Phosphorylation/Dephosphorylation of the Repressor MDBP-2-H1 Selectively Affects the Level of Transcription from a Methylated Promoter in Vitro

Phosphorylation/dephosphorylation of the repressor

MDBP-2-H1 selectively affects the level of

transcription from a methylated promoter

in vitro

Alain Bruhat

_{and Jean-Pierre Jost*}

Friedrich Miescher Institute, PO Box 2543, CH-4002 Basel, Switzerland

Received February 29, 1996; Revised and Accepted April 3, 1996

ABSTRACT

We have previously shown that in vivo estradiol-dependent dephosphorylation 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/dephosph-orylation of MDBP-2-H1 on the expression of the avian vitellogenin II gene, we optimised an in vitro transcrip-tion system using HeLa nuclear extracts. We show that in the absence of the phosphorylated form of MDBP-2-H1 from rooster, methylation of the vitellogenin II promoter does not affect the transcription. Addition of purified MDBP-2-H1 from rooster to the in vitro transcription system inhibits transcription more efficiently from a methylated than an unmethylated DNA template. Dephosphorylation of rooster MDBP-2-H1 by phosphatase treatment or estradiol treatment of rooster lead to the loss of inhibitory activity of the protein when added to the in vitro transcription assays. These findings indicate that the phosphorylation of MDBP-2-H1 is essential for the repression of the transcription. Taken together these results establish the relationship between the dephosphorylation of MDBP-2-H1 caused by estradiol, the down regulation of its binding activity to methylated DNA and the derepression of vitellogenin II transcription.

INTRODUCTION

DNA methylation which occurs in almost every higher organism, appears to be an important component in the regulation of vertebrate gene expression (1). Several laboratories have shown that the methylation of cytosines situated within CpG dinucleotides resulted in the inhibition of transcription from viral, plant and animal tissue-specific genes. Methylated and non-methylated cloned DNA has been introduced into cells via transfection (2,3) or microinjection (4,5) or has been tested in in vitro transcription systems (6–9). Although it is assumed that DNA methylation affects protein–DNA interaction and induces some conformational changes of the gene (1), the mechanism of transcriptional inhibition is poorly understood. Two models have been proposed to explain how promoter methylation causes transcriptional

repression. 5 methyl-cytosine may inhibit directly the binding of transcription factors to DNA (10,11) or it may favour the binding of specific repressor proteins that render the DNA inaccessible to transcription factors (12). However, the detailed mechanism of how these proteins interact with methylated DNA and how precisely this interaction affects gene expression remains unknown. For example methyl-CpG binding proteins (MeCP1 and MeCP2), that form complexes with a variety of unrelated sequences that are methylated at CpG, have been described to be associated with centromeric heterochromatin (13). One of the methylated-DNA binding proteins, MDBP-2-H1, belongs to the histone H1 family and binds in vivo and in vitro with high affinity (Kd 10–9 M) to the methylated promoter region of the avian vitellogenin II gene (14,15). The DNA binding activity of this protein is down regulated in egg laying hens and estradiol treated roosters (16). MDBP-2-H1 is a phosphoprotein and its state of phosphorylation decreases in response to estradiol treatment (17). Importantly, phosphorylation of this protein is essential for its in vitro binding to methylated DNA.

Using an in vitro transcription assay, the present study provides evidence that the inhibitory effect of DNA methylation on the transcription of the avian vitellogenin II gene is an indirect mechanism and that the phosphorylated form of MDBP-2-H1 selectively inhibits transcription from methylated templates. In addition, we report that the phosphorylation of MDBP-2-H1 is essential for the repression of the transcription. The implication of these findings are discussed in the general context of DNA methylation and gene inactivation.

MATERIALS AND METHODS

Chemicals and enzymes

Heparin–Sepharose was purchased from Pharmacia. [γ-32_P]ATP (3000 Ci/mmol; 1 Ci = 37 GBq) was obtained from Amersham. 17-β-estradiol was purchased from Serva (Heidelberg). CpG methylase (SssI methylase) and S-adenosylmethionine were obtained from New England Biolabs whereas polynucleotide kinase and HpaII endonuclease were from Biofinex Praroman (CH 1724; Switzerland). Ribonuclease-free DNase I and S1 nuclease were purchased from Boehringer Mannheim and calf intestinal phosphatase attached to beaded agarose was from Sigma. HeLa * To whom correspondence should be addressed

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nuclear extracts and ribonucleoside triphosphates were obtained from Promega.

Hormone treatment of chickens

Where indicated, mature or immature white Leghorn roosters received a single intramuscular injection of 17β-estradiol (40 mg/ Kg) dissolved in propylene glycol (40 mg/ml).

Preparation of MDBP-2-H1 from rooster liver nuclear extracts

Liver nuclei from roosters were prepared as described by Sierra (18) with the following modifications: for estradiol-treated rooster livers, the buffer contained 1.9 M sucrose and for the non-treated animals the buffer contained 2 M sucrose. Nuclei were then lysed with 1/10 vol 4 M (NH4)2SO4 for 30 min on ice. After centrifugation for 60 min at 300 000 g to remove chromatin, the supernatant was dialyzed two times for 2 h periods against 100 vol dialysis buffer (25 mM HEPES pH 7.6, 2 mM benzamidine, 100 mM KCl, 1 mM dithiothreitol and 10% glycerol). The nuclear proteins were then fractionated at room temperature by FPLC on 10 ml Heparin– Sepharose columns by step elution gradients (0.1–1 M KCl) (17). Individual fractions were concentrated by means of Centriprep-10 concentrators from Amicon. Aliquots of the 0.5 M KCl fraction, containing MDBP-2-H1, were stored frozen at –80
C. The protein concentration was determined according to Bradford (19) using bovine serum albumin as a standard.

Gel mobility shift assays

Experiments were carried out essentially as described by Pawlac

et al. (14). Sonicated E.coli DNA (1 µg) was used as non-specific

competing DNA. Double-stranded oligonucleotides used for these experiments were end labelled with the polynucleotide kinase reaction or with the Klenow fragment of DNA polymerase I (Boehringer Mannheim).

Phosphatase treatment

The fraction containing MDBP-2-H1 was mixed in a 25 µl reaction with either calf intestinal phosphatase attached to beaded agarose (CIP-agarose) (0.1 U/µg protein) or agarose beads only and stirred gently at 37
C for 30 min. The protein solution was separated from the CIP-agarose or agarose by centrifugation at 10 000 g for 10 min at 4
C. The protein concentration was determined according to Bradford (19) using bovine serum albumin as a standard.

Plasmid DNA constructs

For the construction of the plasmid template (pVT 13) used for

in vitro transcription, a 126 bp EcoRI–HindIII fragment of the

avian vitellogenin II gene, containing 73 bp of the 5′ flanking region and the first 52 bp of the first exon was fused to a 144 bp

HindIII–ClaI fragment of the cauliflower mosaic virus (CaMV)

ORFII protein gene and cloned into pBR322 (Fig. 1). Template DNA (MetpVT13) was methylated with CpG methylase (M.SssI) according to the manufacturer’s instructions. After 2 h incubation at 37
C, the DNA was extracted twice with phenol, once with chloroform, precipitated with 1/10 vol 3 M sodium acetate (pH 7.5) and 3 vol ethanol and the pellet was resuspended in 1× TE (10 mM

Figure 1. (A) Construction of the template for in vitro transcription was carried out as described in methods section. CaMV refers to the HindIII–ClaI fragment of the reporter gene from cauliflower mosaic virus. (*) Represents methylated CpGs at positions –53 and +10 respectively. The 41 nt labeled probe used to detect newly synthetized message is represented by a solid line. (B) Analysis of in vitro transcription products on a 12% polyacrylamide–urea gel. RNA transcripts were detected by an S1 nuclease protection assay. The positions of the undigested labeled probe (probe) and the transcription product (TP) are marked. HeLa nuclear extracts were tested with (+) or without (–) α-amanitin.

Tris–HCl pH 7.5, 1 mM EDTA). The extent of methylation was tested with HpaII endonuclease and analyzed on 1% agarose gel.

Oligonucleotides

Oligonucleotides were synthesized with an Applied Biosystems oligonucleotide synthesizer and gel purified. Equal moles of complementary strands were heated to 90
C for 1 min and annealed by slow cooling to room temperature. The sequence of the methylated oligonucleotide from the avian vitellogenin II gene is 5′-TTCACCTTmCGCTATGAGGGGGATCATACTGGCA-3′ (nucleotide positions +2 to +34) (16).

In vitro transcription

A reaction mixture (final volume 25 µl) contained 19 µg HeLa nuclear extracts, 400 ng supercoiled DNA template in 9 mM HEPES, pH 7.9, 12 mM KCl, 3 mM MgCl2, 0.09 mM EDTA, 0.22 mM dithiothreitol, 9% (vol/vol) glycerol and 0.03 mM phenylmethylsulfonyl fluoride. The negative control contained 2 µg/ml α-amanitin. The reaction mixture was preincubated at 4
C for 10 min, after which transcription was initiated by the addition of 400 µM of each ribonucleoside triphosphates and incubation was continued at 30
C for 60 min. The reaction mixture was then treated with proteinase K (300 µg/ml). The volume was brought up to 300 µl with 1× TE (pH 7.5) and samples were extracted twice with phenol, once with chloroform and ethanol precipitated. A 15 min DNase I (70 U/ml) treatment followed according to the manufacturer’s instructions (Boehringer Mannheim), and the RNA was purified by phenol/chloroform extractions and ethanol precipitated. For detection of the CaMV transcript from vitellogenin II promoter, RNA was heated to 75
C for 10 min in 20 µl of hybridization solution (0.4 M NaCl, 10 mM Tris–HCl pH 7.5 and 1 mM EDTA) and hybridized overnight at 40
C with 1 ng 32P end-labeled oligonucleotide (2.5 × 108 c.p.m./µg) 5′-TCTAAAAGGGATTTTACTT-CCTTTAGTTGG-CTCGAAATCCG-3′. Detection of specific transcript was carried out by S1 nuclease protection assay (20). Hybridized sample was digested for 60 min at 37
C in 300 µl S1 nuclease buffer (0.3 M NaCl, 60 mM sodium acetate pH 4.5, 4 mM ZnSO4 and 7.5 µg salmon sperm DNA) with 660 U/ml S1 nuclease. Following ethanol precipitation the transcription product was separated on a 12% (w/v) polyacrylamide–4 M urea denaturating gel. Radioactive bands were

visualized by using a PhosphorImager and IMAGEQUANT software (Molecular Dynamics). Each in vitro transcription experiment was repeated three times to confirm the reproducibility of the results.

RESULTS

In the absence of the phosphorylated form of MDBP-2-H1, methylation of the promoter region of

vitellogenin II does not affect the transcription

The presence of the phosphorylated form of MDBP-2-H1 in the rooster liver, binding selectively to methylated DNA, has been previously documented by in vitro DNA binding studies (14–16). To study the mode of action of this liver protein more closely, a heterologous in vitro transcription system consisting in HeLa nuclear extracts and a DNA template with vitellogenin II promoter region was optimised. Figure 1A shows the plasmid construct (pVT13) used for the in vitro transcription. The details of the construction are given in the methods section. Figure 1B, lane 1 shows that in the absence of the repressor MDBP-2-H1, newly synthesized transcripts (TP) were detected. The plasmid without the vitellogenin II promoter region gave no transcription (data not shown) whereas the template with vitellogenin II promoter region gave a specific transcription product TP (Fig. 1B, lane 1). The transcripts were generated by RNA polymerase II as demonstrated by their sensitivity to a low concentration of α-amanitin (Fig. 1B, lane 2). To determine the effect of methylation on the transcription from the vitellogenin II promoter, two CpG sites of the promoter were methylated with the CpG methylase SssI (Fig. 1A). Transcription was carried out in vitro with the HeLa transcription system which does not contain any MDBP-2-H1. The absence of MDBP-2-H1 in HeLa nuclear extracts was demonstrated by gel shift assays and by immunoblot analysis with calf thymus histone H1 antibodies (data not shown). Template DNA and nuclear extracts were added at zero time and upon 10 min incubation at 4
C, rNTPs were added to the incubation mixture and the incubation was continued for 60 min at 30
C. Figure 2 shows the effect of increasing concentrations of methylated and non-methylated templates on the transcription. Under our experimental conditions, both the methylated and unmethylated DNA templates gave a comparable level of transcription. These results indicate that in the absence of the phosphorylated form of MDBP-2-H1 there is, in the in vitro transcription system, no difference in the level of transcription from methylated or unmethylated DNA templates, therefore, in this case there is no direct inhibitory effect of methylated DNA on the binding of transcription factors.

The phosphorylated form of MDBP-2-H1 selectively inhibits transcription from methylated templates. To determine whether the phosphorylated form of MDBP-2-H1 from rooster liver preferential-ly inhibits the transcription from a methylated promoter, in vitro transcription assays were performed in the presence of partially purified MDBP-2-H1 from rooster liver (0.5 M KCl Heparin– Sepharose fraction). In these experiments the templates DNA (methylated or non-methylated) were first incubated with increas-ing concentrations of purified repressor MDBP-2-H1 for 5 min at 4
C. Upon addition of the HeLa nuclear extracts the reaction mixture was incubated for another 5 min at 4
C. Transcription was initiated by the addition of rNTPs and the incubation was carried out for 60 min at 30
C. The data presented in Figure 3A (summarized in the graph of Fig. 3B) show that the inhibition of

Figure 2. Transcription of methylated and unmethylated DNA templates in the absence of the phosphorylated form of MDBP-2-H1 from rooster. DNA template and 19 µg HeLa nuclear extracts were pre-incubated for 10 min at 4
C and the transcription reaction was then carried out at 30
C for 60 min as indicated. Increasing amounts (200, 250, 300, 400 and 500 ng) of methylated (M) or unmethylated (UM) templates were added to the in vitro transcription system. The positions of the undigested labeled probe (probe) and the transcription product (TP) are indicated.

transcription occurred at lower concentrations of MDBP-2-H1 for the methylated (lanes 1–5) than for the unmethylated template (lanes 6–10). Transcription from the methylated template was inhibited by 60% with 2 µg of partially purified MDBP-2-H1 (lane 3) while with the same concentration of MDBP-2-H1, the transcription from the ummethylated template remained unchanged (lane 8). Although CpG methylase methylates numerous sites in the plasmid vector, the methylation of the vector alone did not affect the transcription of vitellogenin II in the presence of MDBP-2-H1 (J.P.J., unpublished data). These findings demonstrate that the phosphorylated form of MDBP-2-H1 from rooster liver inhibits preferentially vitellogenin II transcription from methylated templates. It has previously been shown (15) that the binding of MDBP-2-H1 to DNA was methylation dependent and required methylated oligonucleotides >30 bp. We examined the effect of methylated and unmethylated competitor DNAs on the inhibition of vitellogenin II transcription by MDBP-2-H1. The amount of competing DNA required for the specific inhibition of binding to the phosphorylated form of MDBP-2-H1 was determined by cross-competition gel mobility shift assays. Figure 4A shows that 2 pmol methylated competitor DNA was enough to bind to saturation 3 µg partially purified MDBP-2-H1 (lane 4) whilst the unmethylated DNA was a very poor competitor (lane 7). The transcription competition assay was carried out by incubating first the repressor MDBP-2-H1 with increasing concentrations of methylated or non-methylated oligonucleotides at 4
C for 5 min. Then template DNA was added together with the nuclear extracts and the incubation was continued at 4
C for 5 min. Transcription was initiated by the addition of rNTPs and the transcription product was analysed after 60 min of incubation at 30
C. Figure 4B shows that addition of increasing amounts of methylated MDBP-2-H1 binding site oligonucleotides (1–2 pmol) to the transcription reaction alleviated the inhibition of the transcription caused by 3 µg partially purified MDBP-2-H1 (lanes 4 and 5). On the other hand addition of its unmethylated counterpart had no effect on the transcription

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Figure 3. Effect of the addition of the phosphorylated form of MDBP-2-H1 from rooster liver to the in vitro transcription system. (A) Transcription reactions were carried out, as indicated, with 400 ng methylated (M) or unmethylated (UM) template and 19 µg HeLa nuclear extracts. Increasing levels of partially purified MDBP-2-H1 (0.5 M KCl Heparin–Sepharose fraction) from rooster and HeLa nuclear extracts were mixed at 4
C as indicated. The positions of the undigested labeled probe (probe) and the transcription product (TP) are represented. (B) Quantification of the transcription products obtained from methylated (M) or unmethylated (UM) DNA template in the presence of increasing concentration of phosphorylated MDBP-2-H1. The error bars represent two standard deviations from the mean of three experimental repetitions.

(lanes 7 and 8). These results indicate that the block on in vitro transcription by MDBP-2-H1 can be released specifically by the addition of an excess of the methylated MDBP-2-H1 DNA binding site but not by the addition of the unmethylated binding site.

Phosphorylation of MDBP-2-H1 is essential for the repression of vitellogenin II transcription

As reported previously, phosphorylation of MDBP-2-H1 is essential for its binding to methylated DNA (17). To assess the effect of MDBP-2-H1 phosphorylation on transcriptional repression of

vitellogenin II, the partially purified liver MDBP-2-H1 from

rooster was dephosphorylated in vitro with CIP-agarose. To test whether the phosphatase treatment abolished the DNA binding capacity of MDBP-2-H1 without altering the amount of protein, control experiments were carried out using gel mobility shift assays and immunoblot analysis with calf thymus histone H1 antibodies (data not shown). The phosphorylated or dephosphorylated forms of MDBP-2-H1 were incubated with a methylated DNA template and used in the in vitro transcription system (Fig. 5A). DNA template and the repressor MDBP-2-H1 (in the phosphorylated and non-phosphorylated form) were pre-incubated at 4
C for 5 min.

Figure 4. (A) Gel shift competition assay of MDBP-2-H1–DNA complex with increasing concentrations of methylated or unmethylated oligonucleotide (double-stranded oligonucleotide containing the MDBP-2-H1 binding site at nucleotide positions +2 to +34 of vitellogenin II). Protein-bound DNA is in band b, and free DNA is in band f. (B) Transcription competition assay: transcription reactions were carried out as indicated, with 400 ng methylated template, 19 µg HeLa nuclear extracts, 3 µg partially purified MDBP-2-H1 from rooster and increasing concentrations (0.5, 1 and 2 pmol) of methylated (M) or unmethylated (UM) MDBP-2-H1 binding site oligonucleotides. The positions of the undigested labeled probe (probe) and the transcription product (TP) are indicated.

The HeLa nuclear extracts were then added and the incubation was continued for another 5 min at 4
C. Transcription was initiated by the addition of rNTPs and the incubation proceeded at 30
C for 60 min. Samples were analysed as indicated in the Materials and Methods. Dephosphorylated MDBP-2-H1 (3–4 µg/incubation mixture) had no inhibitory effect on the transcription when compared with the same concentration of the phosphorylated form of MDBP-2-H1 (compare lanes 4 and 5 with 8 and 9). These findings demonstrate that, in vitro, the phosphorylated form of MDBP-2-H1 is essential for the repression of vitellogenin II transcription. In our previously published results (17) we showed that estradiol triggered a partial dephosphorylation of MDBP-2-H1 in the liver of rooster which was correlated with the loss of in vitro preferential binding to methylated DNA. In the following experiment we tested whether the in vivo estradiol-dependent dephosphorylation of MDBP-2-H1 had any effect on the inhibition of transcription. As shown in Figure 5B, at high concentrations of MDBP-2-H1 (3 and 4 µg) the phosphorylated form from rooster gave a complete inhibition of transcription whereas the partially dephosphorylated form from estradiol treated rooster gave much less inhibition of transcription (compare lanes 4 and 5 with 8 and 9). The same result was observed when

Figure 5. (A) Effect of the addition of dephosphorylated MDBP-2-H1 to the in vitro transcription system. Partially purified MDBP-2-H1 from rooster was treated with or without calf intestinal phosphatase as described in the Materials and Methods. Transcription reactions were carried out as indicated, with 400 ng methylated template and 19 µg HeLa nuclear extracts. Increasing levels of partially purified phosphorylated or dephosphorylated MDBP-2-H1 and HeLa nuclear extracts were added at 4
C as indicated. The positions of the undigested labeled probe (probe) and the transcription product (TP) are shown. (B) Effect of the addition of MDBP-2-H1 from rooster treated with estradiol to the in vitro transcription system. Transcription reactions were carried out as indicated above. Increasing amounts of partially purified MDBP-2-H1 from rooster or rooster treated with estradiol and HeLa nuclear extracts were incubated as indicated. The positions of the undigested labeled probe (probe) and the transcription product (TP) are indicated.

MDBP-2-H1 from egg-laying hen was added to the in vitro transcription system (data not shown). These findings indicate that the in vivo dephosphorylation of MDBP-2-H1 caused by estradiol may be a crucial event for the derepression of vitellogenin II transcription.

DISCUSSION

In recent years evidence has accumulated that histone H1, the internucleosomal histone of higher eukaryotes, acts in vitro and

in vivo as a transcriptional repressor of specific genes (21–24). Histone H1 has been shown to be enriched in inactive chromatin (25) and associated with nucleosomes containing 5 methyl-cytosine (26). Conversely, it is depleted in active genes (27) and is absent in CpG-rich islands from active housekeeping genes (28). Several recent reports have established a correlation between DNA methylation, binding of histone H1 and repression of gene transcription (8,9). In one case, Johnson et al. (9) showed that the histone variant H1c had a preferential inhibition of transcription from methylated DNA. Our results show that MDBP-2-H1, also a member of the histone H1 family, selectively inhibits in vitro

transcription from the methylated vitellogenin II promoter. In the absence of this H1 variant, methylation of the promoter region does not affect the transcription. A gel mobility shift assay carried out with methylated and unmethylated labeled oligonucleotides could not demonstrate the presence of a specific methylated DNA binding protein in total nuclear extracts prepared from HeLa cells and in the 0.5 M KCl fraction eluted from Heparin–Sepharose column (data not shown). These finding demonstrate that DNA methylation confers its inhibitory effect on vitellogenin II transcrip-tion indirectly by a mechanism that involves MDBP-2-H1. However, we do not know how the interaction of MDBP-2-H1 with methylated DNA affects gene transcription. We propose that the binding of MDBP-2-H1 to several sites in the methylated promoter of vitellogenin II (for example nucleotide positions +2 to +34) could lead to a steric occlusion of the basic transcriptional machinery and/or of transcription factors from their binding sites on the DNA. Since histone H1 plays a crucial role in the chromatin structure (29), it is reasonable to assume that DNA methylation and subsequent binding of MDBP-2-H1 to vitellogenin II promoter favor the formation of heterochromatin. The relation between DNA methylation, chromatin structure and the gene activity could be examined by in vitro transcription of chromatin reconstituted in the absence or presence of MDBP-2-H1 (30). Furthermore, previous work from our laboratory has shown that MDBP-2-H1 mediates the binding of another nuclear protein that does not itself bind to methylated DNA (15,31). The identity and the role of this protein in the transcriptional regulation of vitellogenin II, however, remain to be determined.

The histone H1 family consists of several different somatic variants which are present in different species, cell types and developmental stages (32). This genetic microheterogeneity is increased by several reversible post-translational modifications (33) that can influence H1–DNA or H1–H1 interactions which modulate the chromatin structure. We do not know to which variants MDBP-2-H1 belongs but we do know that this protein is phosphorylated at several residues in the nuclei of rooster liver and that the phosphorylation of MDBP-2-H1 is essential in vitro for its binding to methylated DNA (17). Phosphorylation of histone H1 is a complex modification and the literature is undecided about its role in the chromatin condensation/decondensation (34–36). Our present results show that the phosphorylation of MDBP-2-H1 is essential for the repression of vitellogenin II transcription. We suggest that in our case, DNA methylation and phosphorylated MDBP-2-H1 binding to methylated DNA are two major factors involved in the chromatin condensation and the subsequent repression of vitellogenin II transcription.

Treatment of adult rooster with estradiol triggers a partial dephosphorylation of MDBP-2-H1 (17), and a sharp decrease of its binding activity to a methylated sequence (nucleotide positions +2 to 34) of the vitellogenin II promoter (16). The data of the present study demonstrate that these changes result in the loss of the inhibitory activity of MDBP-2-H1 on the vitellogenin II transcription. In addition, we have previously observed dramatic estradiol-induced changes in the chromatin structure of this region (37,38). These parameters combined with a gradual demethylation of the CpG pair of the MDBP-2-H1 binding site (16) contribute to the maximum stimulation of vitellogenin II transcription. Taken together these results establish clearly the relationship between the dephosphorylation of MDBP-2-H1 caused by estradiol, the down regulation of its binding to methylated DNA, the modification of the chromatin structure and the derepression of vitellogenin II

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transcription. However, the mechanism by which estradiol induces a partial dephosphorylation of MDBP-2-H1 is still unknown. We consider two models that are not mutually exclusive and that could explain the dephosphorylation process. (i) Estradiol could induce directly or indirectly a specific histone H1 phosphatase. (ii) The binding of the estradiol–receptor complex to the vitellogenin II promoter could disrupt the chromatin structure and expose MDBP-2-H1 to a specific histone H1 phosphatase already present in the nuclei. However, the nature and the sequence of these specific events involved in the dephosphorylation of MDBP-2-H1 under estadiol control, remain to be established. Furthermore the entire sequence of MDBP-2-H1 needs to be determined since recently it has been conclusively shown that the total population of histone H1 does not have any preferential binding to methylated DNA (39; J.P.J., unpublished data) and that the methylation of CpG sequences does not influence the binding of total histone H1 to nucleosomes (40).

ACKNOWLEDGEMENTS

We are grateful to Michel Siegmann for the technical help in preparation of MDBP-2-H1 from rooster liver nuclear extracts and to Drs Jerzy Paszkowski, Edward Oakeley and Jean Louis Couderc for critically reading the manuscript.

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