| Nucleic Acids Research | Pages |
Prothymosin [alpha] modulates the interaction of histone H1 with chromatin
Introduction
Materials And Methods
Protein and peptide work
Chromatin reconstitution
Immunochemical techniques
Protein bandshifting on agarose gels
Results
Isolation of the ProT[alpha]-H1 complex from NIH 3T3 cell extracts
Identification of interacting sites between ProT[alpha] and histone H1
ProT[alpha] does not interact with H1 in chromatin
ProT[alpha] modulates the interaction of H1 with chromatin
ProT[alpha] extracts a fraction of histone H1 from chromatin but not from free DNA
Discussion
Acknowledgements
References
Prothymosin [alpha] modulates the interaction of histone H1 with chromatin
ABSTRACT
INTRODUCTION
Prothymosin [alpha] (ProT[alpha]) is a highly conserved acidic polypeptide (1-3) localized in the cell nucleus (4-6). The physiological role of this protein remains unclear, despite its wide distribution and abundance (3,7,8). Several pieces of evidence suggest a link between ProT[alpha] and cell proliferation. ProT[alpha] expression is elevated in proliferating cells (9-11) and in response to increased levels of c-myc (12). A correlation between ProT[alpha] and c-myc mRNA expression has been found in human colon cancers (13) and in other in vivo systems (14), while high protein levels have been reported in intestine and breast malignant tissues (15). ProT[alpha] mRNA is present throughout the cell cycle, increases at S/G2 phase and can be induced by transcription factor E2F (16). Moreover, antisense oligonucleotides directed against ProT[alpha] mRNA inhibit cell division in myeloma cells (17), suggesting an essential role in cell proliferation.
The unusual structure of ProT[alpha] might be indicative of its function. Calf thymus ProT[alpha] (12.5 kDa) contains an acidic region in the centre of the molecule (18). Histidine, aromatic and sulphur amino acids are entirely absent, whereas there are only seven widely dispersed hydrophobic residues. Based on these sequence features, an extended non-folded conformation is expected that might be favourable for interaction of the protein with other cellular components (4,19). Nucleoplasmin and high mobility group protein 1 (HMG1) also contain extended acidic domains and have well-documented functions related to the organization of chromatin (20).
ProT[alpha] binds selectively to the linker histone H1 through its acidic domain (21). Histone H1 is responsible for the organization and stabilization of nucleosomal arrays into higher order structures and there is substantial evidence that the differential association of H1 affects gene expression (for a review see 22). A number of biochemical studies have shown that active chromatin in nuclei is partially depleted of linker histone H1 (23,24), suggesting that H1 may exhibit reduced affinity for these regions. However, H1 may also be found associated with active genes, but an altered mode of interaction has been proposed (24-26). Currently, evidence is accumulating that H1, rather than being a global repressor of transcription, affects the expression of selected genes (27-30). The factors that modulate the stoichiometry of H1 association or its mode of interaction with chromatin at a given genomic site are unknown. Conceivably, H1 interactions may be affected by binding of non-chromosomal proteins (20,31). In this vein, the study of proteins such as ProT[alpha] that interact with histone H1 may help to elucidate the regulatory mechanisms of H1 distribution.
In this study we present a detailed analysis of the interaction between ProT[alpha] and histone H1 and its effect on the association of H1 with chromatin and DNA. On the basis of our results, we propose a role for ProT[alpha] in chromatin, where it may modulate the extent and/or mode of interaction of H1 with nucleosomal fibres.
MATERIALS AND METHODS
Protein and peptide work
ProT[alpha] was purified by a new method, based on published procedures (1,4), as follows. Freshly excised calf thymus was immediately frozen in liquid nitrogen and stored at -80°C. Frozen tissue (30 g) was pulverised in liquid nitrogen and immediately added to 400 ml boiling water. The suspension was homogenized with a Polytron homogenizer and acidified with 1 vol buffer A (1 M HCOOH, 0.2 M pyridine, pH 2.8). The acidic extract was centrifuged at 10 000 g for 25 min at 4°C and the supernatant was kept. The pellet was resuspended in an equal volume of buffer A and centrifuged as before. The two supernatants were combined, concentrated to a jelly-like material and dissolved in 5 ml 7.5 mM sodium borate, 1 mM DTT, pH 8.8. The sample was applied to a CM-Sephadex G-25 column (2.2 × 39 cm) pre-equilibrated with the same buffer. The column was run at 18 ml/h and ProT[alpha] was recovered in the first 80 ml of the flow-through volume. The pooled fractions were concentrated to dryness in a Speed-Vac, dissolved in 3 ml 5 mM HCl, 0.02% NaN3 and dialysed over the same solution for 3 h. Subsequently, the sample (6 ml) was concentrated, dissolved in 1 ml 10 mM sodium acetate, 8 M urea, 0.02% NaN3, pH 4.5 and applied to a SP-Sephadex G-25 column (1.0 × 43 cm), previously equilibrated with 10 mM sodium acetate, 0.02% NaN3, pH 4.5. ProT[alpha] was eluted in the first fractions (9-13, 1.2 ml each) of the flow-through volume. All steps were carried out at room temperature, unless indicated.
The preparation was analysed by 15% SDS-PAGE and stained as a single protein band with silver. High performance liquid chromatography (HPLC) gave a single peak and the amino acid analysis was in agreement with the published values (3,18). This method yields ~1.5 mg ProT[alpha]/30 g tissue.
ProT[alpha] concentration was determined by amino acid analysis (3).
Drosophila histone H1 was purified as described (32). Histone H1 and core histones from calf thymus were obtained from Boehringer Mannheim. The globular region of histone H1 was prepared by partial trypsinolysis of 630 µg H1 in 630 µl 20 mM Tris-HCl, 1 M NaCl, pH 7.4, for 20 min at 20°C (33).
Histone H1 (2 µg) was phosphorylated with 0.024 U protein kinase C (Boehringer Mannheim) in the presence of 20 µCi [[gamma]-32P]ATP, 50 µM ATP, 0.2% BSA, 1% phospholipids for 30 min at 37°C.
Thymosin [alpha]1, acidic peptide and ct peptide corresponding to residues 1-28, 52-69, 86-109 respectively of bovine thymus ProT[alpha] (18) were synthesized by the EMBL Protein Sequencing and Peptide Synthesis Facility. Peptide biotinylation has been described elsewhere (34).
Crosslinking experiments using dimethylpimelimidate (DMP; Pierce) were performed as follows. Aliquots of 1 µg histone H1 were incubated with different amounts of ProT[alpha], thymosin [alpha]1, the acidic peptide or the ct peptide in 10 µl 0.1 M sodium phosphate buffer, pH 6.7, for 10 min at room temperature. Then, 10 µl 0.2 M sodium borate buffer, pH 9.0, and 2.2 µl 0.2 M DMP were added and the samples were further incubated for 30 min. The reaction was stopped by addition of 2 µl 1 M Tris-HCl, pH 7.4, 7 µl Laemmli buffer and boiling.
Chromatin reconstitution
Chromatin assembly extract was prepared from Drosophila embryos as described (35). Plasmid immobilization (pdHSP70 XX3.2) on paramagnetic beads, chromatin assembly reactions and micrococcal nuclease analysis were performed as described (29). One unit of H1 is the amount needed to increase the repeat length of chromatin from 180 to 200 bp (29). Incorporation of H1 into chromatin was as follows. H1 (2 U) was mixed with assembly extract prior to addition of DNA beads. Chromatin was assembled for 6 h at 25°C and beads were concentrated in a magnetic field. Immobilized chromatin was washed with 650 mM NaCl to strip bound H1, then three times with 100 mM NaCl, and finally equilibrated in binding buffer (20 mM Tris-HCl, 100 mM NaCl, 1 mM MgCl2, 0.02% NP-40, pH 7.4). Then, histone H1 and traces of 32P-labeled H1 were added to the binding buffer (15 µl total volume) and incubated with the chromatin beads for 30 min at 25°C. Histone H1-DNA complexes were formed in a binding buffer consisting of 20 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl2, 0.02% NP-40, pH 7.4, in 15 µl total volume.
To assay the interaction of ProT[alpha] with H1-containing chromatin, chromatin was reconstituted in the presence of 2 U histone H1 on 100 ng DNA which had been immobilized on paramagnetic beads. Magnetically purified chromatin was incubated with 0.4 µg ProT[alpha] in 100 µl 20 mM Tris-HCl, 150 mM NaCl, pH 7.4. After incubation, the supernatant was removed and the chromatin beads were subjected to salt extractions with 50, 400 and 650 mM NaCl. The proteins were precipitated with 6 vol acetone overnight at -20°C and analysed by western blotting using an anti-ct antibody.
Immunochemical techniques
Anti-H1 antibodies were raised in rabbits according to Srebreva and Zlatanova (36) and the serum was affinity-purified over immobilized H1.
Antibodies against peptide ct were raised in rabbits and were affinity-purified as described (21). For construction of the immunoaffinity and the non-specific IgG columns, 1 mg affinity-purified anti-ct or non-immune IgGs were bound to 0.7 ml protein A-Sepharose. The antibodies were covalently coupled to the beads using DMP as a crosslinker (37). ProT[alpha]-agarose was prepared by coupling 220 µg of the protein to 1.0 ml Affigel 15 (BioRad).
Immunoisolation of the ProT[alpha]-H1 complex. NIH 3T3 cells were grown as monolayers in DMEM supplemented with 10% fetal calf serum. Samples of 50 × 106 cells were lysed with 3 ml 20 mM Tris-HCl, pH 7.4, 800 mM NaCl, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 0.5 mM dithiothreitol, protease inhibitors (pepstatin, leupeptin, aprotinin, 1 µg/ml each) and incubated on ice for 15 min. After centrifugation at 10 000 g for 10 min at 4°C, the supernatant was adjusted to 150 mM NaCl, divided into four portions (corresponding to 12.5 × 106 cells) and incubated with (i) anti-ct protein A-agarose, (ii) IgG protein A-agarose or (iii) ProT[alpha]-agarose beads for 3 h at 4°C and further for 1 h at 25°C, with gentle rocking. Then, the beads were poured into columns, washed with 10 ml 20 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 1 mM EDTA, 1 mM PMSF and protease inhibitors and then with 0.3, 0.6 and 1.0 M NaCl (1.5 ml each). The fractions were dialysed over 1 mM phosphate buffer, pH 7.4, 1 mM EDTA, 1 mM PMSF, concentrated, divided into two and analysed by western blotting for histone H1 and ProT[alpha].Western Blotting for ProT[alpha]. Samples were resolved by 15% SDS-PAGE (0.75 mm thickness) and, after electrophoresis, the gels were equilibrated in 20 mM sodium acetate, pH 4.5 (300 ml with three changes, 15-20 min). Electrotransfer was performed onto activated nitrocellulose treated with glutaraldehyde (21,38) at 7 V, 45 mA, for 7 h in the above buffer. The membranes were washed with TBST (20 mM Tris, 0.9% NaCl, 0.1% Tween 20, pH 7.4) and incubated in blocking buffer (TBST with 0.1% gelatin) for 2-12 h. Finally, the filters were incubated with anti-ct antibody (1:1000), then with anti-rabbit IgG-peroxidase (1:5000) and detected by enhanced chemiluminescence (ECL; Amersham Life Science).Protein bandshifting on agarose gels
ProT[alpha] was incubated with histone H1 or with the GH1 peptide in 20 mM NaH2PO4/Na2HPO4, pH 6.7, for 30 min at 25°C in 23 µl total volume. After addition of the sample buffer (2 µl glycerol, 4 µl bromophenol blue), the samples were loaded on an agarose gel (0.75% in 20 mM NaH2PO4/Na2HPO4, pH 6.7) and electrophoresed at 34 V, 15 mA, for 3 h at room temperature, using 0.1 M NaH2PO4/Na2HPO4, pH 6.7, as the running buffer. The gels were stained with Coomassie blue for 20 min (1 vol Coomassie blue diluted in 3 vol destaining solution), destained in 25% methanol, 7% acetic acid for 3 h and finally equilibrated in distilled H2O for 3-12 h. Ligand blotting assays were performed as described (21).
SDS-polyacrylamide gels (39) were stained with silver as described (40).
RESULTS
Isolation of the ProT[alpha]-H1 complex from NIH 3T3 cell extracts
To obtain further evidence for the interaction between ProT[alpha] and histone H1, we have attempted to co-isolate ProT[alpha] and histone H1 from crude cell extracts by immunoaffinity chromatography. Affinity-purified anti-ProT[alpha] antibodies (anti-ct) were covalently linked to protein A-agarose and the column matrix was incubated with extracts of NIH 3T3 cells (for details see Materials and Methods). The bound proteins were eluted by applying to the column 0.3, 0.6 and 1.0 M NaCl and analysed by western blotting using the affinity-purified anti-ct and anti-H1 antibodies. ProT[alpha] was detected in the 0.6 M fraction of the immunoaffinity column (Fig.
Figure 1. Immunoisolation of the ProT[alpha]-histone H1 complex from NIH 3T3 cell extracts. (A) Western blotting analysis of the material eluted from the anti-ct protein A-agarose column. The fractions eluted from the anti-ct protein A-agarose column with 0.3, 0.6 and 1.0 M NaCl (lanes 1-3) or the non-specific IgG protein A-agarose column with 0.6 and 1.0 M NaCl (lanes 4, 5) were divided into two and analysed as follows. (Upper) The samples were fractionated by 15% SDS-PAGE and transferred onto glutaraldehyde-activated nitrocellulose membranes as described in Materials and Methods. The blot was probed with affinity-purified anti-ct antibody (1:1000) and then with anti-rabbit IgG-peroxidase (1:5000). Lane PT[alpha] indicates 0.1 µg purified ProT[alpha] as a control. (Lower) The second part of the samples was analysed by western blotting using affinity-purified anti-H1 antibody (1:20) and then with anti-rabbit IgG-peroxidase as described in Materials and Methods. Lane H1 contains 0.2 µg histone H1 as a control. (B) Western blotting analysis of the material eluted from the ProT[alpha]-agarose column with 0.3, 0.6 and 1.0 M NaCl (lanes 1-3). The blot was probed with affinity-purified anti-H1 antibody as described above. (C) Specificity of the affinity-purified anti-H1 and anti-ct antibodies. An aliquot of 0.2 µg H1 (lane 1) and cell extract from 8 × 105 NIH 3T3 cells (lane 2) were fractionated by 15% SDS-PAGE, electrotransferred onto nitrocellulose membranes and probed with affinity-purified anti-H1 antibody (1:20). For detection of ProT[alpha], 0.2 µg ProT[alpha] and NIH 3T3 cell extract (8 × 105 cells) were electrotransferred onto glutaraldehyde-activated nitrocellulose membranes and probed with affinity-purified anti-ct antibody (lanes 3 and 4), as described in Materials and Methods. Reactions were revealed by ECL.
Identification of interacting sites between ProT[alpha] and histone H1
To further characterize the interaction of ProT[alpha] with histone H1, we employed a bandshifting assay. Due to their charge characteristics, histone H1 and ProT[alpha] migrate to opposite electrodes when electrophoresed on an agarose gel at neutral pH (Fig.
Figure 2. ProT[alpha] interacts with the globular domain of histone H1. (A) Purity of histone H1 and GH1 derived from H1 by trypsin digestion. Aliquots were resolved by 15% SDS-PAGE and stained with Coomassie blue. M, molecular weight markers. (B) An electrophoretic mobility shift assay reveals contacts between ProT[alpha] and GH1. The following amounts of proteins were incubated as detailed in Materials and Methods and electrophoresed on a 0.75% agarose gel. Lane 1, 3 µg H1; lane 2, 1.5 µg H1 + 6 µg ProT[alpha]; lane 3, 3 µg H1 + 6 µg ProT[alpha]; lanes 4-6, 6 µg ProT[alpha] + 6, 3 or 1.5 µg cytochrome c (Cyt) respectively; lane 7, 3 µg cytochrome c; lanes 8 and 11, 6 µg ProT[alpha]; lane 9, GH1 peptide equimolar to 8 µg H1 + 6 µg ProT[alpha]; lane 10, GH1 as in lane 9. In order to gain an insight into the binding site of ProT[alpha] within the H1 molecule, we treated H1 with trypsin. Limited trypsinization of H1 results in degradation of the non-folded protein tails, while the central globular domain (GH1) resists degradation (33; Fig. In a previous work we suggested that the acidic domain of ProT[alpha] was involved in the interaction with H1, based on the inhibitory effect of polyglutamic acid on ProT[alpha]-H1 binding (21). In order to test this prediction directly, we used as diagnostic tools three peptides modeled after the published amino acid sequence of bovine thymus ProT[alpha] (18): the acidic peptide, comprising the first 18 amino acid residues of the acidic region (residues 52-69), and the peptides thymosin [alpha]1 and ct (residues 1-28 and 87-109 respectively), which served as controls. Binding of these peptides to histone H1 was analysed by two independent methods: (i) ligand blotting assays, employing biotinylated peptides as probes; (ii) chemical crosslinking using DMP as a crosslinker. Ligand blotting assays showed that only the acidic peptide bound to histone H1 in a dose-dependent manner (Fig. Figure 3. Identification of interacting sites within ProT[alpha]. (A) Far western analysis. Aliquots of 1 µg histone H1 (H1) and 40 µg total nuclear protein (Nu) were resolved by 15% SDS-PAGE and electrotransferred onto nitrocellulose membranes. The filters were incubated with the indicated concentrations of biotinylated ProT[alpha] or the biotinylated peptides acidic peptide, thymosin [alpha]1 and ct. Bound peptides were visualized with a streptavidin-peroxidase conjugate and the ECL technique. (B) Crosslinking analysis. An aliquot of 1 µg histone H1 (lane 1) was incubated with the indicated amounts of ProT[alpha] (lanes 2-5), thymosin [alpha]1 (lanes 6-8), acidic peptide (lanes 9-12) or ct peptide (lanes 13-15). Interacting partners were crosslinked with DMP and analysed by SDS-PAGE and Coomassie blue staining. Numbers to the left indicate the molecular weight markers in kDa. (C) Species specificity of the interaction. Aliquots of 0.1, 0.2 and 0.4 µg calf H1 (lanes 1-3) and 0.05, 0.1, 0.2 and 0.3 µg Drosophila histone H1 (lanes 4-7) were separated by 15% SDS-PAGE and transferred onto nitrocellulose filters. The membranes were incubated in blocking buffer (TBST with 0.2% gelatin) for 12 h and then with 0.2 µg/ml ProT[alpha] for 1.5 h at room temperature. The filters were probed with affinity-purified anti-ProT[alpha] antibody (anti-ct) and anti-rabbit IgG-peroxidase conjugate (panel ProT[alpha]). Reactions were revealed by ECL. Subsequently the membrane was stained with india ink to check for protein loading (panel ink). The interaction between histone H1 and ProT[alpha] displayed a remarkable species specificity: ProT[alpha] exhibited considerably reduced affinity for Drosophila H1 compared with calf H1 (Fig. 
ProT[alpha] does not interact with H1 in chromatin
Our analysis of the ProT[alpha]-histone H1 interaction in chromatin was based on a chromatin reconstitution system using extracts derived from Drosophila embryos (35). Because of the low affinity binding of ProT[alpha] to Drosophila histone H1, we used calf histone H1 for all chromatin reconstitution experiments. Faithful incorporation of calf H1 into chromatin was verified by visualizing the characteristic increase in nucleosome repeat length (NRL) (29,35,41). Analysis of reconstituted chromatin by partial digestion with micrococcal nuclease (MNase) demonstrated a gradual increase in NRL from 185 to 205 and 215 bp when increasing amounts of calf histone H1 were titrated (Fig.
Figure 4. ProT[alpha] does not interact stably with H1-containing chromatin. (A) Reconstitution of chromatin with varying stoichiometries of H1. Plasmid DNA was assembled into chromatin with Drosophila extracts without H1 (0 units) or in the presence of the indicated units of calf histone H1. Incorporation of H1 was determined from the accompanying change in nucleosome repeat length (185, 205 and 215 bp). Reconstituted chromatin was digested with MNase for 0.5, 1 or 5 min. Purified DNA was analysed on a 1.3% agarose gel and stained with ethidium bromide. White circles mark the tetranucleosome-derived DNA, visualizing the increase in NRL. M, 123 bp ladder (BRL). (B) ProT[alpha] does not interact stably with H1-containing chromatin. Chromatin was reconstituted in the presence of 2 U calf histone H1 on 100 ng linearized plasmid DNA, immobilized onto paramagnetic beads. Magnetically purified chromatin was incubated with ProT[alpha] and purified again. Proteins in the supernatant were precipitated with acetone and the chromatin beads were subjected to salt extraction with 50, 400 and 650 mM NaCl. Western blot analysis with anti-ct antibody detects ProT[alpha] exclusively in the supernatant (lane 1) and not in the salt-extracted proteins or the remaining chromatin beads (lane 5). Lane 6 contains 0.2 µg ProT[alpha] as a positive control (C). In order to investigate whether ProT[alpha] was able to associate directly with H1-containing chromatin, we reconstituted chromatin on immobilized DNA (29), purified it magnetically and incubated it with ProT[alpha]. The chromatin beads were re-isolated from the reaction and the associated non-histone proteins were eluted with buffer containing 50, 400 or 650 mM NaCl. Immunoblotting analysis detected ProT[alpha] exclusively in the supernatant of the binding reaction (Fig. 
ProT[alpha] modulates the interaction of H1 with chromatin
The fact that the GH1 domain of H1 is a potential binding site for ProT[alpha] raises the possibility that ProT[alpha] may actually modulate the interaction of H1 with chromatin. To visualize this competition, Drosophila chromatin was first assembled on immobilized DNA in the presence of histone H1, which was subsequently washed off the beads again with 650 mM NaCl, in order to create a nucleosomal array with available binding sites for H1. After equilibration at a lower salt concentration, the chromatin beads were incubated with 5 U H1 (or 2 U, data not shown) in the absence or presence of increasing concentrations of ProT[alpha] (see Materials and Methods). When ProT[alpha] was titrated into the reaction it efficiently prevented interaction of bulk H1 with chromatin, which was recovered in the supernatant (Fig.
Figure 5. ProT[alpha] affects the binding of histone H1 to chromatin. Chromatin with free H1 binding sites was reconstituted on immobilized DNA and then incubated with 5 U calf histone H1, in the absence of ProT[alpha] (panel 0) or in the presence of ProT[alpha] at ProT[alpha]:H1 ratios of 1, 2 and 5 (w/w) (panels 1, 2 and 5). Supernatants were removed and the beads were washed three times with 20 mM Tris-HCl, 100 mM NaCl, 0.2% NP-40. Proteins in the supernatant (S), the last wash (W) and remaining on the beads (B) were fractionated by 15% SDS-PAGE and stained with silver. Numbers to the left indicate the molecular masses in kDa. The arrow points to histone H1. In a second approach, ProT[alpha] was directly added to a chromatin assembly mixture with or without histone H1. This time incorporation of H1 was followed by the characteristic change in NRL (Fig. Figure 6. ProT[alpha] modulates the interaction of H1 with chromatin. DNA was assembled into chromatin in the Drosophila extracts without histone H1 (panels 1 and 2) or with 2 U histone H1 (panels 3 and 4) in the absence (panels 1 and 4) or presence of 6 µg ProT[alpha] (panels 2 and 3). The NRL was determined as in Figure 4A.
ProT[alpha] extracts a fraction of histone H1 from chromatin but not from free DNA
Finally, we asked whether ProT[alpha] could detach histone H1 from chromatin or free DNA. Chromatin was assembled with 5 U histone H1, purified magnetically and excess histone H1 was washed off the beads. Incubation of these beads with increasing concentrations of ProT[alpha] released significant amounts of H1 from chromatin (Fig.
| Figure 7. ProT[alpha] strips histone H1 from chromatin. (A) Chromatin was assembled on immobilized DNA with 5 U histone H1 and traces of 32P-phosphorylated H1, purified magnetically, washed with 100 mM NaCl and incubated with buffer alone (panel 0) or increasing amounts of ProT[alpha] [indicated ratios of ProT[alpha]:H1 (w/w)]. Chromatin was isolated again and the proteins in the supernatant (S), those removed by washing with 100 mM salt (W) and those remaining stably bound on the beads (B) were separated by 15% SDS-PAGE. The gel was autoradiographed (upper) and stained with silver (lower). Numbers to the left indicate positions of molecular weight markers in kDa. The arrow marks the position of H1. (B) As (A) with the following modifications. Chromatin was reconstituted with 1, 2 and 5 U histone H1 and challenged with ProT[alpha] at a ProT[alpha]:H1 ratio of 5 (w/w). (C) As (B) except that Drosophila H1 was substituted for calf H1. In contrast to calf H1, Drosophila H1 is not released from chromatin in the presence of ProT[alpha] (see B). |
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Importantly, the H1 molecules that were stripped from chromatin by ProT[alpha] did not represent linker histones bound to non-nucleosomal DNA, because ProT[alpha] was unable to dissociate histone H1 from naked DNA. H1-DNA complexes were formed after incubation of 1.0 µg immobilized DNA with 1.3 µg histone H1 and challenged with increasing concentrations of ProT[alpha] (Fig.
Figure 8. ProT[alpha] does not extract histone H1 from naked DNA. Histone H1 (1.3 µg) was incubated with 1.0 µg DNA to form H1-DNA complexes (see Materials and Methods). After incubation, the beads were washed with 150 mM NaCl and incubated with buffer alone (panel 0) or with ProT[alpha], at ProT[alpha]:H1 ratios of 1, 2 and 5 (w/w). Proteins in the supernatant (S), those released during the last wash step (W) and those remaining on the beads were analysed by 15% SDS-PAGE and stained with silver. (C) Input of histone H1 (1.3 µg). These results point to a particular mode of interaction of H1 with chromatin that is sensitive to extraction by ProT[alpha].
DISCUSSION
ProT[alpha] is amongst the most abundant proteins in the mammalian nucleus (4-6), at amounts matching those of core histones (17). Yet, the function of this conserved, small and acidic polypeptide remains elusive. Using a variety of assays we have demonstrated an interaction between ProT[alpha] and linker histone H1, which is dependent on concentration (0.05-0.2 µg/ml) and temperature (21). In this concentration range ProT[alpha] does not bind to other highly charged proteins, such as core histones (21) or cytochrome c (this study), but in a higher concentration range binding of ProT[alpha] to core histones has been reported (42). It is noteworthy that biotinylated ProT[alpha] also retains its ability to interact with histone H1. In this study, we have isolated ProT[alpha] by immunoaffinity chromatography from crude cell extracts. By analysing the immunopurified material, we have shown that histone H1 has been retained by the anti-ProT[alpha] column. The ability of ProT[alpha] to interact with histone H1 may therefore provide insight into its physiological function.
In this work we analysed the effects of ProT[alpha] on the interaction of histone H1 with chromatin using a cell-free system for chromatin reconstitution under physiological conditions (35) on bead-immobilized DNA (29,43). ProT[alpha] failed to interact stably with chromatin in the presence or absence of H1. In addition, it did not bind DNA or H1-coated DNA directly (not shown). Furthermore, ProT[alpha] and purified calf thymus polynucleosomes did not co-fractionate during sedimentation in a sucrose gradient (data not shown).
Incorporation of increasing amounts of histone H1 into chromatin results in a characteristic increase in the nucleosome repeat length (NRL), a measure of the linker length that separates adjacent nucleosomes (35,41). If H1 is titrated into a chromatin assembly reaction, the NRL is increased to the physiological value of ~200 bp, but further addition of H1 results in even wider nucleosome spacing, with NRLs reaching 220 bp (35). Remarkably, the presence of ProT[alpha] during chromatin assembly did not affect incorporation of sufficient H1 to increase the NRL to the physiological value of ~200 bp, but prevented association of excess H1. In a complementary experiment, ProT[alpha] was able to detach a fraction of H1 from chromatin if excess H1 (over the amount required to establish the physiological spacing, i.e. 4-5 U) had been incorporated. In contrast, the amount of H1 required to increase the NRL to 200 bp resisted extraction by ProT[alpha]. These results suggest the presence of at least two distinct interaction modes of H1 with chromatin that can be distinguished by their sensitivity to ProT[alpha] extraction.
Since substoichiometric amounts of H1 gradually increase the nucleosomal spacing (44), it is difficult to derive information about the H1/nucleosome stoichiometry from NRL measurements. However, there is evidence for varying H1 stoichiometry in vivo with values exceeding one per nucleosome (26) and several studies have suggested that NRLs beyond 200 bp may reflect the binding of a second H1 molecule to a nucleosome core (45,46). Recently, Nightingale et al. (47) showed that more than one molecule of histone H1 may associate with a nucleosome, with secondary sites having a lower affinity, comparable with that of the association of histone H1 with naked DNA. Importantly, despite the lower affinity of H1 for naked DNA compared with chromatin (48), ProT[alpha] was not able to release H1 from naked DNA. This suggests that in vitro association of excess H1 with chromatin is not simply due to binding to naked DNA, but is rather due to a different type of interaction. ProT[alpha] may, therefore, regulate the extent and mode of association of H1 with chromatin.
The structural and functional significance of varying histone H1 stoichiometry in chromatin is presently unclear. However, given the fact that H1 is a major determinant of chromatin fibre folding (25,49-51) and that H1-nucleosome interactions are profoundly altered in active chromatin (24), varying H1 stoichiometry may indicate distinct higher order structures and/or functional states. Evidence is accumulating for a key role of histone H1 in transcriptional repression of a selected group of genes (22,27,28,30,52). Gene activation during development or in rapid response to inducers may require the selective dissociation of H1 from specific target sites. While our data do not support a role of ProT[alpha] in active release of all histone H1 from chromatin, they are compatible with a role as an acceptor for H1 molecules that are stripped from chromatin by other activities. Recently, Dimitrov and Wolffe (53) showed that nucleoplasmin, an abundant protein in early developmental stages of Xenopus, was able to release histone H1 from chromatin. Interestingly, ProT[alpha] resembles nucleoplasmin with respect to the highly acidic region that associates with histone H1.
Another potential function for ProT[alpha] is suggested from the observation that its concentration is particularly high in proliferating cells (3,9-11,17). The assembly of newly replicated DNA into chromatin in dividing cells requires the faithful incorporation of histone H1. By analogy to the core histones, which are kept soluble in complexes with acidic carriers that ensure their faithful assembly into nucleosomes (54-57), it is possible that freshly synthesized linker histones associate with carrier molecules that ensure the proper association of H1 with nucleosomal fibres. The highly charged linker histone, if not safeguarded by a protein, is expected to associate with DNA and other proteins in a manner that could be harmful to the cell. We have shown that ProT[alpha] will allow association of sufficient H1 to increase the NRL to the physiological value of 200 bp, but will prevent further association of H1. It will also detach H1 from low affinity binding sites on chromatin that differ from naked DNA. These results are compatible with a role of ProT[alpha] as an H1 carrier molecule. This hypothesis is supported by recent studies which showed that ProT[alpha] is present in the cell nucleus throughout the cell cycle, with highest levels at S phase, and its gene promoter can be strongly induced by transcription factor E2F (16). E2F is an important component of the mechanism which controls progression of cells into S phase, while the targets for its action include genes encoding proteins directly involved in DNA replication and cell cycle regulation (58).
ACKNOWLEDGEMENTS
We thank K.Nightingale for critical reading of the manuscript and many helpful suggestions and Dr S.Georgatos for useful discussions. R.S. acknowledges the receipt of an EMBO post-doctoral fellowship. This work was supported in part by PENED 289/1995 to T.P. C.-Y.L. is a Fulbright Senior Scholar
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G. Covelo, C. S. Sarandeses, C. Diaz-Jullien, and M. Freire
Prothymosin {alpha} Interacts with Free Core Histones in the Nucleus of Dividing Cells
J. Biochem.,
November 1, 2006;
140(5):
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[Abstract]
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