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An altered conformation of nucleosomal core particle in the active chromatin of Physarum polycephalum
Biochimica et Biopt~vsica Acta 866 (1986) 252-257 Elsevier
252
BBA 91569
An altered conformation of nucleosomal core particle in the active chromatin of Physarumpolycephalum Marta Czupryn, Lilianna Solnica and Kazimierz Toczko * Institute of Biochemistry. Warsaw University, AI. Zwirki i Wigurv 93, 02-089 Warsaw {Poland)
(Received September 26th, 1985)
Key words: Transcriptionally active chromatin; Nucleosomestructure; ( P. polycephalum) We show that nucleosomal particles from transcriptionally active chromatin (MgCl2-soluble fraction) of
Physarum polycephalum have lower electrophoretic mobility than those of the bulk chromatin (MgCI 2-insoluble fraction). Altered electrophoretic behaviour is observed even at the level of the nucleosomal core, and is shown not to result from its different DNA, RNA or protein content, nor to be a consequence of the process of D N A replication. Analysis of nucelosomes from both fractions on density gradient gels reveals that the slow migration of active nucleosomes is due to increased friction, i.e., more asymmetrical (unfolded) conformation of the nucleosomal core.
Introduction Experimental evidence, derived in the most part from animal systems, has shown that chromatin regions containing active genes differ from the nontranscribed chromatin in higher order structures of the chromatin fibre as well as in nucleosomal organization (for a review see Ref. 1). Work from our laboratory has demonstrated in recent years that the transcriptionally active chromatin of a lower eukaryote Physarum polycephalum shares many structural and compositional characteristics with transcribed chromatin from animal cells. The fraction of active chromatin in Physarum is organized in a nucleosome-type structure [2,3], distinguished by an increased susceptibility to nuclease digestion [2], enrichment in the HMG-like proteins [4] and depletion in histone H1, which is
* To whom correspondence should be addressed. Abbreviations: PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate; bp, base pairs; HMG, high mobility group.
replaced by specific for this fraction 37 and 39 kDa nonhistone proteins [3]. There are data suggesting that active nucleosomes may undergo structural changes, resulting in unfolding (linearization) of the core particle [5-8]. However, little is known about the active nucleosome structure and the molecular mechanisms of the conformational change. In this work we present evidence that nucleosomal core particles of transcriptionally active chromatin from P. polycephalum have a markedly increased asymmetry, supporting further the hypothesis that the activation process involves linearization of a nucleosome structure. Materials and Methods
Growth of plasmodia and in vivo labelling Microplasmodia of P. polycephalum, strain M3CIV, grown in submerged shaken cultures as described by Daniel and Baldwin [9], were used, For pulse-labelling of DNA, plasmodia were incubated at 26°C with 5 / t C i / m l of [3H]thymidine (43 C i / m o l ) for 3 or 10 rain.
0167-4781/86/$03.50 ~c21986 Elsevier Science Publishers B.V. (Biomedical Division)
253
Isolation of nuclei and chromatin fractionation Nuclei were isolated according to the method of Jockusch and Walker [10]. All buffers contained 0.1 mM PMSF to prevent proteolysis. Transcriptionally active and inactive chromatin fractions were prepared by a modified procedures of Bloom and Anderson [11]. Briefly, isolated nuclei were suspended in 0.25 M sucrose/50 mM KC1/1 mM MgClz/10 mM Tris-HC1 (pH 7.8) and digested with micrococcal nuclease (EC 3.1.31.1) (10-100 units/200 ttg DNA) for 10 min at 37°C. The reaction was terminated by chilling the tubes on ice. To the nuclease digest 0.1 M MgCI 2 was added dropwise, with rapid stirring, to a final concentration of 5 mM. After 20 min on ice, the suspension was centrifuged at 14000 × g for 15 min. The supernatant, containing transcriptionally active chromatin, was designated fraction S r The pellet was resuspended by gentle homogenization for 10 min on ice in 3 vol. 5 mM EDTA (pH 7.8) and was centifuged as above. The second supernatant, containing transcriptionally inactive chromatin, was designated fraction S2. Micrococcal nuclease digestion products were precipitated by addition to supernatant fractions of 2 vol. 96% ethanol for 24 h a t - 2 0 ° C . Resulting precipitates were collected by centrifugation. In experiments on the distribution of RNA, prior to ethanol precipitation, chromatin fractions were incubated at 37°C for 10 min with RNAase A (100 /~g/100 #g DNA). In order to determine the radioactivity of samples containing DNA labelled with [3H]thymidine, chromatin fractions were precipitated at 4°C with 10% (w/v) trichloroacetic acid. Precipitates were sedimented by centrifugation and washed twice with cold 5% (w/v) trichloroacetic acid. Resulting pellets were hydrolysed in 0.5 M HCIO 4 (30 rain, 80°C), DNA content was estimated according to Burton [12], and 3H radioactivity was determined in a Packard liquid scintillation counter in KochLight Unisolve 1.
Polyacrylamide gel electrophoresis Nucleoprotein digestion products were separated electrophoretically using e i t h e r 0.5% agarose/2.5% acrylamide [13] or 4% acrylamide [14] gels. Step gradient gels (17 x 17 × 0.2 cm) were prepared from 3, 3.5, 4, 4.5 and 5% acryl-
amide solutions according to Bode [15]. Isolation and electrophoresis of DNA fragments were as described earlier [8]. For analysis of DNA fragments in mononucleosomes, individual bands were cut from the 4% acrylamide gels and the DNA was extracted [3]. The DNA lengths were estimated by comparisons of fragment mobilities with marker AluI restriction fragments of pBR 322 run on the same gel. Nucleoprotein and DNA gels were stained with ethidium bromide (2.5 ~g/ml) in 2 mM EDTA, and photographed under ultraviolet light using an orange filter. Proteins of electrophoretically separated nucleoprotein particles were displayed by performing a second step electrophoresis as described elsewhere [8]. 0.1% SDS/15% acrylamide slab gels, prepared according to Laemmli [16] were used. The gels were stained with silver [17]. Densitomettic scans were taken from the gel photographs. Results
When chromatin of P. polycephalum is fractionated by the micrococcal nuclease-MgCl 2 method [11] about 15-20% of the total chromatin is recovered in the MgCl2-soluble fraction (fraction S~). We have previously shown that this fraction, similarly to the situation in animal cells, contains transcriptionally active chromatin, whereas the remaining MgClz-insoluble chromatin, which can be solubilized over 80% by extraction with EDTA buffer (fraction $2), represents the transcriptionally inactive component {2]. As shown in Fig. 1, DNA fragments isolated from fraction S~ and electrophoresed on a polyacrylamide gel were distributed in over a typical ladder pattern (lane d), and their electrophoretic mobilities were similar to those of homologous fragments from fraction S2. This is consistent with our previous statement [2,3] that the DNA of transcriptionally active chromatin is organized into nucleosomes showing the same spacing as in the bulk inactive chromatin. Electrophoretic patterns of nucleoprotein particles of fraction S~, analyzed in two different non-denaturing gel systems, are shown in Fig. 2. It is evident that, in contrast to the DNA pattern, mono-, di- and oligonucleosomes of fraction S~ have a substantially lower electrophoretic mobility
254
&b
c
d
B
A
910 659 655 521 403 281 257
$2
10 50 100
10 50 100
?
o
226
$1
D
D
M-
1,4 M
100
S2
SI
m
SI S2
SI S 2
Fig. 1. Electrophoretic patterns of DNA fragments isolated from whole fractions S I (lane d), S 2 (lane b) and from electrophoretically separated mononucleosomal particles S~ (lane c) and S 2 (lane a). m, marker DNA fragments of pBR 322 DNA digested with Alul; sizes given in bp. Fig. 2. Electrophoretic patterns of nucleoprotein particles released by micrococcal nuclease from fractions S 1 and S2. (A) 0,5% agarose, 2.5% polyacrylamide gel; (B) 4% polyacrylamide gel. M, D, O designate positions of monomer, dimer and oligomer particles, respectively, identified by estimation of their DNA fragment lengths. Fig. 3. Effect of the extent of micrococcal nuclease digestion on electrophoretic profiles of nucleoprotein particles from fractions S~ and S 2. Electrophoresis was carried out in 4% polyacrylamide gel. Numbers refer to the concentration of micrococcal nuclease (units/200 /tg DNA). For further details see Fig. 2.
than their counterparts from fraction S2, independent of the gel system used. As illustrated in Fig. 3, the observed difference in electrophoretic mobility was also independent of the extent of nuclease digestion. In an attempt to determine the molecular basis for the differential electrophoretic behaviour of nucleosomal particles released from fractions St and $2, we compared mainly monomer particles, because of their structural simplicity. Monomer particles of fractions S~ and S2 had an average DNA fragment length of 154 + 2 and 157 _+ 4 (S.D.) bp, respectively, as shown by analysis of DNA fragments both whole fractions and from isolated monomer particles (see Fig. 1), i.e., much less than that of the DNA repeat in Physarum, about 181 bp [18]. Thus, they are equivalent to core particles rather than complete nucleosomes. This is in agreement with the results
of analysis of their protein components. As shown in Fig. 4, monomer particles of both fractions contained a full complement of the core histones, but lacked the proteins known to be associated with nucleosome linker DNA histone H1 in fraction S2, and 37 and 39 kDa proteins in fractions S~ [3]. On an electrophoretogram of S~ monomers (Fig. 4), minor nonhistone protein species, including HMG-1P and HMG-2P, are also visible; however, their concentrations are relatively low. The similar protein and DNA content of the $1 and S2 monomers indicates that their different electrophoretic mobility cannot result from a difference in molecular weight of these particles. The lower migration rate of St monomers also cannot be ascribed to the presence of RNA, which might be associated with nucleosomes of actively transcribed chromatin [19]. As seen in Fig. 5, extensive digestion with RNAase A had no effect
255
$1 ::~ " r "'r
$2
f
0 I
Ig ~r
N
-,q
Z nO 0') m
M
S2
1
2
3
1
2
3
Fig. 5. Effect of RNAase digestion on electrophoretic mobility of S] a/ad S2 nucleosomes. 1, control; 2, incubation at 37°C for 10 rain without RNAase; 3, incubation at 37°C for 10 rain with RNAase (100 #g/100 #g DNA) Electrophoresis was carried out in 4% polyacrylamide gel.
2.0
MIGRATION Fig. 4. Proteins associated with monomer particles from fractions S1 and S2. Densitometer scans of second dimension SDS/polyacrylamide gel electrophoretograms are shown. Band X is an artifact due to 2-mercaptoethanol [23]. Micrococcal nuclease digestion products were electrophoresed on 4% polyacrylamide gels (first dimension), gel strips containing monomer particles were cut and than run in the second-dimension SDS/polyacrylamide gel system.
S e,i
b t5
S2
E L~ O ..J
S1 o n the e l e c t r o p h o r e t i c m o b i l i t y o f e i t h e r S~ or S 2 particles. T o e x c l u d e t h e p o s s i b i l i t y t h a t S~ n u c l e o s o m e s represent structurally altered 'immature' particles excised from newly replicated regions of chrom a t i n , we e x p o s e d m i c r o p l a s m o d i a to [ 3 H ] t h y m i d i n e pulses a n d a n a l y z e d n a s c e n t D N A c o n t e n t in f r a c t i o n s S 1 a n d S 2. W e f o u n d that a f t e r a 3 m i n
1.0
~ a t
a J "~11
1
4
5
c
(%}
2
3
Fig. 6. Electrophoretic mobility of S] and S2 monomer particles as a function of a gel density. Plot of the logarithm of the migration distance (log m) against the acrylamide concentration (c).
256
pulse of [-~H]thymidine, the radioactivity per mg DNA was 38200 cpm for fraction S 1 and 38800 cpm for fraction S2, and after a 10 rain pulse, 130300 cpm and 135 300 cpm, respectively. From these results it is evident that fraction S~ is not enriched in freshly replicated DNA. In order to check whether the different electrophoretic behaviour of nucleosomal particles released from transcriptionally active chromatin could be due to their altered conformation, we performed electrophoretic analysis of S~ and S2 nucleosomes on a gel with a horizontal gradient of acrylamide concentration. This system permits the detection of differences in shape of the particles studied [15,20]. Fig. 6 shows a plot of the logarithm of the migration distance of monomer particles, as a function of the gel density. As can be seen, the straight lines obtained for S~ and S2 particles have clearly different slopes. Identical results were also obtained for di- and oligonucleosomes (data not shown). From this analysis it can be concluded that the different electrophoretic mobility of S~ and $2 particles results primarily from the difference in their shape; however, a difference in their effective electrical charge cannot be excluded. The relatively steeper slope observed for S~ particles is indicative of the increased frictional coefficient, i.e., the higher degree of assymetry of these particles in comparison to cannonical nucleosomes (S 2 particles).
Discussion In this work we have continued our studies on a structural basis for transcriptional activation of genes in Physarum. We have adopted the micrococcal nucleose-MgC12 method of Bloom and Anderson [11] to obtain the fraction of transcriptionally active chromatin [2]. In Physarum, the active MgC12-soluble chromatin fraction amounts up to 20% of the total genome, which is in good correspondance with the amount of transcribed DNA in this organism (12-18% as calculated from data in Refs. 6 and 21). In addition to the peculiarities of the structural organization of Physarum active chromatin reported earlier [2-4,8], we now present data indicating an altered conformation of its nucleosomes,
resulting from increased asymmetry of the nucleosomal core. This structural change in the active nucleosomal core cannot be attributed to the depletion of any of the core histones, or to the presence of specific nonhistone proteins or RNA, nor is it a consequence of the process of D N A replication. These data further support the idea that the process of transcriptional activation of genes involves loosening or opening of the nucleosome structure [5-8,22]. An advanced model of this type of structural transition has been proposed by Prior et al. [7] for active nucleosomes of ribosomal gene chromatin of P. polycephalum. According to this model, the cores of active nucleosomes (called the lexosomes) are symmetrically unfolded particles, having 3-4-times greater diameter (length) than compact nucleosomes, and consisting of two 'half-nucleosomes' connected by a 50 bp nucleoprotein bridge. Their structure is stabilized by two highly abundant nonhistone proteins (LP 30 and LP 32), associated with the connecting nucleoprotein bridge [7]. Although the open conformation of the active nucleosomal cores observed by us shares certain characteristics with the lexosome structure, we could not find any proteins which, like LP 30 and LP 32, could be responsible for the maintenance of the relaxed structure of the particle. Therefore, we postulate that the process of unfolding (linearization) of the nucleosomal core does not necessarily result from association with specific nonhistone proteins, but rather might be triggered by some modifications of the core histones. Our observations indicate that the active nucleosome structure is sensitive to Mg 2+. At a very low Mg 2+ concentration (0.1 mM), nucleosomal cores may undergo complete linearization, increasing the accessibility of intracore DNA to interaction with macromolecules. This is reflected by the disruption of the normal micrococcal nuclease cutting pattern seen when active chromatin is digested with this enzyme [8].
Acknowledgement We thank Mrs Aleksandra Bakula for technical assistance and Mr Artur Szymafiski for photographic prints.
257
References 1 Reeves, R. (1984) Biochim, Biophys. Acta 782, 343-393. 2 Czupryn, M. and Toczko, K. (1982) Acta Biochim. Polon. 29, 17-26. 3 Czupryn, M., Solnica, L. and Toczko, K. (1985) FEBS Lett. 189, 89-91 4 Czupryn, M. and Toczko, K. (1984) FEBS Lett. 169, 174-178. 5 Lohr, D.E. (1984) Cell Biophys. 6, 87-102. 6 Scheer, U., Zentgraf, H. and Saner, H.W. (1981) Chromosoma 84, 279-290. 7 Prior, C.P., Cantor, C.R., Johnson, E,M., Littau, V.C. and AUfrey, V.G. (1983) Cell 34, 1033-1042. 8 Czupryn, M. and Toczko, K. (1985) Eur. J. Biochem. 147, 575-580. 9 Daniel, J.W. and Baldwin, H.H. (1964) in Methods in Cell Physiology (Prescott, D., ed.), Vol. 1, pp. 9-41, Academic Press, New York. 10 Jockusch, B.M. and Walker, I.O. (1974) Eur. J. Biochem. 48, 417-425.
11 Bloom, K.S. and Anderson, J.N. (1978) Cell 15, 141-150. 12 Burton, K. (1956) Biochem. J. 62, 315-323. 13 Sahasrabuddhe, C.G. and Saunders, G.F. (1977) Nucleic Acids Res. 4, 853-866. 14 Bode, J., G6mez-Lira, M.M. and Schri~ter, H. (1983) Eur. J. Biochem. 130, 437-445. 15 Bode, J. (1984) Arch. Biochem. Biophys. 228. 364-372. 16 Laemmli, U.K. (1970) Nature 227, 680-685. 17 Wray, W., Boulikas, T., Wray, V.P. and Hancock, R. (1981) Anal. Biochem. 118, 197-203. 18 Stalder, J. and Braun, R. (1978) FEBS Lett. 62, 251-254. 19 Gottesfeld, J.M. and Butler, P.J.G. (1977) Nucleic Acids Res. 4, 3155-3173. 20 Albanese, I. and Weintraub, H. (1980) Nucleic Acids Res. 8, 2787-2805. 21 Hardman, N., Jack, P.L., Fergie, R.C. and Gerrie, L.M. (1980) Eur. J. Biochem. 103, 247-257. 22 Tsanev, R. (1983) MoL Biol. Rep. 9, 9-17. 23 Tasheva, B. and Dessev, G. (1983) Anal Biochem. 129, 98-102.
252
BBA 91569
An altered conformation of nucleosomal core particle in the active chromatin of Physarumpolycephalum Marta Czupryn, Lilianna Solnica and Kazimierz Toczko * Institute of Biochemistry. Warsaw University, AI. Zwirki i Wigurv 93, 02-089 Warsaw {Poland)
(Received September 26th, 1985)
Key words: Transcriptionally active chromatin; Nucleosomestructure; ( P. polycephalum) We show that nucleosomal particles from transcriptionally active chromatin (MgCl2-soluble fraction) of
Physarum polycephalum have lower electrophoretic mobility than those of the bulk chromatin (MgCI 2-insoluble fraction). Altered electrophoretic behaviour is observed even at the level of the nucleosomal core, and is shown not to result from its different DNA, RNA or protein content, nor to be a consequence of the process of D N A replication. Analysis of nucelosomes from both fractions on density gradient gels reveals that the slow migration of active nucleosomes is due to increased friction, i.e., more asymmetrical (unfolded) conformation of the nucleosomal core.
Introduction Experimental evidence, derived in the most part from animal systems, has shown that chromatin regions containing active genes differ from the nontranscribed chromatin in higher order structures of the chromatin fibre as well as in nucleosomal organization (for a review see Ref. 1). Work from our laboratory has demonstrated in recent years that the transcriptionally active chromatin of a lower eukaryote Physarum polycephalum shares many structural and compositional characteristics with transcribed chromatin from animal cells. The fraction of active chromatin in Physarum is organized in a nucleosome-type structure [2,3], distinguished by an increased susceptibility to nuclease digestion [2], enrichment in the HMG-like proteins [4] and depletion in histone H1, which is
* To whom correspondence should be addressed. Abbreviations: PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate; bp, base pairs; HMG, high mobility group.
replaced by specific for this fraction 37 and 39 kDa nonhistone proteins [3]. There are data suggesting that active nucleosomes may undergo structural changes, resulting in unfolding (linearization) of the core particle [5-8]. However, little is known about the active nucleosome structure and the molecular mechanisms of the conformational change. In this work we present evidence that nucleosomal core particles of transcriptionally active chromatin from P. polycephalum have a markedly increased asymmetry, supporting further the hypothesis that the activation process involves linearization of a nucleosome structure. Materials and Methods
Growth of plasmodia and in vivo labelling Microplasmodia of P. polycephalum, strain M3CIV, grown in submerged shaken cultures as described by Daniel and Baldwin [9], were used, For pulse-labelling of DNA, plasmodia were incubated at 26°C with 5 / t C i / m l of [3H]thymidine (43 C i / m o l ) for 3 or 10 rain.
0167-4781/86/$03.50 ~c21986 Elsevier Science Publishers B.V. (Biomedical Division)
253
Isolation of nuclei and chromatin fractionation Nuclei were isolated according to the method of Jockusch and Walker [10]. All buffers contained 0.1 mM PMSF to prevent proteolysis. Transcriptionally active and inactive chromatin fractions were prepared by a modified procedures of Bloom and Anderson [11]. Briefly, isolated nuclei were suspended in 0.25 M sucrose/50 mM KC1/1 mM MgClz/10 mM Tris-HC1 (pH 7.8) and digested with micrococcal nuclease (EC 3.1.31.1) (10-100 units/200 ttg DNA) for 10 min at 37°C. The reaction was terminated by chilling the tubes on ice. To the nuclease digest 0.1 M MgCI 2 was added dropwise, with rapid stirring, to a final concentration of 5 mM. After 20 min on ice, the suspension was centrifuged at 14000 × g for 15 min. The supernatant, containing transcriptionally active chromatin, was designated fraction S r The pellet was resuspended by gentle homogenization for 10 min on ice in 3 vol. 5 mM EDTA (pH 7.8) and was centifuged as above. The second supernatant, containing transcriptionally inactive chromatin, was designated fraction S2. Micrococcal nuclease digestion products were precipitated by addition to supernatant fractions of 2 vol. 96% ethanol for 24 h a t - 2 0 ° C . Resulting precipitates were collected by centrifugation. In experiments on the distribution of RNA, prior to ethanol precipitation, chromatin fractions were incubated at 37°C for 10 min with RNAase A (100 /~g/100 #g DNA). In order to determine the radioactivity of samples containing DNA labelled with [3H]thymidine, chromatin fractions were precipitated at 4°C with 10% (w/v) trichloroacetic acid. Precipitates were sedimented by centrifugation and washed twice with cold 5% (w/v) trichloroacetic acid. Resulting pellets were hydrolysed in 0.5 M HCIO 4 (30 rain, 80°C), DNA content was estimated according to Burton [12], and 3H radioactivity was determined in a Packard liquid scintillation counter in KochLight Unisolve 1.
Polyacrylamide gel electrophoresis Nucleoprotein digestion products were separated electrophoretically using e i t h e r 0.5% agarose/2.5% acrylamide [13] or 4% acrylamide [14] gels. Step gradient gels (17 x 17 × 0.2 cm) were prepared from 3, 3.5, 4, 4.5 and 5% acryl-
amide solutions according to Bode [15]. Isolation and electrophoresis of DNA fragments were as described earlier [8]. For analysis of DNA fragments in mononucleosomes, individual bands were cut from the 4% acrylamide gels and the DNA was extracted [3]. The DNA lengths were estimated by comparisons of fragment mobilities with marker AluI restriction fragments of pBR 322 run on the same gel. Nucleoprotein and DNA gels were stained with ethidium bromide (2.5 ~g/ml) in 2 mM EDTA, and photographed under ultraviolet light using an orange filter. Proteins of electrophoretically separated nucleoprotein particles were displayed by performing a second step electrophoresis as described elsewhere [8]. 0.1% SDS/15% acrylamide slab gels, prepared according to Laemmli [16] were used. The gels were stained with silver [17]. Densitomettic scans were taken from the gel photographs. Results
When chromatin of P. polycephalum is fractionated by the micrococcal nuclease-MgCl 2 method [11] about 15-20% of the total chromatin is recovered in the MgCl2-soluble fraction (fraction S~). We have previously shown that this fraction, similarly to the situation in animal cells, contains transcriptionally active chromatin, whereas the remaining MgClz-insoluble chromatin, which can be solubilized over 80% by extraction with EDTA buffer (fraction $2), represents the transcriptionally inactive component {2]. As shown in Fig. 1, DNA fragments isolated from fraction S~ and electrophoresed on a polyacrylamide gel were distributed in over a typical ladder pattern (lane d), and their electrophoretic mobilities were similar to those of homologous fragments from fraction S2. This is consistent with our previous statement [2,3] that the DNA of transcriptionally active chromatin is organized into nucleosomes showing the same spacing as in the bulk inactive chromatin. Electrophoretic patterns of nucleoprotein particles of fraction S~, analyzed in two different non-denaturing gel systems, are shown in Fig. 2. It is evident that, in contrast to the DNA pattern, mono-, di- and oligonucleosomes of fraction S~ have a substantially lower electrophoretic mobility
254
&b
c
d
B
A
910 659 655 521 403 281 257
$2
10 50 100
10 50 100
?
o
226
$1
D
D
M-
1,4 M
100
S2
SI
m
SI S2
SI S 2
Fig. 1. Electrophoretic patterns of DNA fragments isolated from whole fractions S I (lane d), S 2 (lane b) and from electrophoretically separated mononucleosomal particles S~ (lane c) and S 2 (lane a). m, marker DNA fragments of pBR 322 DNA digested with Alul; sizes given in bp. Fig. 2. Electrophoretic patterns of nucleoprotein particles released by micrococcal nuclease from fractions S 1 and S2. (A) 0,5% agarose, 2.5% polyacrylamide gel; (B) 4% polyacrylamide gel. M, D, O designate positions of monomer, dimer and oligomer particles, respectively, identified by estimation of their DNA fragment lengths. Fig. 3. Effect of the extent of micrococcal nuclease digestion on electrophoretic profiles of nucleoprotein particles from fractions S~ and S 2. Electrophoresis was carried out in 4% polyacrylamide gel. Numbers refer to the concentration of micrococcal nuclease (units/200 /tg DNA). For further details see Fig. 2.
than their counterparts from fraction S2, independent of the gel system used. As illustrated in Fig. 3, the observed difference in electrophoretic mobility was also independent of the extent of nuclease digestion. In an attempt to determine the molecular basis for the differential electrophoretic behaviour of nucleosomal particles released from fractions St and $2, we compared mainly monomer particles, because of their structural simplicity. Monomer particles of fractions S~ and S2 had an average DNA fragment length of 154 + 2 and 157 _+ 4 (S.D.) bp, respectively, as shown by analysis of DNA fragments both whole fractions and from isolated monomer particles (see Fig. 1), i.e., much less than that of the DNA repeat in Physarum, about 181 bp [18]. Thus, they are equivalent to core particles rather than complete nucleosomes. This is in agreement with the results
of analysis of their protein components. As shown in Fig. 4, monomer particles of both fractions contained a full complement of the core histones, but lacked the proteins known to be associated with nucleosome linker DNA histone H1 in fraction S2, and 37 and 39 kDa proteins in fractions S~ [3]. On an electrophoretogram of S~ monomers (Fig. 4), minor nonhistone protein species, including HMG-1P and HMG-2P, are also visible; however, their concentrations are relatively low. The similar protein and DNA content of the $1 and S2 monomers indicates that their different electrophoretic mobility cannot result from a difference in molecular weight of these particles. The lower migration rate of St monomers also cannot be ascribed to the presence of RNA, which might be associated with nucleosomes of actively transcribed chromatin [19]. As seen in Fig. 5, extensive digestion with RNAase A had no effect
255
$1 ::~ " r "'r
$2
f
0 I
Ig ~r
N
-,q
Z nO 0') m
M
S2
1
2
3
1
2
3
Fig. 5. Effect of RNAase digestion on electrophoretic mobility of S] a/ad S2 nucleosomes. 1, control; 2, incubation at 37°C for 10 rain without RNAase; 3, incubation at 37°C for 10 rain with RNAase (100 #g/100 #g DNA) Electrophoresis was carried out in 4% polyacrylamide gel.
2.0
MIGRATION Fig. 4. Proteins associated with monomer particles from fractions S1 and S2. Densitometer scans of second dimension SDS/polyacrylamide gel electrophoretograms are shown. Band X is an artifact due to 2-mercaptoethanol [23]. Micrococcal nuclease digestion products were electrophoresed on 4% polyacrylamide gels (first dimension), gel strips containing monomer particles were cut and than run in the second-dimension SDS/polyacrylamide gel system.
S e,i
b t5
S2
E L~ O ..J
S1 o n the e l e c t r o p h o r e t i c m o b i l i t y o f e i t h e r S~ or S 2 particles. T o e x c l u d e t h e p o s s i b i l i t y t h a t S~ n u c l e o s o m e s represent structurally altered 'immature' particles excised from newly replicated regions of chrom a t i n , we e x p o s e d m i c r o p l a s m o d i a to [ 3 H ] t h y m i d i n e pulses a n d a n a l y z e d n a s c e n t D N A c o n t e n t in f r a c t i o n s S 1 a n d S 2. W e f o u n d that a f t e r a 3 m i n
1.0
~ a t
a J "~11
1
4
5
c
(%}
2
3
Fig. 6. Electrophoretic mobility of S] and S2 monomer particles as a function of a gel density. Plot of the logarithm of the migration distance (log m) against the acrylamide concentration (c).
256
pulse of [-~H]thymidine, the radioactivity per mg DNA was 38200 cpm for fraction S 1 and 38800 cpm for fraction S2, and after a 10 rain pulse, 130300 cpm and 135 300 cpm, respectively. From these results it is evident that fraction S~ is not enriched in freshly replicated DNA. In order to check whether the different electrophoretic behaviour of nucleosomal particles released from transcriptionally active chromatin could be due to their altered conformation, we performed electrophoretic analysis of S~ and S2 nucleosomes on a gel with a horizontal gradient of acrylamide concentration. This system permits the detection of differences in shape of the particles studied [15,20]. Fig. 6 shows a plot of the logarithm of the migration distance of monomer particles, as a function of the gel density. As can be seen, the straight lines obtained for S~ and S2 particles have clearly different slopes. Identical results were also obtained for di- and oligonucleosomes (data not shown). From this analysis it can be concluded that the different electrophoretic mobility of S~ and $2 particles results primarily from the difference in their shape; however, a difference in their effective electrical charge cannot be excluded. The relatively steeper slope observed for S~ particles is indicative of the increased frictional coefficient, i.e., the higher degree of assymetry of these particles in comparison to cannonical nucleosomes (S 2 particles).
Discussion In this work we have continued our studies on a structural basis for transcriptional activation of genes in Physarum. We have adopted the micrococcal nucleose-MgC12 method of Bloom and Anderson [11] to obtain the fraction of transcriptionally active chromatin [2]. In Physarum, the active MgC12-soluble chromatin fraction amounts up to 20% of the total genome, which is in good correspondance with the amount of transcribed DNA in this organism (12-18% as calculated from data in Refs. 6 and 21). In addition to the peculiarities of the structural organization of Physarum active chromatin reported earlier [2-4,8], we now present data indicating an altered conformation of its nucleosomes,
resulting from increased asymmetry of the nucleosomal core. This structural change in the active nucleosomal core cannot be attributed to the depletion of any of the core histones, or to the presence of specific nonhistone proteins or RNA, nor is it a consequence of the process of D N A replication. These data further support the idea that the process of transcriptional activation of genes involves loosening or opening of the nucleosome structure [5-8,22]. An advanced model of this type of structural transition has been proposed by Prior et al. [7] for active nucleosomes of ribosomal gene chromatin of P. polycephalum. According to this model, the cores of active nucleosomes (called the lexosomes) are symmetrically unfolded particles, having 3-4-times greater diameter (length) than compact nucleosomes, and consisting of two 'half-nucleosomes' connected by a 50 bp nucleoprotein bridge. Their structure is stabilized by two highly abundant nonhistone proteins (LP 30 and LP 32), associated with the connecting nucleoprotein bridge [7]. Although the open conformation of the active nucleosomal cores observed by us shares certain characteristics with the lexosome structure, we could not find any proteins which, like LP 30 and LP 32, could be responsible for the maintenance of the relaxed structure of the particle. Therefore, we postulate that the process of unfolding (linearization) of the nucleosomal core does not necessarily result from association with specific nonhistone proteins, but rather might be triggered by some modifications of the core histones. Our observations indicate that the active nucleosome structure is sensitive to Mg 2+. At a very low Mg 2+ concentration (0.1 mM), nucleosomal cores may undergo complete linearization, increasing the accessibility of intracore DNA to interaction with macromolecules. This is reflected by the disruption of the normal micrococcal nuclease cutting pattern seen when active chromatin is digested with this enzyme [8].
Acknowledgement We thank Mrs Aleksandra Bakula for technical assistance and Mr Artur Szymafiski for photographic prints.
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References 1 Reeves, R. (1984) Biochim, Biophys. Acta 782, 343-393. 2 Czupryn, M. and Toczko, K. (1982) Acta Biochim. Polon. 29, 17-26. 3 Czupryn, M., Solnica, L. and Toczko, K. (1985) FEBS Lett. 189, 89-91 4 Czupryn, M. and Toczko, K. (1984) FEBS Lett. 169, 174-178. 5 Lohr, D.E. (1984) Cell Biophys. 6, 87-102. 6 Scheer, U., Zentgraf, H. and Saner, H.W. (1981) Chromosoma 84, 279-290. 7 Prior, C.P., Cantor, C.R., Johnson, E,M., Littau, V.C. and AUfrey, V.G. (1983) Cell 34, 1033-1042. 8 Czupryn, M. and Toczko, K. (1985) Eur. J. Biochem. 147, 575-580. 9 Daniel, J.W. and Baldwin, H.H. (1964) in Methods in Cell Physiology (Prescott, D., ed.), Vol. 1, pp. 9-41, Academic Press, New York. 10 Jockusch, B.M. and Walker, I.O. (1974) Eur. J. Biochem. 48, 417-425.
11 Bloom, K.S. and Anderson, J.N. (1978) Cell 15, 141-150. 12 Burton, K. (1956) Biochem. J. 62, 315-323. 13 Sahasrabuddhe, C.G. and Saunders, G.F. (1977) Nucleic Acids Res. 4, 853-866. 14 Bode, J., G6mez-Lira, M.M. and Schri~ter, H. (1983) Eur. J. Biochem. 130, 437-445. 15 Bode, J. (1984) Arch. Biochem. Biophys. 228. 364-372. 16 Laemmli, U.K. (1970) Nature 227, 680-685. 17 Wray, W., Boulikas, T., Wray, V.P. and Hancock, R. (1981) Anal. Biochem. 118, 197-203. 18 Stalder, J. and Braun, R. (1978) FEBS Lett. 62, 251-254. 19 Gottesfeld, J.M. and Butler, P.J.G. (1977) Nucleic Acids Res. 4, 3155-3173. 20 Albanese, I. and Weintraub, H. (1980) Nucleic Acids Res. 8, 2787-2805. 21 Hardman, N., Jack, P.L., Fergie, R.C. and Gerrie, L.M. (1980) Eur. J. Biochem. 103, 247-257. 22 Tsanev, R. (1983) MoL Biol. Rep. 9, 9-17. 23 Tasheva, B. and Dessev, G. (1983) Anal Biochem. 129, 98-102.