Nucleic Acids Research, Vol. 19, No. 21 5999-6006

.7.1 1991 Oxford University Press

ATP dependent histone phosphorylation assembly in a human cell free extract

and nucleosome

Subhasis Banerjee, Gordon R.Bennion1, Martin W.Goldberg1 and Terry D.Allen1 Department of Pathology, The Royal Veterinary College, Royal College Street, University of London, London NW1 OTU and 1CRC Paterson Institute for Cancer Research, Manchester M20 9BX, UK Received June 19, 1991; Revised and Accepted September 25, 1991

ABSTRACT Physiologically spaced nucleosome formation in HeLa cell extracts is ATP dependent. ATP hydrolysis is required for chromatin assembly on both linear and covalently closed circular DNA. The link between the phosphorylation state of histones and nucleosome formation has been examined and we demonstrate that in the absence of histone phosphorylation no stable and regularly spaced nucleosomes are formed. Phosphorylated H3 stabilizes the nucleosome core; while phosphorylation of histone H2a is necessary to increase the linker length between nucleosomes from 0 to 45 bp. Histone Hi alone, whether phosphorylated or unphosphorylated, does not increase the nucleosome repeat length in the absence of core histone phosphorylation. Phosphorylations of Hi and H3 correlate with condensation of chromatin. Maximum ATP hydrolysis which is necessary to increase the periodicity of nucleosomes from 150 to 185 bp, not only inhibits Hi and H3 phosphorylation but facilitates their dephosphorylation. -

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INTRODUCTION The nucleosomes in eukaryotic chromatin are spaced at regular intervals with a distinct periodicity which can vary from 160 bp in yeast (1) to 250 bp in sea urchin sperm (2, 3). Besides species specificity, the repeat length of nucleosomes has been shown to alter depending upon the type of tissue and the transcriptional state of the chromatin. For example, the periodicities of nucleosomes in neural and glial cells of rabbit and bovine cerebral cortex are 160 and 200 bp respectively (4, 5); the spacing in transcriptionally active mouse ,B-globin or immunoglobulin (cit) genes is larger than their inactive counterparts and bulk chromatin (6); whereas transcriptionally active ribosomal genes (rDNA) of Tetrahymena thermophilia have shorter repeat lengths compared to adjacent non-transcribing spacer DNA (7). Moreover, the nucleosome periodicity fluctuates significantly during chicken erythropoiesis (8) or spermatogenesis (9) and throughout the early development of sea urchin (10-12). Historically, by virtue of its interaction with linker DNA (13, 14), lysine-rich histone HI was suggested to be the major determinant of spacer length (15, 16). There are experimental reports (17-19) which are consistent with such a hypothesis.

However, several recent reports suggest that HI alone is not sufficient to specify the periodicity of nucleosomes. For example, contrary to earlier reports, Sun et al. (20) showed that replacement of HI by H5 was not adequate to increase the repeat length of nucleosomes in adult chicken erythrocytes, while in polyspermic sea urchin embryos, substitution of sperm-specific HI by cleavage-stage specific HI was not sufficient to reduce the spacing immediately following fertilization (10). Additionally, chromatin containing physiologically spaced nucleosomes assembled in Xenopus oocyte extracts (21) does not contain HI. Chromatin reconstituted on natural DNA from purified components contains either irregularly spaced or close-packed (0 linker length) nucleosomes (20, 23; for a review see 24). However, naked DNA irrespective of its source can be assembled into chromatin with physiologically spaced nucleosomes in cellfree extracts from Xenopus oocytes (25, 26), Drosophila embryos (27) and HeLa cells (28). Both Xenopus oocyte and HeLa cell extracts require ATP hydrolysis for the formation of stable and physiologically spaced nucleosomes. An energy dependent topoisomerase II activity was claimed to be necessary for regular spacing of nucleosomes (26), but remained controversial (29) until recently, when topoisomerase I was shown to be sufficient for chromatin assembly of covalently closed circular DNA in Xenopus extracts (30). We reported (28) that the energy requirement for physiologically spaced nucleosome formation in HeLa cell extract was independent of DNA topology and suggested that ATP hydrolysis was necessary for phosphorylation of histone and non-histone structural proteins of the chromatin. Here we test that possibility and demonstrate that in the absence of histone phosphorylation, no stable and correctly spaced nucleosomes are formed. An increase in the periodicity of nucleosomes, from 150 to 185 bp, is observed when H2a is phosphorylated; while phosphorylation of HI and H3 correlates with condensation of the chromatin. Phosphorylation of HI and H3 is inhibited when energy release is maximum; such energy conditions also favor their dephosphorylation. -

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MATERIALS AND METHODS HeLa cell extracts and chromatin assembly The extracts were prepared from suspension cultures (5-8 x 106 cells per ml, S3 type) exactly as described previously (28) except final dialysis was in Spectra/por 3 dialysis bags (Mr cut off 3500) for 8-10 hrs. The efficiency of nucleosome assembly

6000 Nucleic Acids Research, Vol. 19, No. 21 increased considerably compared to our earlier extracts where dialysis was carried out in Spectra/por 2 tubes with Mr cut off of 10-12,000. However, extracts used in this work contain more endogenous ATP and Mg+ + than our previous preparations. This endogenous ATP hydrolysis can be competitively inhibited by 5' AMP-PNP. The DNA used in this work was a pGEM derivative of SV40 (nucleotide 5083 to nucleotide 1303). The supercoiled plasmid was relaxed by calf thymus topoisomerase I (Bethesda Research Laboratory) in 20 mM Tris-Cl (pH 8.0), 150 mM NaCl at 37°C for 1 hr. The chromatin assembly efficiency of each extract was determined as follows: nucleosome assembly was carried out in 50 1l reaction containing 20 Al extract (300-400 /Ag of total protein), 15 mM HEPES-Cl, (pH 7.9), 150 mM KCl, 1 mM ATP, 6 mM Mg++, 100 ltg/ml of poly L-glutamic acid (ICN, Mr 108,000) and variable amounts of covalently closed relaxed DNA at 37°C for 1 hr. The extent of nucleosome assembly was determined by supercoiling assay in 1.5% agarose gels. One yl (15-20 Ag of protein) of extract could assemble 25-40 ng of DNA.

Micrococcal nuclease digestion of chromatin and gel electrophoresis For micrococcal nuclease digestion the assembly reaction was scaled up to 250-300 yl. Following assembly at 37°C, the reactions were stopped on ice. An aliquot was taken to determine the state of assembly and CaCl2 and MNase were added to 3 mM and 40 U per 100 IL reaction respectively, to rest of the reactions. Digestion was carried out at 300 or at 37°C. At 37°C, digestion was completed (mononucleosomes) within 10-12 min whereas at 30°C up to 30 min. The efficiency of chromatin assembly of linear and covalently closed circular DNA was comparable as determined by MNase digestion of the chromatin. The deproteinized DNA was resolved in 2% agarose or 4% nondenaturing polyacrylamide gels with appropriate DNA standards. Ethidium stained gels were destained and photographed under UV illumination with polaroid type 55 film. The negatives were scanned in a DU 8 Beckman spectrophotometer with gel scanning accessory to determine the internucleosomal distance.

Sucrose gradient isolation, electrophoresis and electron microscopy of the chromatin Chromatin assembled in 750-1000 IL reaction (10-12 ytg of DNA) was gently layered over 11 ml of 10-30% (10-40% for the experiments in Figure 2) linear sucrose gradients containing 20 mM HEPES-C1, pH 7.9, 150 mM KCI, 0.1 mM EDTA, 1 mM DTT in Beckman SW 41 centrifuge tubes. Control gradients with no DNA, 30S and 50S subunits of E. coli ribosomes in 1 mM Mg++ were run in parallel gradients. The chromatin was separated by centrifugation in a Beckman SW 41 rotor at 35,000 rpm for 4-6 hrs. Twenty fractions ( - 600 ILI per fraction) were collected from each tube. About 20 yl of each fraction was loaded directly on 1 % low ionic strength agarose DNP gels (31) where the gel and the electrophoresis buffer contained 20 mM Tris-Cl, pH 7.6 and 2 mM EDTA. The buffer was recirculated during electrophoresis at 2.5 V per cm for 6 hrs. The gels were stained with 0.5 ,tg per ml of ethidium bromide and photographed under UV illumination without destaining. In some experiments, 100

yd of each or alternate fractions was deproteinized and the DNA was resolved in 1.5% agarose gels and type 55 polaroid negatives were densitometrically scanned at 540 nm to determine the distribution of chromatin in the gradients.

For electron microscopy, 20 ll of appropriate fraction was diluted 10-fold in 20 mM HEPES-Cl, pH 7.9, 0.1 mM EDTA, 150 mM KCl or in distilled water, fixed with 0.1 % glutaraldehyde, adsorbed to freshly prepared carbon-coated grids. The grids were shadowed with platinum-palladium at an angle of 8° as described (32). The micrographs were taken at a magnification of 45,000 and in some cases were reverse printed.

Extraction of phosphorylated histones from purified chromatin and gel electrophoresis Chromatin was assembled in the presence or in absence of exogenous ATP, Mg+ + and y32P ATP (3,000 Ci/mM, Amersham) at a final concentration of 0.5-0.8 ttM. The preparative reactions, sequence and time of addition of 732p ATP, MNase digestion of aliquots containing radiolabelled chromatin and any alteration of reaction protocol are described in individual figure legend. The radiolabelled chromatin was separated in linear 10-30% sucrose gradients by centrifugation in a Beckman SW41 rotor at 35,000 rpm for 4-6 hrs, the DNP containing fractions were pooled, layered over 0.8 ml of 40% sucrose and centrifuged at 40,000 rpm for 20 hrs in a SW 50.1 rotor (Beckman). The chromatin pellet was extracted with 0.25 N HCI for 30 min on ice and the acid soluble proteins were precipitated with 20% Trichloroacetic acid (TCA). The precipitated proteins were washed once with acidified acetone, twice with acetone and were resolved in 18% discontinuous polyacrylamide-SDS gels under reducing conditions or in 15% acid-urea-Triton (AUT) gels with appropriate protein standards and and HeLa or calf thymus histones. The AUT gels were as described (33) except the stacking gels contained 0.375 M potassium acetate (pH 4.0) and the resolving gels were prerun at 10 mA (constant current) for 3 hrs before pouring 8% stacking gel. Electrophoresis was at 6 mA (constant current) for 50-60 hrs; gels were stained with coomassie blue or amido black, photographed, dried and autoradiographed.

RESULTS Stability and increase in spacing of nucleosomes are ATPdependent Although linear and circular DNA can be assembled in HeLa cell extract (28), the former was chosen in order to avoid any problem of interpretation of the results related to DNA topology. To study the role of ATP hydrolysis in specifying the repeat length of nucleosomes, linear chromatin assembled in the presence or absence of exogenous ATP and Mg+ + was digested with MNase, and deproteinized DNA was resolved in agarose or non-denaturing polyacrylamide gels. The periodicities of the nucleosomes were 150-165 and 180-195 bp (Figure la), depending upon the absence or presence of exogenous ATP and Mg ++ respectively. Besides increasing the internucleosomal distance, ATP hydrolysis increased the stability of the nucleosome (Figure lb) as judged by its sensitivity to MNase. In order to test at what phase of chromatin assembly ATP hydrolysis was needed to increase the nucleosome periodicity, ATP and Mg+ + were added at different time points in separate reactions during assembly. When ATP and Mg++ were added following one hr of incubation and incubated for a further 30 min, chromatin was more stable and nucleosome spacing appeared to increase in a large fraction of the chromatin (data not shown). To further control this experiment, chromatin was assembled in the absence

Nucleic Acids Research, Vol. 19, No. 21 6001

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Figure 1. ATP hydrolysis is necessary to stabilize the nucleosome core and to increase the periodicity of nucleosomes from 150 to 185 bp. MNase digestion of linear chromatin assembled (8 yg DNA in 500 I reaction) in the presence or absence of 1 mM ATP, 6 mM Mg+ + and 5 mM creatine phosphate (CP). One half of the deproteinized DNA was separated in a) 1.5% agarose and the other half was resolved in b) 4% non-denaturing polyacrylamide gel. c) The spacing of close-packed nucleosomes increased only in one of the three aliquots (the experimental outline is shown above the gel) that received ATP, Mg+ + and CP before further incubation. d) Nucleosome assembly in the presence of 1 mM ATP, 6 mM Mg++, 5 mM CP, 5 mM glucose and 25 or 50 units HK mn- DNA standards were 123 bp, 1 kb ladders (Bethesda Research Laboratory) and HpaII digested pBR322. Nucleosome ladders from HeLa chromatin were generated following MNase digestion of isolated nuclei. -

of exogenous ATP and Mg+ +; subsequently divided into three and ATP and Mg+ + were added in one of the three aliquots before further incubation for an hr (see the experimental outline above the gel, Figure ic). It is evident (Figure ic), that an increase in periodicity of nucleosomes from 150 bp to 185 bp is ATP dependent. Although in every experiment the spacing of close-packed nucleosomes increased following incubation at optimum ATP and Mg++ concentrations, the shift from closepacked to physiologically spaced nucleosome ladders was not always quantitative (data not shown). The reason for such variations in different extracts is not clear. To further confirm the role of ATP hydrolysis in chromatin assembly, exogenously added ATP was depleted (34) by glucose and hexokinase (HK). The MNase digestion of the chromatin formed at two concentrations of HK revealed a close-packed and diffuse -

nucleosome ladder (Figure id), suggesting that a low level of endogenous ATP hydrolysis was necessary to stabilize the nucleosome cores, whereas an increase in nucleosome spacing required high energy.

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Densely packed and physiologically spaced nucleosomes are structurally different A distinct difference in spacing of nucleosomes in chromatin assembled in the presence or absence of exogenous ATP and Mg++ led us to examine their structural properties. For such studies, the assembled chromatin was sedimented in a 10-40% linear sucrose gradient and an aliquot of each fraction was assayed directly in a low ionic strength DNP gel (31) or following deproteinization in an agarose gel. The chromatin assembled in the presence of ATP alone

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6002 Nucleic Acids Research, Vol. 19, No. 21 NP; ATP |

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and electron microscopy (EM) of chromatin. Chromatin assembled (8 Acg DNA in 700 Al reaction) under the conditions Figure 2. Sucrose gradient sedimentation indicated (250 AuM ATP, 6 mMv Mg + , 5 mM CP, 5 mMv glucose and 25 U/mil hexokinase) was sedimented in 10 -40 % linear sucrose gradients in SW 41 rotor at 35,000 rpm for 3 hrs and 20 fractions were collected from each tube. Twenty Acl of each fraction was a) separated in low ionic strength DNP gels (31) or 100 pattern Al of alternative fractions was deproteinized and DNA was resolved in b) 1. 2% agarose gel ( left to right is top to bottom of the gradients). c) Sedimentation with scanning of chromatin as determined by densitometric scanning of DNA gels (polaroid negatives, Type 55) at 540 nm in a DU-8 (Beckman) spectrophotometer ++ accessory; left to right is top to bottom of the gradients. d -g) Electron micrographs of chromatin formed d) in the presence of ATP, Mg at 150 mM K +; e) in the absence of ATP and Mg+ + at 150 mM K+; f) in the presence of ATP, Mg+ + and CP or g) by ATP depletion at 15 mM K+. For EM, sucrose gradient fractions were diluted 10-fold in reaction buffer (150 mM K+) or in water (15 mM K+), fixed with glutaraldehyde and the rest was as described (32).

spread in the gradient compared to that formed of ATP and Mg+ + (Figure 2a and b). A densitometric quantitation of the DNA gels (Figure 2c) showed that the sedimentation velocities of the chromatin assembled in the absence of exogenous Mg++ and ATP or in the presence of ATP alone were slightly faster and that sedimentation profiles was more widely in the presence

were bell-shaped compared to a sharp peak in the presence of ATP and Mg++. Electron microscopic studies showed that chromatin assembled in the presence of ATP and Mg +I+ had beaded-string structures including condensed forms at 150 mM K+ concentration (Figure 2d). At this salt concentration, closepacked chromatin assembled in the absence of ATP and Mg"±

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Figure 3. ATP dependent phosphorylation of histones which affect nucleosome spacing and chromatin structure. Chromatin (17.5 itg DNA in 1 ml reaction) was assembled under conditions (1 mM ATP, 6 mM Mg+', 5 mM CP, 5 mM glucose and 25 U/ml HK) indicated in the presence of 7y32P ATP (3000 Ci/mMole) at a final concentration of 0.8 jiM; a), an aliquot (250 jl) of each reaction was digested with MNase to examine nucleosome spacing. The rest was layered over 11 ml 10-30% linear sucrose gradients in SW 41 rotor at 35,000 rpm for 5 hrs and the DNP containing fractions were pooled and chromatin was pelleted by further centrifugation in SW 50.1 rotor. The pellet was extracted with 0.25 N HCI, acid soluble proteins were precipitated with 20% TCA. The precipitated proteins were washed with acidified acetone, acetone twice and were resolved in 18% discontinuous polyacrylamide-SDS gels under reducing conditions; b), Coomassie stained gel; lane 1; protein standards; lane 4, calf thymus histones; lane 7, HeLa HI; lane 8, a mixture of calf thymus high mobility group (HMG) proteins 1 and 2, and mouse HMG 14 and 17; c) an autoradiogram of b); d), Coomassie stained gel of an experiment as a) except ATP concentration was 250 zM instead of 1 mM; lane 1, calf thymus histones; lane 2, ATP, Mg+ + and CP; lane 3, ATP alone; lane 4, no ATP and no Mg+ '; lane 5, same as lane 1 except 50 U of HK ml-l; e) a relevant part of the autoradiogram of d), lanes 1-4 corresponds to lanes 2-5 of d) respectively. Histone phosphorylation was quantitated by densitometric scanning at 540 nm.

was totally condensed (Figure 2e). However, at 15 mM K+, only chromatin formed in the presence of ATP and Mg++ or under ATP depleted conditions was unfolded (Figure 2f and g). These results suggest that condensed chromatin generated in the absence of exogenous ATP and Mg++ or in presence of ATP alone could not be unfolded by reducing the ionic strength.

ATP dependent phosphorylation of Hi, H3 and H2a in chromatin Energy dependent topoisomerase II is not required (28) for physiologically spaced nucleosome formation in this extract. Thus we examined the possibility of whether ATP was required for phosphorylation of histones. In pilot studies we noted that HI, H3, and H2a (which was mosfly ubiquitinated), were phosphorylated when the extract was incubated under assembly conditions in the absence of DNA (data not shown). To examine histone phosphorylation, chromatin was assembled under four

conditions in the presence of trace amounts of y32P ATP as follows: no ATP and no Mg++; ATP alone; ATP, Mg++ and creatine phosphate (CP); ATP, Mg++, CP, glucose and HK. At the end of incubation, an aliquot was taken from each reaction for MNase digestion to examine nucleosome spacing. The rest of the reactions were separated in linear 10-30% sucrose gradients. The fractions containing DNP were resedimented, acid extracted and acid soluble proteins were resolved in 18% polyacrylamide-SDS gel, stained, dried and autoradiographed. Agarose gel analysis of MNase digested chromatin revealed that ATP alone increased the periodicity of nucleosomes from 150 to 170 bp (Figure 3a). Electrophoresis of proteins extracted from chromatin showed (Figure 3b) that besides core histones and HI, the chromatin contained many unidentified proteins; some of these comigrate with high mobility group proteins. An autoradiogram of the gel (Figure 3c) showed that in the presence of trace amounts of oy32P ATP (no exogenous ATP and Mg+ +), -

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6004 Nucleic Acids Research, Vol. 19, No. 21

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Figure 4. Phosphorylation of H2a is necessary to increase the repeat length of nucleosomes from 150 bp to 185 bp. a) Thirty yig of linear DNA was assembled in a 3 ml reaction in the absence of ATP and Mg+ + for I hr at 37°C. A 250 Id aliquot was taken for MNase digestion. The rest was divided into two halves and incubated for 1 hr more in the presence of 250 FM ATP, -y32P ATP (final concentration 0.5 FM), 6 mM Mg+ + and 5 mM CP or -y32P ATP alone. At the end of second incubation aiiquots (250 ul) were taken from each reaction for MNase digestion and histones were extracted from chromatin in rest of reactions and resolved in 18% polyacylamide-SDS gel and autoradiographed; lane 1, Y32P ATP alone; lane 2, 250 FM ATP, 6 Mg++ and 6 mM CP during second incubation; b), same as a) except ey2P ATP (at a concentration of 0.5 uM) was added at the beginning of assembly, divided into three where one reaction was held in ice, and the other two were further incubated in the presence or absence of 250 FM ATP, 5 mM CP and 6 mM Mg+ + as described in Figure lc; lanes 1 & 2 are incubations for 1 & 2 hrs respectively in the presence of 7y32P ATP alone; lane 3, ATP, Mg+ + and CP were added following I hr incubation; lane 4, chromatin was assembled in the presence of 250 FiM ATP, -y32P ATP, 6 mM Mg+ + and 6 mM CP for 2 hrs; lane 5 is shorter exposure of lane 4; c) a similar experiment as b) except 250 FtM unlabeled and y32P ATP were added at the beginning of incubation and Mg++ plus CP were added subsequently in one of the three aliquots (lane 3); lanes 1 and 2, chromatin formed in the presence of ATP alone for 1 and 2 hrs respectively, lane 4 is same as lane 4 of b); lanes 5 & 6 are longer exposures of lanes 3 & 4 respectively. The acid soluble proteins were separated in 15% polyacrylamide acid-urea-Triton (AUT) gel (see Materials and Methods) with calf thymus and HeLa cell histone markers; uH2a, ubiquitinated H2a; X and Z are heteromorphous variants of H2a as described by West and Bonner (54). -

HI and H3 were phosphorylated. Notably the specific activity of oy32P ATP (lane C) is at least 1,000-fold higher than under the other three conditions described in this experiment. In the presence of ATP alone, HI phosphorylation was inhibited with concomitant phosphorylation of H2a and H3 at the ratio of 1:3 (1:10); at high energy conditions (ATP, Mg"+ and CP), H2a and H3 were phosphorylated at the ratio of 9.2:1 (11:1); when ATP was depleted (lane D), phosphorylation in general was severely inhibited; however, under these conditions HI and H3 phosphorylations could be detected in longer exposure of the autoradiogram (data not shown). The ratios in parentheses represent the results (Figure 3c and d) from a similar experiment as described above except that the ATP concentration was 250 AM instead of 1 mM. These results suggest that stable nucleosome cores are formed when H3 is phosphorylated; a progressive increase in the ratio of H2a to H3 phosphorylation correlated with an increase in periodicity of the nucleosomes from 150 bp to 170 bp and finally to 195 bp. Moreover, a very low level of H2a phosphorylation in the presence of ATP alone (lanes 1 and 2 of Figure 3c and e respectively) facilitated an increase in spacing by 20 bp. Interestingly, low energy release favoured HI and H3 phosphorylation which was inhibited by high ATP hydrolysis. -

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The ratio of H2a to H3 phosphorylation is critical for nucleosome spacing The experiments described above suggested that H2a was predominantly phosphorylated in chromatin having physiologically spaced nucleosomes, although a low level of H 1 and H3 phosphorylation also occurred under these conditions. To further examine the specific effect of H2a and H3 phosphorylation on nucleosome spacing several two-step experiments as described above (Figure lc) were carried out.

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In these experiments, the spacing of nucleosomes in close-packed chromatin was increased by further incubating under optimum ATP hydrolysis conditions. In one case, -y32P ATP plus unlabelled ATP, Mg+ + and CP were added following assembly in the absence of exogenous cofactors, and in another, only oy32p ATP was incorporated during first incubation and Mg+ +, unlabelled ATP plus CP were added at the onset of the second incubation. The aim of the first experiment was to examine if H2a was phosphorylated during the second incubation to increase spacing of nucleosomes; whereas the second shift experiment would allow monitoring of the fate of phosphorylated H 1 and H3 in addition to H2a phosphorylation during second incubation. The acid extracted histones were separated in polyacrylamide-SDS or acid-urea-Triton (AUT) gels. The latter gel system establishes that the phosphorylated histone migrating below H3 in SDS-polyacrylamide gels is H2a. The results of these two-step experiments (Figure 4a, b) demonstrate that H2a was the only core histone that was newly phosphorylated with simultaneous increase in spacing of the nucleosomes. The unexpected result of the second two-step experiment (Figure 4b) was that H3 was dephosphorylated when preassembled chromatin was subjected to high ATP hydrolysis conditions. An appearance in H2a phosphorylation with simultaneous dephosphorylation of H3 in the two-step experiment suggests that besides H2a, the relative phosphorylation of core histones H2a and H3 might be important for nucleosome spacing. The dephosphorylation of H3 during second incubation under high energy conditions (Figure 4b) was rather surprising. We suspected that this could be due to the difference in specific activity of ATP during first and second period of incubation in above experiment. To control that, both unlabelled and -y32P ATP were added during the first incubation, and Mg+ + plus CP were added at the second step. The results (Figure 4c) show that both H 1 and H3 are

Nucleic Acids Research, Vol. 19, No. 21 6005 ?Jmin

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absence of cofactors (Figure Ic). This increase in linker length correlates with increased H2a phosphorylation. Moreover, HI and H3 were dephosphorylated when shifted to high energy conditions (Figure 4b and c). These results led us to ask if phosphorylated H2a could be dephosphorylated by depleting ATP in the assembly reaction. Chromatin was assembled in the presence of ATP and Mg++ ( see the experimental outline above Figure 5a), and subsequently ATP was depleted by adding glucose and HK. The MNase digestion of clhromatin showed (Figure 5a) no significant difference in spacing of nucleosomes compared to controls (panels A & B). However, the ATP depleted chromatin became increasingly resistant to MNase,; twice the amount of MNase was needed to achieve digestion (panels C & D) compared to the control. An analysis of histones extracted from chromatin revealed that once phosphorylated, H2a could not easily be dephosphorylated by depleting ATP. This result is consistent with the data (Figure 5a) showing that physiological spacing is maintained following ATP depletion. Interestingly, ATP depletion induced HI and H3 phosphorylation. This result is also consistent with a low ATP hydrolysis requirement for HI and H3 phosphorylation. Moreover, electron microscopic analysis revealed that ATP depletion resulted in condensation of chromatin (Figure 5c-f) suggesting that Hla and H3 phosphorylation is necessary for chromatin condensation.

DISCUSSION iw~~~~~~~~~~~~~~~~~~/.

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Figure 5. ATP depletion fails to reduce nucleosome spacing, to dephosphorylate H2a but enhances Hla and H3 phosphorylation. Chromatin (50 Ag DNA in 4 ml reaction) was assembled in the presence of 250 AM ATP, 6 mM Mg+ +, 5 mM CP and 7y32P ATP at a final concentration of 0.6 AM for 1 hr and subsequently divided into four aliquots as described in the a) experimental outline; at the end of second incubation a portion (250 yd) of each was digested with MNase (87.5 and 150 U for panels A, B and panels C, D respectively). b) Phosphorylation of histones with and without ATP depletion; lanes 5 & 6 are shorter exposures of lanes 3 & 4 respectively. Extraction of histones and their separation in AUT gel are same as in Figure 4. Chromatin containing sucrose gradient fractions of each condition was diluted 10-fold in water before fixing with glutaraldehyde; c - f) are electron micrographs of chromatin assembled under conditions A to D respectively. The micrographs in c) and d) are reverse printed.

dephosphorylated with detectable H2a phosphorylation during second incubation under high energy conditions. These data further indicate that the relative phosphorylation of H2a and H3 affects nucleosome spacing and that increased phosphorylation of H 1 and H3 are not necessary to alter the periodicity of nucleosomes. The dephosphorylation of Hl and H3 (compare lanes 1 & 2 with 3, Figure 4b and c) was possibly due to the activation of ATP-dependent phosphatases. We conclude that high ATP hydrolysis not only inhibits HI and H3 phosphorylation, but also favours their dephosphorylation.

In this report, we asked why ATP hydrolysis is necessary for chromatin assembly in a human cell-free extract. Our results suggest that ATP hydrolysis facilitates phosphorylation of HI, H2a and H3 which affect the stability and spacing of nucleosomes as well as chromatin structure. When ATP was depleted by glucose plus hexokinase or endogenous ATP hydrolysis was inhibited by 5' AMP-PNP (S. Banerjee, unpublished data), neither stable nucleosome core was generated, nor H3 was phosphorylated. These results suggest that the stability of nucleosome cores in densely-packed chromatin is dependent on H3 phosphorylation. The -NH2 terminal region of H3 contains the phosphorylation site at serine 10 residue (35) and plays a fundamental role in the maintenance of chromatin superstructure (36-38). Indeed, compared to other core histones, H3 in chicken chromatin is highly sensitive to trypsin suggesting that it is most exposed in nucleosome cores (36, 37). This notion is supported by our data where compared to H2a, H3 in chromatin could be easily phosphorylated and dephosphorylated by altering the energy conditions. The spacing of the nucleosomes is not only tissue specific (5), but varies during differentiation and development (8, 10, 11). In these reports alterations in repeat length of nucleosomes have been correlated with the variants of H2a, H2b and Hi, their chemical modifications and the metabolic state of the cells. How could H2a phosphorylation increase the spacing of the nucleosomes? H3 and H2b of the core histone octamer, besides laterally shielding superhelical core DNA, due to spatial proximity, can extend to the adjacent superhelix of spacer DNA (39). It is possible that H2a phosphorylation indirectly facilitates the ionic interactions of H2b and H3 with spacer DNA. Notably spacer length in sea urchin sperm increases with concurrent

6006 Nucleic Acids Research, Vol. 19, No. 21 dephosphorylation of HI and H2b (40), whereas in cultured cells H2a remains phosphorylated throughout the cell cycle (41, 42). Moreover, HI appears not to be necessary (21) for -200 bp regular spacing of nucleosomes in chromatin assembled in Xenopus extracts and replacement of HI by H5 in adult chicken erythrocytes is not sufficient (20) to increase the linker length. Our data provide possible explanations of these results suggesting the vital role of modified core histones in determining nucleosome spacing. Our inability to dephosphorylate H2a indicates a more defined nucleosome assembly system is required to understand the effect of relative phosphorylation of core histones on nucleosome spacing. HI subfractions might play different roles depending upon their state of phosphorylation (43). Our results provide a correlation between Hi and H3 phosphorylation and chromatin condensation (Figure 2) which is independent of linker length (Figure 5), a result which agrees with a recent report (20). Histone HI alone, phosphorylated or unphosphorylated, cannot increase the spacing of the nucleosomes in the absence of core histone phosphorylation. Nevertheless, our experiments fail to define the specific role of phosphorylated HI in the absence of core histone phosphorylation on nucleosome periodicity. The low ATP requirement for Hi and H3 phosphorylation, inhibition of phosphorylation or their dephosphorylation at high energy conditions are also novel observations in this report. Interestingly, HI and H3 phosphorylation are the hallmark of mitosis (35, 42-46). A mammalian cell undergoes dramatic structural reorganization during mitosis. Metabolically the cell is incapable of moving, transcribing RNA and secreting proteins (47, 48). The major structural changes of nuclear membrane and chromatin are brought about by phosphorylation and dephosphorylation of multiple proteins (49-51). The energy state (especially ATP synthesis and transport to the nucleus) of a cell might play an important role in regulating such a kinase-phosphatase network. It has been shown (52) that growth associated HI kinase, a human homolog of p34cdc2 kinase, which is inactivated (53) by phosphorylation at interphase, phosphorylates HI at mitosis. Future experiments are necessary to examine whether such reciprocal relationships in the state of HI and p34Cdc2 phosphorylation could be established by altering energy conditions in vitro. Note added: Kleinschmidt and Steinbeisser (EMBO J., 10, 3043 -3050, 1991) made similar observations, as reported here, that phosphorylation of H2a correlated with physiological spacing of nucleosomes in chromatin assembled in Xenopus laevis oocyte extracts.

ACKNOWLEDGEMENTS S.B. thanks Charles R. Cantor in whose lab this work was

initiated, Cancer Research Campaign and SmihKline Foundation for support; Stephen Spoulding, Raymond Reeves for providing HMG proteins and Ron Laskey for discussions, communicating unpublished results; Graham Goodwin and Steve Dilworth for critical reading and comments on the manuscript.

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