Prothymosin α binds histones in vitro and shows activity in nucleosome assembly assay

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ELSEVIER

Prothymosin

Biochimica et Biophysica Acta 1296 (1996) 219-227

Biochi~ic~a et BiophysicaAEta

binds histones in vitro and shows activity in nucleosome assembly assay

Cristina Diaz-Jullien, Antonio P6rez-Est6vez, Guillermo Covelo, Manuel Freire

*

Departamento de Bioqufmica y Biologfa Molecular, Facultad de Biolog&, Universidad de Santiago, 15706, Santiago de Compostela, Spain Received 9 January 1996; revised 22 March 1996; accepted 26 April 1996

Abstract

Prothymosin c~ (ProTc~) is a polypeptide which appears to be involved in cell proliferation, though its precise function has yet to be identified. Here, we report experiments which show that calf ProTa selectively binds to core histones and histone H1 in vitro. Characterization of these interactions by various procedures (including affinity chromatography on ProTa-Sepharose columns, immunoblotting assay and investigation of the behaviour of mixtures of ProTc~ and histones in solution) indicated that ProTa has higher affinity for core histones (particularly H3 and H4) than for HI. Similarities between the histone-binding patterns of ProTa and of poly(glutamic acid) suggest that the observed histone-binding capacity resides largely in the acidic central region of ProTc~. However, all five histones were also bound by Ta 1 (a peptide corresponding to the first 28 amino acids of ProTa); histone binding by the N-terminal region of ProTa thus cannot be ruled out. Phosphorylation of ProTet does not appear to affect these interactions. In accordance with the observed capacity for histone binding, ProTa (in conjunction with ATP and some ProTa-binding factor/s in a thymocyte extract) was able to induce in vitro nucleosome assembly. We discuss the possibility that ProTa plays a role in chromatin remodelling. Keywords: Prothymosin a; Histene binding; Nucleosome assembly; (Bovine)

I. Introduction

Prothymosin c~ (ProTc~), a polypeptide with a highly conserved structure (109-111 amino acids) [1] which is widely distributed in animals and especially abundant in lymphoid tissues of mammals [2-4], epitomizes the difficulties often encountered in elucidating the biological role of proteins. Characterized as the precursor of thymosin ~ 1 (ToL 1) [5-7] more than 15 years ago, its function remains largely unknown, though the patterns of expression of the P r o T a gene suggest a role in the cell cycle [3,8,9]; indeed, translation-inhibition studies using antisense RNA indicate that it is essential for cell proliferation [10]. Furthermore, ProToL gene expression is regulated by the c-myc protein [11,12], likewise supporting a role in the cell cycle. However, ProTo~ is characteristically present at high concentrations (particularly in mammalian cells; see [2,4]), which argues against a direct regulatory role. Certain

Abbreviations: ProTa, prothymosin ~; Ta 1, thymosin ~j; CK-2, casein kinase II; PGA, poly(glutamic acid); APTCE, affinity-purified thymocyte cytosolic extract. * Corresponding author. Fax: + 34 81 596094.

structural characteristics of P r o T a - - namely the karyophilic signal [13] and central acidic domain [1], together with immunolocalization data [14] and results indicating nuclear targeting [15-17] - - all suggest that P r o T a exercises its function in the nucleus. P r o T a contains nine phosphorylation sites for caseinkinase-like enzymes. We have previously demonstrated that P r o T a is phosphorylated in vitro by casein kinase 2 (CK-2) at Ser and Thr residues between amino-acid positions 1 and 14 inclusive [18]. Subsequently, we showed that the same 14-residue fragment is phosphorylated in vivo in various cell systems, with a phosphorylation activity which is positively correlated with cell proliferation activity [19]. However, these results suggested that in vivo (unlike in vitro) only Thr residues are phosphorylated, indicating that CK-2 may not be responsible for the observed in vivo phosphorylation. A subsequent study by Sburlati et al. [20] broadly confirmed our results, though these authors reported in vivo phosphorylation of Ser rather than Thr residues. Controversy as regards the pattern of phosphorylation of P r o T a , however, remains peripheral to the main problem, that of elucidating its role in cell division. One

0167-4838/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 8 3 8 ( 9 6 ) 0 0 0 7 2 - 6

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possible approach to this problem, currently being explored in our laboratory, is to use affinity chromatography to identify cell components which interact specifically with ProTot in vitro. Here, we report results which indicate that histones from calf lymphocytes bind selectively to ProTot. We also report experiments aimed at characterization of the observed in vitro interaction between ProTot and histones, and at elucidation of possible functional implications.

2. Materials and methods 2.1. Materials

Individual calf thymus histones were purchased from Boehringer-Mannheim. Poly(L-glutamic acid) was from ICN (mol. mass over 60 kDa) and from Sigma (mol. mass 12 kDa). CNBr-activated Sepharose 4B was from Pharmacia. The anti-histone monoclonal antibody, ECL Western blotting detection reagents and DNA topoisomerase I were from Amersham. Synthetic thymosin otl ( T a l ) was kindly provided by Dr. E.P. Heimer (Peptide Research Department, Hoffman-La Roche, Nutley, N J, USA) 2.2. Isolation o f prothymosin a

ProTot was isolated from calf thymocytes by HPLC as described previously [21]. The isolated ProTot was then treated with alkaline phosphatase to remove phosphate residues and finally purified again by HPLC. Phosphorylated ProToL was obtained from calf thymocytes stimulated with Con A and IL-2 as reported previously [19]. 2.3. Preparation o f thymosin otl antiserum

Rabbits were injected with synthetic Ta~ coupled to keyhole limpet haemocyanin as described previously [22]. Antisera were assayed by peroxidase-linked immunosorbent assay for anti-ProTa and anti-Ta 1 antibodies [23]. 2.4. Preparation o f cytosolic and nuclear extracts

Calf thymocytes or splenic lymphocytes obtained as described previously [19] were suspended in ice-cold 20 mM Tris buffer (pH 7.6) containing 150 mM NaC1, 1.5 mM MgCI2, 0.5% NP-40 and 1 Ixg/ml each of pepstatin and leupeptin, and mechanically disrupted with a Potter homogenizer (3 strokes). Nuclei were pelleted by centrifugation at 2000 × g for 10 min, and the supernatant was centrifuged at 100000 X g for 1 h to obtain the cytosolic fraction. Nuclei were then fractionated as described by Davis and Blobel [24]. Briefly, the nuclear pellet was resuspended in 10 mM Tris buffer (pH 8.5) containing 10% sucrose, 0.1 mM MgC12, lmM DTT and 0.5 mM PMSF, then treated for 1 h with 160 Ixg/ml and 20

~ g / m l of DNase and RNase respectively. After centrifugation at 2 0 0 0 0 × g for 10 min, the supernatant was collected for use as the nuclear fraction. 2.5. Affinity chromatography

ProTa, T~I,, BSA and poly(glutamic acid) (mol. mass 12 kDa) were conjugated to Sepharose by the standard procedure (Pharmacia) at a protein concentration of 1 m g / m l of matrix. The coupling efficiency, as determined by colorimetric assay [25] of non-bound protein, was always over 85%. Before ProTot-affinity chromatography of cytoplasmic and nuclear extracts, the extract was passed through a BSA-Sepharose column (about 10 mg of extract per mg of Sepharose-bound BSA). The flow-through fraction was then loaded onto a ProTa-Sepharose column containing about 0.85 mg of matrix-bound ProTot. Retained proteins were eluted with 0.3 M then 1 M NaC1 in column buffer (20 mM Tris, pH 7.6, containing 5% glycerol). The amount of eluted protein was determined by colorimetric assay after dialysis and concentration by centrifugation in Amicon microconcentrators. Analysis was done by SDS-PAGE [26], and gels were stained with Coomassie brilliant blue. For ProTa-affinity chromatography of histones, total histone mixtures, in PBS, were loaded onto a ProTo~-Sepharose column (1 ml bed volume) at various concentrations with respect to the amount of matrix-bound ProTot, and the column was then sequentially eluted with 0.2 M, 0.5 M and 2 M NaC1 in PBS. The eluates were dialysed and concentrated as above, and analysed by SDS-PAGE according to Hames [27], in order to obtain a better resolution of the five histones. Chromatography on Tot l-Sepharose and poly(glutamic acid)-Sepharose columns was as described for the ProTotSepharose column. All operations were carried out at 4°C. 2.6. Electroblotting and immunodetection

For immunoblotting assays of the cytosolic and nuclear components purified by ProTo~-Sepharose chromatography, the purified extracts were separated by SDS-PAGE under the conditions described by Hames [27] and transferred onto nitrocellulose membranes in a semi-dry Novablot transfer system (Pharmacia) at 10 V for 1 h at room temperature. Blots were blocked with PBS containing 5% non-fat dry milk and 0.02% sodium azide, then incubated overnight with anti-histone monoclonal antibodies (5 p~g/ml in blocking buffer) and peroxidase-linked antimouse IgG. Detection was performed with ECL detection reagents according to the manufacturer's instructions. For immunodetection of ProTa- or T a 1-histone interactions, histone H1 and core histones (10 Ixg each) were resolved in SDS-PAGE and transferred onto nitrocellulose membranes as described above. Blots were incubated with ProTa (25 or 50 ~ g / m l in blocking buffer) or Tot I (25 txg/ml) for 1 h at room temperature, washed four times

C. D{az-Jullien et al. / Biochimica et Biophysica Acta 1296 (1996) 219-227

with blocking buffer, incubated with anti-Tet 1 (1:100) for 1 or 2 h, or non-immune serum for 2 h, and further incubated with peroxidase-coupled anti-rabbit IgG for 1 h. Detection was with 3,3'-diaminobenzidine. 2.7. Binding o f ProT~ to histones in solution

Total histone mixtures (leach containing 10 Ixg of HI and of each of the core histones in 10 txl of PBS) were mixed with 2.5, 5, 10, 25 or 50 Ixg of ProTa, in PBS, to a final volume of 30 Ixl. The mixtures were incubated for 10 min at room temperature then microfuged for 5 min. Supernatants were collected and the pellets washed with 20 Ixl of PBS. Both fractions were analysed by SDS-PAGE as described in Section 2.6. 2.8. Nucleosome assembly assay

Covalently closed circu]lar DNA (0.2 Ixg) of pBR322 was preincubated with 15 units of topoisomerase I for 30 min at 37°C in 15 Ixl of 35 mM Tris-HC1 (pH 8), 72 mM KCI, 5 mM MgC12, 5 n ~ l DTF, 5 mM spermidine and 0.01% BSA as recommended by the enzyme supplier. A mixture of core histones (0.1 ixg each) was preincubated with ProTa - - in the presence or absence of (a) ATP (8 I~M) a n d / o r (b) ProTon-affinity-purified cytosolic or nuclear extracts from calf thymocytes or splenic lymphocytes - - for 15 min at 37°C in 15; Ixl of 10 mM Tris-HC1 (pH 8), 1 mM EDTA, 150 mM NaC1 and 100 p,g/ml of BSA [28]. The DNA-containing and h!istone-containing mixtures were then combined and the mixture was incubated for a further 45 min. After this time, SU,S and proteinase K were added (to final concentrations of 0.2% and 100 i~g/ml, respectively), and the mixture was incubated for 15 min. DNA was purified by phenol/chloroform extraction and electrophoresed in 1% agarose gel in an 89 mM Tris, 89 mM borate, 2.5 mM EDTA buffer system. DNA molecules that form nucleosomes gain nelgative superhelical turns, while those that fail remain as relaxed circles [29].

3. Results 3.1. Binding o f lymphocyte components to ProTa

In the first series of experiments, we used affinity chromatography of cell extracts to identify cell components which interact with ProTe~. Thymic and splenic lymphocyte extracts were used, since in mammals ProTa concentration is higher in these cell types than in cells from other tissues [3,4]. Nuclear and cytosolic extracts were previously passed through BSA-Sepharose columns to eliminate non-specific interactions; the flow-through fractions from these columns were then loaded onto ProTot-Sepharose columns and the retained components eluted with increasing concentrations of NaCI. As can be

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seen from Fig. 1, most proteins in the thymic extracts were not retained (lanes 1), whereas a few showed moderate (lanes 2) or strong (lanes 3) affinity for ProTet. Similar results were obtained with splenic lymphocyte extracts (not shown). ProTa-binding proteins made up about 0.56% of the total protein content of the thymocyte cytosolic extract (see Fig. 1A, lanes 2 and 3), and about 0.12% of the total protein content of the thymocyte nuclear extract (see Fig. 1B, lane 3). Among the ProTa-binding components detected in both the cytosolic and the nuclear extracts are a group of proteins with molecular mass in the 15-22 kDa range, similar to that of core histones. Analysis of these proteins by SDS-PAGE under conditions in which histones are well resolved (see Section 2.5) revealed electrophoretic mobilities identical to those of core histones, while corresponding bands were not detected in SDS-PAGE analysis of the flow-through fraction (not shown). Immunoblotting assays with an anti-histone monoclonal antibody confirmed the presence of core histones among the cytosolic and nuclear components with affinity for ProToL (Fig. 1C). Note that the detergent-based extraction procedure used for lymphocyte disruption means that some nuclear components may have ended up in the cytosolic fraction, as may have occurred with the group of proteins at about 105 kDa (Fig. 1, lane 3) and with the core histones. 3.2. ProTa-histone interactions

To characterize the observed ProTa-histone interactions in greater detail, we performed experiments based on affinity chromatography and immunoblotting, using commercially supplied calf histones. In the first experiment, a mixture of H1 and core histones was chromatographed on a ProTa-Sepharose column, with a 1:1 ( w / w ) ProTa-tohistone loading ratio. Electrophoretic analysis of the various eluate fractions (Fig. 2A) confirmed that ProTe~ binds to all five histones, since no histones were detected in the non-retained fraction (lane 1). However, binding affinity differed among histones: HI was eluted with 0.2 M NaC1, whereas elution of H2A and H2B required 0.5 M NaC1 and elution of most of the H3 and H4 required 2 M NaC1 (Fig. 2A, lanes 2-4). Similar patterns were obtained with higher ProTo~-to-histone loading ratios; with lower ratios, however, histones were detected in the non-retained fraction (results not shown). In control experiments on a BSA-Sepharose column, with various BSA-to-histone loading ratios, none of the histones were retained (results not shown). ProTa-histone interaction was also confirmed by immunoblotting analyses using an antibody raised against synthetic T~ l (i.e., the first 28 amino acids of ProTtx [7]). The reactivity of this antibody with ProTtx has been conclusively demonstrated [21,23]. The results of these analyses (Fig. 2B, lanes 2-4) confirm that ProTa, at concentrations ranging between 25 and 1 ~ g / m l , binds to all five histones. In the range of 0.1 Ixg/ml of ProTe~, no

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Fig. 1. Affinity chromatography of cell extracts. Ten mg of protein of the flow-through fractions from BSA-Sepharose chromatography of cytosolic (Panel A) or nuclear (Panel B) extracts of calf thymic lymphocytes was chromatographed on a ProTa-Sepharose column; the retained components were eluted with increasing concentrations of NaCI. An aliquot of the non-retained components, and the total amount of retained components were analysed by SDS-PAGE according to Laemmli procedure [26]. Lanes 1: cytosolic (35 ~g) and nuclear (20 o,g) components in the non-retained fractions. Lanes 2: cytosolic (20 p,g) and nuclear (2 txg) components in fractions eluted with 0.3 M NaC1. Lanes 3: cytosolic (30 I~g) and nuclear (10 p,g) components in fractions eluted with 1 M NaC1. Panel C shows the immunoblotting analysis (see Section 2) of 10 ~g of cytosolic (lane 2) and 10 I~g of nuclear (lane 3) components strongly retained in ProTct-Sepharose columns transferred onto nitrocellulose membranes and immunodetected with anti-histone antibodies. Lane 1 shows a mixture of the five histones (12 Ixg) runned in parallel and stained with Amido black.

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Fig. 2. Interaction of ProTct with histones. (A) Affinity chromatography. A mixture of the five histones (50 Ixg of each) was chromatographed on a ProTc~-Sepharose column. The retained components were eluted stepwise with increasing concentrations of NaCI in PBS, and analysed by SDS-PAGE as described in Fig. 1. Lane 1, nonretalned fraction; lanes 2 to 4, fractions eluted with 0.2 M, 0.5 M and 2 M NaC1, respectively. (B) Immunodetection of ProTct-histone interactions. The five histones (10 ~g of each) were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The blots were incubated with 25 txg/ml of ProTct (lanes 2 and 7), 10 I.Lg/ml of ProTet (lanes 3 and 5), 1 i~g/ml (lane 4) or without ProTet (lane 6), washed, and then incubated with anti-Ttx i for 2 h (lanes 2, 3,4 and 6) or 1 h (lane 5) or with non-immune serum for 2 h (lane 7). Finally, peroxidase-coupled anti-rabbit IgG was added and the reaction was revealed with 3,3'-diaminobenzidine (see Section 2). Lane 1 shows Amido black staining of the membrane.

C. Diaz-Jullien et al. / Biochimica et Biophysica Acta 1296 (1996) 219-227 interactions were immunodetected. The binding pattern observed when the time of incubation with anti-Tot 1 was reduced from 2 to 1 h (Fig. 2B, lane 5) indicated that P r o T a shows a greater affinity for H3 and H4 than for the other histones, as was suggested by the results of affinity chromatography. No interaction was observed when 1 I x g / m l of ProTot was used in the 1 h incubation. In control experiments without P r o T a (Fig. 2B, lane 6) or using non-immune serum instead of anti-Ta 1 (Fig. 2B, lane 7), no histone bands were stained. 3.3. Location o f histone-binding sites on ProTa Structural similarities between ProTot and histone-binding proteins [13] suggest that the P r o T a domain most likely to be responsible for the observed interaction with histones is the 42-residue ce,ntral acidic region (see Fig. 3). However, in immunoblotting-based experiments with thymosin et ~ (Te~ 1), which con:esponds to the first 28 residues of P r o T a (see Fig. 3), this peptide ( p I 4.2) appeared to bind to all five histones (results not shown), suggesting that the N-terminal region of ProTa may interact with histones. To investigate this possibility, and also to assess

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the possible influence of the acidic region on interaction with histones, we performed affinity chromatography experiments using Sepharose columns to which T a 1 or poly(glutamic acid) - - w h i c h mimics the central acidic region of ProTe~ - - had been coupled. Experimental conditions were as for ProTet-affinity chromatography. The results of these analyses (Fig. 3) indicated that both T a I and poly(glutamic acid) are capable of interacting with all five histones. Tet 1, unlike P r o T a , showed similar affinity for each of the five histones (Fig. 3A). In addition, the amount of histones in the non-retained fraction (Fig. 3A, lane 1) suggests that the capacity of Tct I to bind histones (at a 1:1 loading ratio) is about half that of ProTet. The pattern of binding of poly(glutamic acid) to histones, on the other hand, was similar to that of ProTet, both in terms of amounts bound and relative affinities for the different histones (Fig. 3B). 3.4. Interaction o f ProTa with histones in solution In experiments initially designed to investigate interactions between ProTot and histones in solution, we observed appreciable precipitation on mixing solutions of lO

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Fig. 3. Interactions of Tot t and poly(glutamicacid) (PGA) with histones in affinity chromatography.A mixture of the five histones (40 p,g of each) was chromatographed on a TarSepharose column (panel A) or a PGA-Sepharose column (panel B). The retained components were eluted stepwise with increasing concentrations of NaCl in PBS, and analysed by SDS-PAGE as described in Section 2. Lane 1, non-retained fraction; lanes 2 to 4, fractions eluted with 0.2 M, 0.5 M and 2 M NaC1, respectively. The amino-acid sequence of ProTot is shown at the top of the figure: residues 1-28 (the N-terminal region correspondingto Tot1) are indicated by bold type, and residues 40-83 (the central acidic region) by italics.

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Fig. 4. ProTc~-histone interactions in solution. Solutions of the five histones (10 t-~g of each in 10 ILl of PBS) were mixed with solutions of ProT~ to a final volume of 30 Ixl. The ProTct solutions contained 2.5 ~g (A), 5 ~g (B), 10 Ixg (C) or 25 p~g (D) of the protein. Precipitated material (P) was separated from the soluble fraction (S) by centrifugation, and both fractions were analysed by SDS-PAGE as described in Section 2.

ProTc~ and histones at w / w ratios of between 1:20 and 1:2. We thus decided to analyse the pellet and supernatant fractions of various such mixtures, using SDS-PAGE. The results (Fig. 4) indicate that ProTot has a high capacity to insolubilize core histones (particularly H3 and H4) when

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Elution volume (ml) Fig. 5. Size exclusion chromatography of ProTct-histone mixtures in solution. A solution of the five histones (40 I-tg of each) was mixed with 200 I~g of ProTo~ and 100000 cpm [32p]ProTtx, and the mixture was chromatographed on a 40 x 1.2 cm Sephacryl S-200 column previously equilibrated in 10 mM Tris (pH 7.6) containing 0.15 M NaC1. Absorbance ( - 0 - ) and radioactivity in 300 ~1 aliquots ( - • - ) were determined in succesive 1 ml outflow fractions. Inset shows an SDS-PAGE analysis of the three outflow fractions with highest absorbance at 220 nm. The following molecular weight markers were passed through the column: alcohol dehydrogenase (A), bovine serum albumin (B), chymotrypsinogen (Q) and cytochrome c (C). Absorbance peaks for ProTct alone (P) and histones alone (H) are also shown.

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ProTet was very difficult to detect in these experiments, since under the electrophorelfic conditions used (optimized for separation and visualization of histones) the ProTet band was scarcely visible when the amount used in the assay was 10 Ixg or less (Fig. 4, lanes A, B and C). This was the case even in parallel runs of ProTe~ without histones (results not shown). Even when a larger amount was used (Fig. 4, lane D), the ProTet band was very weakly stained and difficult Io distinguish from that of H3. We thus carried out parallel insolubilization assays using various amounts of [32 P]ProTa obtained from proliferating thymocytes as described previously [19]. In these assays (results not shown), the distribution of radiolabelled ProTot between the pellet and sediment fractions was proportional to that of core bistones. Tot 1 did not induce insolLubilization of histones over a wide range of Tct 1-to-histone ratios, whereas poly(glutamic acid) induced insolubilization with a similar pattern (in terms of effect of varying ratio and of relative capacities to insolubilize specific histones) to that observed with ProTa (results not shown). With a view to characterizing ProTa-histone interactions in solution, 1:1 mixtures of ProTtx and all five histones were analysed by gel filtration at physiological ionic strength. As noted above, it is difficult to detect ProTet under SDS-PAGE conditions optimized for resolution of histones; 32P-labelled ProTc~ was thus added to the mixtures as a tracer. As shown in Fig. 5, the radioactive ProTo~ coeluted with a single peak with an apparent molecular mass of 100 kDa. Electrophoretic analysis of eluate fractions with highest absorbance (Fig. 5, inset) indicated that H1 and all c.ore histones comigrated with ProTc~. Since the apparent molecular weight of ProToL chromatographed alone in ~Le same conditions (see Fig. 5) is about 60 kDa (consistent with a pentameric configuration [30]), the result of this experiment is thus compatible with an equimolar interaction between ProTo~ and the five histones when all components are in solution in a 1:1 w / w ratio. 3.5. Effect o f ProTa on nucleosome assembly in vitro

The observed ability of F'roTa to bind histones in vitro led us to speculate a possible role in the structural organization of the chromosome, and specifically in nucleosome assembly. Similar acidic proteins with histone-binding ability have been implicated in nucleosome assembly [31]. To explore this hypothesis, we used the assay developed by Laskey and Earnshaw [32], which permits identification of factors enabling in vitro assembly of nucleosome-like structures. When the only candidate assembly factor supplied was ProTot (whether phosphorylated or unphosphorylated), nucleosomes did not form (Fig. 6, lanes 3 and 4). In a preliminary search for possible cofactors, we performed assays in which unphosphorylated ProTo~ was supplied together with ATP a n d / o r thymocyte cytosolic

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Fig. 6. Nucleosome assembly assay. Mixtures of the four core histones (0.1 Ixg of each) were incubated in the presence or absence of ProTc< and of various other components for 15 min at 37°C. A preincubated mixture containing relaxed pBR322 DNA (0.2 Ixg) and topoisomerase I was then added and the incubation continued for 45 min. The DNA was then purified and analysed by electrophoresis in a 1% agarose gel. For full details see Section 2. Lane 1: input DNA only. Lane 2: relaxed DNA only. Lanes 3-11: DNA from reaction mixtures containing the following components. Lane 3: phosphorylated ProTa (0.4 p,g). Lane 4: unphosphorylated ProT~ (0.4 p~g). Lane 5: ProTet-affinity-purified thymocyte cytosolic extract (0.6 p~g). Lane 6: as lane 5 plus unphosphorylated ProTet (0.4 Ixg). Lane 7: as lane 6 plus ATP (8 IxM).Lane 8: unphosphorylated ProTa (0.4 Ixg) plus ATP (8 IxM) plus affinity-purified thymocyte cytosolic extract (0.3 p,g). Lane 9: as lane 8 but with 0.6 Ixg of affinity-purified thymocytecytosolic extract. Lane 10: as lane 8 but with 0.9 p~g of affinity-purified thymocyte cytosolic extract. Lane 11: poly(glutamic acid) (mol. mass 60 kDa) (0.4 ~g). The positions of the relaxed (I) and supercoiled (II) forms of pBR322 DNA are indicated on the left of the figure.

components with strong affinity for ProTc~ (see Fig. 1A, lane 3). Neither affinity-purified thymocyte cytosolic extract (APTCE) alone (Fig. 6, lane 5) nor APTCE plus ProToL (Fig. 6, lane 6) induced nucleosome assembly. Likewise, no assembly activity was observed with APTCE plus ATP (result not shown). However, ProTtx plus APTCE plus ATP induced assembly (Fig. 6, lane 7). Similar results were obtained with phosphorylated ProTcx (results not shown). Assembly activity was affected by protein concentration in the affinity-purified thymocyte cytosolic extract (Fig. 6, lanes 8, 9 and 10). Other characteristics of the extract (perhaps related to extraction method a n d / o r differences between cell types) also seemed to affect assembly activity, since neither ProTe~-affinity-purified thymocyte nuclear extracts nor ProToL-purified splenic lymphocyte extracts (whether cytosolic or nuclear) induced nucleosome assembly (results not shown). Poly(glutamic acid) alone showed nucleosome assembly activity (Fig. 6, lane 11).

4. Discussion ProTct was first identified in mammalian cells about 15 years ago [5-7], and there is strong evidence that it has a role in cell proliferation [3,8,9]. Despite this, its precise function has yet to be identified. One approach to this problem is to identify cell components which show strong and selective in vitro interactions with ProTa. In the present study, we found that ProTe~ interacts in such a way with core histones. This is in accordance with the known structural similarities between

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ProTa and histone-binding proteins [13], and may be relevant to understanding of the functional role of ProTc~ in the nucleus. It has previously been reported, on the basis of immunoblotting assays, that ProTa binds H1 but not core histones [33]. The apparent disagreement with our results is perhaps attributable to these authors' use of an antibody directed against the C-terminal region of ProTa (residues 97-109) rather than against the N-terminal region (as in the present study). The reactivity with ProTa of the antibody used by us has been conclusively demonstrated [23]. Regardless of the possible reasons for Papamarcaki et al.'s findings, however, our results (based on a number of different techniques) clearly demonstrate a strong in vitro interaction between ProTc~ and core histones, and a weaker but still considerable in vitro interaction between ProTa and HI. ProTa appears to show especially high affinity for H3 and H4, as has been reported for the acidic nuclear proteins N1 and N2 of Xenopus eggs [34]. However, we did not detect any preferential association between ProTc~ and core histones either a) in gel filtration of ProToL and histone solutions which had been mixed at a 1:1 w / w ratio (such that all components remained in solution) (present results) or b) in gel filtration of calf thymocyte extracts (results not shown); this contrasts with results obtained for N 1 / N 2 and for nucleoplasmin (another acidic protein from Xenopus, which shows preferential association with H2A and H2B) [35]. Evidently, we were unable to investigate binding behaviour in solution at ProTc~-to-histone ratios of 1:2 or lower, since core histones were insolubilized under these conditions. Our results indicate that poly(glutamic acid) shows similar histone-binding characteristics to ProTet, suggesting that the higher affinity for core histories (and particularly H3 and H4) is due to the acidic region of ProTa. This is in accordance with the sequence homology of this region to the histone-binding regions of other nuclear proteins such as nucleoplasmin [36] and N1 and N2 [37]. However, affinity chromatography and immunoblotting assay indicated that Te~ 1 is also able to bind histones, making the N-terminal region of ProTa a candidate for histone binding. Note that it is of course possible that both regions bind histones in vivo. The results of our in vitro nucleosome assembly assays constitute preliminary evidence that ProTa may be involved in the structural organization of chromosomes in mammalian cells. The apparent requirement for various factors for nucleosome assembly is in accordance with the characteristics of the chromatin remodelling process. The Xenopus proteins nucleoplasmin and N 1 / N 2 both cooperate in the in vitro assembly of nucleosomes [35]. The apparent requirement for ATP is compatible with findings which indicate that chromatin remodelling is an energy-dependent process [38]. Induction of nucleosome assembly by nucleoplasmin and N 1 / N 2 likewise requires ATP [35],

as does induction of nucleosome assembly by certain yeast proteins [39]. The ProTa-binding cofactor/s apparently necessary for nucleosome assembly were isolated only from the thymocyte cytosolic extract, not from the thymocyte nuclear extract. As noted above, in detergent-based extraction procedures of the type used by us, nuclear components may end up in the 'cytosolic' fraction of the extract; a nuclear origin for the putative cofactor/s can therefore not be ruled out. Phosphorylation of ProToL has been shown to occur when cells are induced to proliferate [19,20], though its functional implications have yet to be elucidated. The results of our experiments to investigate histone insolubilization by [32p]ProTa, and of our nucleosome assembly assays, suggest that phosphorylation state does not affect the interaction of ProTo~ with histones. In conclusion, the results of the present study indicate that ProTa interacts in vitro with H1 and all four core histones. Whether such interactions occur in vivo, and if so whether they are functionally significant, remains to be determined. However, our results - - together with the structural similarity between ProTa and nuclear proteins whose activity is mediated via interactions with histones [31], and the reported association between ProTa and chromatin in intestinal cells [14] - - raise the possibility that the apparent interaction of ProTc~ with histones is important for understanding the function of this protein in the nucleus. Further research will clearly be necessary to confirm this hypothesis.

Acknowledgements We are grateful to Dr. E.P. Heimer (Hoffman-La Roche) for providing us synthetic TC~l. This work was supported by 'Direcci6n General de Investigaci6n Cientffica y T6cnica' Grant P B 4 3 / 0 5 1 8 and 'Consellerfa de Ordenaci6n Universitaria' Grant XUGA 20005B93 from the Spanish 'Ministerio de Educaci6n y Ciencia' and 'Xunta de Galicia', respectively.

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