP-2-H1 combined with gel mobility shift assay indicates that phosphorylation of MDBP-2-H1 is essential for the binding to methylated DNA and that the dephosphorylation can occur on the protein bound to methylated DNA causing its release from DNA. Thus, these results suggest that in vivo modification of the phosphorylation status of MDBP-2-H1 caused by estradiol treatment may be a key step for the down regulation of its binding to methylated DNA.
Bruhat
A
A
Friedrich Miescher Institute, Basel, Switzerland.
Jost
J P
JP
D000818:Animals; D001483:Base Sequence; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000378:metabolism*; D002645:Chickens; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004958:Estradiol / Q000494:pharmacology*; D005260:Female; D008099:Liver / Q000378:metabolism*; D008297:Male; D008745:Methylation; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010449:Peptide Mapping; D010710:Phosphates / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010768:Phosphoserine / Q000032:analysis; D012097:Repressor Proteins / Q000378:metabolism*; D014357:Trypsin
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D010710:Phosphates; D010748:Phosphopeptides; D012097:Repressor Proteins; D010768:Phosphoserine; D004958:Estradiol; D004247:DNA; D014357:Trypsin