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 mem ber 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-H 1 from rooster liver nuclear extracts is a phosphoprotein. Phosphoamin o acid analysis reveals that the phosphorylation occurs exclusively on serine residues. Two-dimensional gel electrophoresis and tryptic phos phopeptide analysis show that MDBP-2-H1 is phosphorylated at several s ites. Treatment of roosters with estradiol triggers a dephosphorylatio n of at least two sites in the protein. Phosphatase treatment of purif ied rooster MDBP-2-H1 combined with gel mobility shift assay indicates that phosphorylation of MDBP-2-H1 is essential for the binding to met hylated DNA and that the dephosphorylation can occur on the protein bo und to methylated DNA causing its release from DNA. Thus, these result s suggest that in vivo modification of the phosphorylation status of M DBP-2-H1 caused by estradiol treatment may be a key step for the down regulation of its binding to methylated DNA.