PHOSPHORYLATION DEPHOSPHORYLATION OF THE REPRESSOR MDBP-2-H1 SELECTIVELY AFFECTS THE LEVEL OF TRANSCRIPTION FROM A METHYLATED PROMOTER IN-VITRO/

We have previously shown that in vivo estradiol-dependent dephosphoryl ation of MDBP-2-H1 (a member of the histone H1 family) correlates with the loss of in vitro preferential binding to methylated DNA, To study the effects of the phosphorylation/dephosphorylation of MDBP-2-H1 on the expression of the avian vitellogenin II gene, we optimised an in v itro transcription system using HeLa nuclear extracts, We show that in the absence of the phosphorylated form of MDBP-2-H1 from rooster, met hylation of the vitellogenin II promoter does not affect the transcrip tion, Addition of purified MDBP-2-H1 from rooster to the in vitro tran scription system inhibits transcription more efficiently from a methyl ated than an unmethylated DNA template, Dephosphorylation of rooster M DBP-2-H1 by phosphatase treatment or estradiol treatment of rooster le ad to the loss of inhibitory activity of the protein when added to the in vitro transcription assays, These findings indicate that the phosp horylation of MDBP-2-H1 is essential for the repression of the transcr iption, Taken together these results establish the relationship betwee n the dephosphorylation of MDBP-2-H1 caused by estradiol, the down reg ulation of its binding activity to methylated DNA and the derepression of vitellogenin II transcription.