187,16-24

EXPERIMENTALCELLRESEARCH

(1990)

Characterization and Nucleosomal Core Localization of Achlya Histones Involved in Stress-Induced Chromatin Condensation DAVID H. PEKKALA Division

of Life Sciences and Department

of Microbiology,

Scarborough

Campus, University

INTRODUCTION The filamentous fungus Achlya ambisexualis exhibits several features of chromatin organization which differ somewhat from those of most higher eucaryotes. These include: a short nucleosomal DNA repeat length; the apparent absence of condensed chromatin in either interphase or mitotic nuclei; a somewhat unusual histone complement marked by the presence of a unique histone, designated CY,and by the apparent absence of histone Hl [l, 21. A histone complement and chromatin structure nearly identical to that of Achlya is seen also in the closely related oomycete fungus Saprolegnia ferax [3]. The nucleosomal linker-associated histone Hl is widely believed to mediate, and to be required for, condensation of chromatin. However, despite the apparent absence of an Hl-like histone, under heat-shock conditions, Achlya chromatin becomes markedly condensed and refractory to DNase I digestion [4]. Concomitant with these changes, marked increases in the phosphorylation of Achlya histones H3 and (Y are observed [4]. These observations suggest that divergence may exist in the Oomycetes not only with respect to certain histones

0014.4827/90 $3.00 Copyright 0 1990 by Academic Press, All rights of reproduction in any form

and reprint

MATERIALS

Ontario, Canuda Ml C lA4

AND METHODS

Cell Culture Asexual spores were obtained from A. ambisexuulis J. Raper strain E87 using the method of Griffin and Breuker [5]. Spores were inoculated into liquid peptone-yeast extract-glucose medium [6] to a final concentration of 1 X lo4 viable spores per milliliter and grown for 16 h at 28°C on a rotary shaker (Orbit Environ-Shaker, Lab Line) at 140 rpm. Cultures were then diluted into 10 vol of Barksdale’s mating medium (BMM), pH 7.0 [7], and grown for 18 h at 28°C and 120 rpm. All experiments were carried out using 18-h cultures in BMM. Isolation

of Achlya Nuclei

Achlya mycelia were recovered from the medium by gentle suction filtration through Miracloth filters (Johnson and Johnson) and the harvested cells were rinsed with doubly distilled water. The cells were broken open with a Willems Polytron homogenizer (Brinkmann Instruments) at 70% maximum rated speed (approximately 15,400 rpm) for 3 to 5 s in the buffer system described by Silver [l]. The sample container was immersed in ice during disruption of the cells. The cell homogenate was filtered through Miracloth and the filtrate was collected. The mycelium was resuspended in buffer, disrupted again with the Polytron, and filtered as described above. The filtrates were pooled and centrifuged at 65OOg for 15 min to obtain the crude

requests should be ad-

16 Inc. reserved.

of Toronto, Scarborough,

(e.g., Hl and (Y) but also with respect to the packaging of chromatin into nucleosomes and higher-order structures. Further analysis of Achlya histones and chromatin could therefore enhance our understanding of strategies of chromatin organization and packaging. To further investigate the Achlya histones and to determine their relationship to one another and to the well-characterized histones of higher eucaryotes, Achlya histones were analyzed in several 2D gel electrophoresis systems and specific Achlya histones were also analyzed by one-dimensional peptide mapping. In addition, the nucleosomal location (i.e., core versus linker) of each Achlya histone was determined. In these studies, no major nuclear protein analogous to the histone Hl of higher eucaryotes was found to be associated with the nucleosoma1 linker regions of Achlya chromatin. In addition, the electrophoretic behavior on several 2D systems and the partial peptide map of Achlya histone CYwere found to differ from those of known histones. This novel Achlya histone was found to be a component of the nucleosomal core region.

To better understand the basis for heat shock-induced chromatin condensation in Achlya, a further characterization of the histones of this organism was carried out. The nucleosomal location (i.e., core vs linker), partial peptide map, and electrophoretic behavior of each Achlya histone was determined and compared to the well-characterized histones of rabbit kidney. The results of this and previous studies suggest that in Achlya, no nucleosome linker-associated histone analogous to histone Hl of higher eucaryotes is observed and that the Achlya histone designated a is a novel nucleosomal core histone. These observations may reflect the existence of a mechanism of stress-induced chromatin condensation which does not involve histone Hl. 0 1990 Academic Press, Inc.

1 To whom all correspondence dressed.

C. SILVER’

AND JULIE

FUNGAL

NUCLEOSOMAL

nuclear pellet and the postnuclear supernatant. The crude nuclear pellet was purified by centrifugation through dense sucrose at 161,000g for 60 min as described by Silver [ 11. Nuclei were resuspended in buffer containing 0.5% Nonidet P-40 and centrifuged at 12,000g for 20 min to recover the final nuclear pellet. Preparation

of Achlya Histones

The sucrose- and detergent-washed nuclei were resuspended in 6 vol of 0.4 N HCl and acid-soluble proteins were extracted for 60 min on ice. The acid-insoluble material was sedimented by centrifugation at 12,000g for 15 min. The acid-soluble material was precipitated from the 0.4 N HCl at -20°C for 3 days with 10 vol of acetone containing 0.1 M HCI and 5.0 mM phenylmethylsulfonyl fluoride (PMSF) as a protease inhibitor, Proteins were collected by centrifugation at 12,OOOgfor 20 min, dried under nitrogen gas, and resuspended in appropriate sample buffer for gel electrophoresis. Preparation

of Rabbit Kidney Histones

Rabbit kidneys were collected and homogenized as described by Silver [l]. The homogenate was then filtered through four layers of cheesecloth and centrifuged at 2500g for 10 min. The pellet was resuspended in buffer [l] except that Triton X-100 was added to 0.1% and the sample was centrifuged at 2500g for 10 min to obtain the crude nuclear pellet. The nuclei were further purified by centrifugation through dense sucrose as described by Silver [l] except that the buffer contained 0.1% Triton X-100. Nuclei were resuspended in buffer without Triton X-100 and centrifuged at 12,000g for 15 min. To extract histones from the rabbit kidney nuclei, the nuclear pellet was resuspended in 10 vol of 0.3 N HCl and the acid-soluble proteins were extracted for 60 min on ice. Acid-insoluble material was removed by centrifugation at 12,OOOgfor 15 min and the acid-soluble material was precipitated with 10 vol of acetone containing 0.1 M HCl and 5.0 mM PMSF for 24 h at -20°C. Proteins were collected and treated as described for Achlya acid-soluble nuclear proteins. Micrococcal

Nuclease Digestion of Achlya Chromatin

Nuclei were isolated from cells grown in BMM at 28°C as described above. The nuclei were resuspended in 1.0 mM Tris-HCl, pH 8.0,O.l mM MgClz, 0.25 M sucrose (150 ~1 of buffer/100 ODzM, units of sample). The samples were warmed at 37°C for 2 min and made up to 0.5 mM with CaCl,. Aliquots of chromatin were digested for 7 min at 37-C with 0.06 units of micrococcal nuclease (Sigma) per ODzM) unit of the nuclear suspension. Digestion was stopped by placing the samples on ice and adding an equal volume of 0.02 M Tris-HCl, pH 8.0, 0.06 M Na,EDTA. The samples were dialyzed for 18 h at 4°C against 10 n&f Tris-HCl, pH 7.8,1.0 mM Na,EDTA, 0.1 mM PMSF, and clarified by centrifugation at 12,000g for 15 min. The micrococcal nuclease digestion products were electrophoresed immediately on polyacrylamide-agarose gels as described below. This was followed by either the isolation of the nucleosomal DNA fragments or the isolation of nucleosomal proteins. Preparation

of Nucleosomal

DNA Fragments

Nucleosomes were prepared from Achlya nuclei by micrococcal nuclease digestion as described above and analyzed in 2.5% polyacrylamide-0.5% agarose gels. The position of specific nucleosome bands were detected by staining adjacent sample tracks with ethidium bromide and viewing under ultraviolet light. Gel pieces containing nucleosomes of different sizes were individually excised from unstained tracks. Each gel piece was finely chopped with a razor blade and incubated for 15 h at 37°C with agitation in 20 mM Tris-HCl, pH 7.8, 20 mM Na,EDTA, 0.2 M NaCl, 1.0% SDS. Gel pieces were removed by centrifugation at 12,000g for 15 min and the supernatant was recovered and mixed with an equal volume of phenol saturated with 20 mM

CORE

HISTONES

17

Tris-HCl, pH 7.8, 20 mM Na,EDTA. After separation, the aqueous phase containing DNA was recovered and DNA was precipitated overnight at -20°C by the addition of 2 vol of absolute ethanol followed by centrifugation at 12,000g for 30 min. The DNA was resuspended in 10 mM Tris-HCl, pH 7.8, 20 mM NaeEDTA, 100 mM NaCl, 10% glycerol, 0.05% bromophenol blue, 0.5% SDS prior to electrophoresis in polyacrylamide-agarose gels with size standards for determination of DNA fragment lengths. Electrophoretic

Procedures

Polyacrylumide-agarose (PA) gel electrophoresis of nudeosomes or DNA. Gels composed of a mixture of polyacrylamide and agarose were used for the analyses of nucleoprotein particles (i.e., nucleosomes) and of DNA. Nucleosome samples were resuspended in 10 mM Tris-HCl, pH 7.8,1.0 n&f Na,EDTA, 0.1 mM PMSF which was made 10% with glycerol prior to loading. DNA samples were resuspended in 10 mM Tris-HCl, pH 7.5,50 mM NaCl, 0.1 mM Na,EDTA which was made up to 0.4% SDS, 0.04% bromophenol blue, 40 mM Na,EDTA, and 20% glycerol prior to loading. Vertical slab gels (1.5 mm thick) of 2.5% polyacrylamide and 0.5% agarose were prepared using GelBond PAG Film (FMC, Marine Colloids Division) as a support. The gel and running buffer was 40 m&f Tris-HCl, pH 7.8, 20 mM sodium acetate, and 2.0 mM NazEDTA (TAE buffer). Gels were electrophoresed for 1 h at 80 V prior to loading of samples and for 4 h at 90 V subsequent to application of the sample. A 123-bp ladder DNA marker (Bethesda Research Laboratories) was electrophoresed alongside DNA samples for the calculation of DNA fragment sizes. One-dimensional SDSpolyacrylnmidegel electrophoresis. Acid-soluble nuclear proteins were precipitated from the extraction buffer and dried under nitrogen gas as described above. The samples were dissolved in SDS sample buffer (0.0625 M Tris-HCl, pH 6.8, 3% SDS, 5% BME) and boiled for 30 s. After boiling, glycerol, SDS, and bromophenol blue were added to each sample to final concentrations of 14, 0.05, and O.Ol%, respectively, and the samples were loaded onto the gel. Proteins were electrophoresed in SDS-polyacrylamide slab gels (17 X 13 X 0.15 cm) as described by Studier [8] using the discontinuous buffer system of Laemmli [9]. The gels consisted of either 15% polyacrylamide or of gradients of 7-17% or 8-15% polyacrylamide. SDS gels were electrophoresed at constant voltage (90 V) until bromopheno1 blue tracking dye reached the bottom of the gel (approximately 22 h). Molecular weight markers used as reference standards in SDS gels were phosphorylase a (94,000) bovine serum albumin (68,000), rabbit muscle actin (43,000), a-chymotrypsinogen (25,700), myoglobin (17,200), and cytochrome c (12,500). Mammalian histones prepared from rabbit kidney nuclei were used as additional reference standards in analyses of histones. Acetic acid-urea (AU) or Triton X-l 00-acetic acid-urea (TA U) polyacrylumide gel electrophoresis. Acid-soluble nuclear proteins were isolated and dried under nitrogen gas as described above. The samples were dissolved in 8 M urea with 5% BME and the solution was made up to 20% sucrose and 1% Pyronin Y (tracking dye) prior to loading onto the gels. Acetic acid-urea gels composed of 15% polyacrylamide, 5.375% acetic acid, and 2.5 M urea were prepared as described by Panyim and Chalkley [lo] except that slabs (17 X 13 X 0.15 cm) were used instead of tubes. The running buffer was 7% acetic acid containing 0.1% 2-mercaptoethanol. The gels were preelectrophoresed prior to application of the sample, for 3 h at 19 mA of constant current to remove unreacted ammonium persulfate. The buffer was then changed and the samples applied. The gels were electrophoresed at 12 mA of constant current for 22 h at 4’C in a cold room to reduce heating of the gels. Triton X-loo-acetic acid-urea gels composed of 15% polyacryamide, 0.625% Triton X-100,5.375% acetic acid, and 2.5 M urea were prepared as described by Zweidler [ll]. The gels were preelectrophoresed prior to the application of samples as described for AU gels. After

18

PEKKALA

application of the samples, the gels were electrophoresed at 19 mA constant current for 7 to 8 h at room temperature. Two-dimensional gel electrophoresis. For AU:SDS, TAU:SDS, or PA:SDS two-dimensional gel electrophoresis, gel tracks or pieces were excised from the first-dimension AU, TAU, or PA gels, respectively. Each gel piece was then equilibrated for 30 min in three changes of SDS running buffer [9] made to 1.0% SDS, 5% BME, and 0.01% bromophenol blue. The gel pieces were then loaded onto 15% polyacrylamide SDS slab gels (17 X 13 X 0.2 cm). Electrophoresis procedures and reference standards (markers) were as described above.

Partial Peptide Mapping

of

Histones

Achlya histones were compared to one another and to mammalian histones by peptide mapping following limited digestion with Staphylococcus aweus V8 protease (Miles) as described by Cleveland et al. [ 121 with the procedural modifications of Fey et al. [ 131 for the analysis of peptides recovered from two-dimensional gels. Essentially, Achlya and rabbit kidney histones were excised from either AUSDS or TAU: SDS two-dimensional gel systems or from SDS one-dimensional gels, as was appropriate. Excised gel pieces approximately 7 X 3 X 1.5 mm in size were dehydrated in methanol and stored at -20°C. Before the gel pieces were used they were dried under nitrogen gas and then incubated at 37°C for 1 h to remove all methanol. The gel pieces were rehydrated in 50 pl of buffer (0.1267 M Tris-HCl, pH 6.8, 11.2% glycerol, 0.02 M Na,EDTA, 0.2% SDS) containing 28 units of V8 protease (Miles). Controls were rehydrated in buffer without protease V8. After incubation at 37°C for 60 min, the gel pieces were finely chopped and loaded onto 15-20% acrylamide gradient SDS gels for electrophoresis. The peptides were detected by either Coomassie blue or silver staining. Gel Staining AU, TAU, and SDS polyacrylamide gels were stained for 2 h with a solution of 0.25% Coomassie blue, 50% methanol, and 10% acetic acid. The gels were destained with 40% methanol and 5% acetic acid, Where greater sensitivity was required, SDS polyacrylamide gels were silverstained using the methods of either Oakley et al. [14] or Wray et al. [15]. DNA or nucleosomes on polyacrylamide-agarose gels were detected by staining with 25 mg/ml of ethidium bromide in TAE buffer (see above) and photographed under short wavelength ultraviolet light.

RESULTS Analysis of the Electrophoretic Achlya Histones

Properties of

Analysis of Achlya histones on acetic acid-urea:SDS two-dimensional polyacrylamide gels. One of the two different 2D electrophoretic systems used to analyze Achlya histones was a gel system consisting of an acetic acid-urea polyacrylamide gel [lo] in the first dimension and an SDS polyacrylamide gel in the second dimension (AU:SDS). For comparison, rabbit kidney histones were analyzed in the same gel system. The designation of the Achlya and rabbit histone markers in the SDS dimension was made possible from their known electrophoretie mobilities on 1D gels [ 1,3]. The relationship of the Achlya histone bands seen on the first-dimension AU gels to those seen on the second-dimension SDS gels was confirmed by cutting out individual stained bands from the first-dimension gels and electrophoresing them separately onto SDS gels (data not shown).

AND

SILVER

As shown in Figs. 1A and lB, several of the rabbit and Achlya histones resolved on the first-dimension AU gels into at least two bands which migrated to the same or similar locations on the second-dimension SDS gels. The order of migration of the Achlya histones on the first-dimension AU gels was /3 < H3 < H2A < cr < H4 (Fig. 1A). The order of migration of the rabbit kidney histones on these gels was Hl < H3 -CH2B < H2A < H4 (Fig. 1B). In the AU:SDS system, the Achlya histone designated /3 was found to migrate on the second-dimension SDS gels to a position similar to that of Achlya histone H3 (Fig. lA, small arrowheads). This suggested that Achlya nuclei might contain two major histone species with the molecular weight of histone H3 but differing with respect to charge. In earlier studies using 1D gels [l, 31, it was suggested that Achlya histone & seen only on AU gels, might possibly migrate to the position of Achlya histone (Yon SDS gels. The AU:SDS gels however, establish that Achlya histones cyand fi are quite distinct proteins, which migrate to very different positions in this 2D gel system. Achlya histone (Y migrated to a position between histones H2A and H4 and could be resolved into at least two components on the first-dimension AU gel, both of which migrated to the same position on the second-dimension SDS gel. Achlya histones H2A and H4 each migrated essentially as single protein species in the AU:SDS gel system (Fig. 1A). No Achlya nuclear protein with the stoichiometry and electrophoretic behavior expected for histone Hl was observed on either the AU or SDS gels. Analysis of Achlya histones on Triton X-IOO-acetic acid-urea:SDS two-dimensional polyacrylamide gels. A second 2D gel system used to analyse Achlya histones consisted of a Triton X-loo-acetic acid-urea polyacrylamide gel in the first dimension and an SDS polyacrylamide gel in the second dimension (TAU:SDS). TAU gels separate proteins primarily on the basis of hydrophobicity [ll]. For comparison, rabbit kidney histone standards were also analyzed in the TAU:SDS system. The slowest migrating Achlya histone band on the firstdimension TAU gel resolved into two components on the second-dimension SDS gels. On the second dimension, one component migrated to the position of histone H3 and the other migrated to the position of histone H2A (Fig. 2A). In contrast, the rabbit histone band which migrated most slowly in the first-dimension TAU gel was found to consist of only histone H2A (Fig. 2B). For both Achlya and rabbit, the histone migrating second from the top on the first-dimension TAU gels migrated to the position of histone H3 on the second-dimension SDS gel (Figs. 2A and 2B). Thus, as was also seen in the AU:SDS system (Fig. lA), two well-resolved Achlya proteins each migrated to the position of histone H3 in the TAU:SDS system (Fig. 2A, small arrowheads). On the basis of these

FUNGAL

NUCLEOSOMAL

H2A D H3 o( I I Ih

AU+

CORE

HISTONES

19

H2B HP

AU

1, JH~ -H2B IH2A -t-i4

B

A

FIG. 1. Analysis of Achlya histones on AU:SDS two-dimensional polyacrylamide gels. Acid-soluble nuclear proteins were prepared from Achlya and from rabbit kidney. The proteins were electrophoresed in a two-dimensional polyacrylamide gel system consisting of an acid-urea (AU) gel in the first dimension and a 15% a&amide SDS gel in the second dimension. The direction of migration in the first and second dimensions is indicated by the large arrows. Proteins were stained with Coomassie blue. The first-dimension AU gels for Achlya and rabbit histones are shown at the top of A and B, respectively. (A) Achlya acid-soluble nuclear proteins. On the left side of the two-dimensional gel are Achlya histone markers electrophoresed on the second-dimension SDS gel only. A separate track of Achlya histone markers from a onedimensional gel is shown for comparison in the insert at the far left. Small arrowheads indicate the positions of the two Achlya histones which migrate to the region of histone H3 on the second dimension gel (see text). (B) Rabbit kidney acid-soluble nuclear proteins. At the right are rabbit histone markers electrophoresed on the second-dimension SDS gel only.

results and peptide mapping (see below) the slower migrating H3 band in TAU gels, previously designated histone @,was designated H3.1 and the faster H3 band was designated H3.2.

Achlya histones H4 and H2A each migrated as single protein species in the TAUSDS gel system (Fig. 2A). Achlya histone CYmigrated slightly faster on the firstdimension gel than did Achlya histone H4. Although his-

H3, H2A

TAU-

H2A 73 HP

I

HI(H2B

.H3 .H2A :;4

FIG. 2. Analysis of Achlya histones on TAUSDS two-dimensional polyacrylamide gels. Acid-soluble nuclear proteins were prepared from Achlya and from rabbit kidney. The proteins were electrophoresed in a two-dimensional polyacrylamide gel system consisting of a triton-acid urea (TAU) gel in the first dimension and a 15% acrylamide SDS gel in the second dimension. The direction of migration in the first and second dimensions is indicated by the large arrows. Proteins were stained with Coomassie blue. (A) Achlya acid-soluble nuclear proteins. The firstdimension TAU gel is shown at the top and Achlya histone markers which were electrophoresed on the second-dimension SDS gel only are shown at the right. Small arrowheads indicate the two Achlya histones which migrated to the position of H3 on the second dimension gel. (B) Rabbit acid-soluble nuclear proteins. The first-dimension TAU gel is shown at the top and rabbit kidney histone markers which were electrophoresed on the second-dimension SDS gel only are shown at the right.

20

PEKKALA

a

b

“34 “3.1 lchlVa

cde

V8

fg

H3.2

H3.1

,H3

Achlya

H2B, rabbit

h

i

V8

L “3

j

HZ6

k

,

V8

rabbit

VE-treated

FIG. 3. Peptide mapping of Achlya histones H3.1 and H3.2 and rabbit kidney histones H3 and H2B isolated from TAUSDS gels. Achlya acid-soluble nuclear proteins were electrophoresed in a TAU:SDS two-dimensional gel system. Specific histones were excised from the second-dimension gel and treated with Staphylococcus oure~(s V8 protease. V8-treated Achlya histones and nontreated controls were electrophoresed in a 20 to 25% acrylamide gradient SDS gel, along with rabbit histones H3 and H2B which had been treated similarly. The peptides were located by silver staining. (a) Untreated Achlya H3.2; (b) untreated Achlya H3.1; (c) V8 only; (d) V8-treated Achlya H3.2; (e) V8-treated Achlya H3.1; (f) V8-treated rabbit H3; (g) VB-treated rabbit H2B; (h) V8 only; (i) untreated rabbit H3; (j) untreated rabbit H2B; (k) V8 only.

tone QI migrated as one large spot in the TAU:SDS gel system (Fig. 2A) this histone could be resolved into at least two components in the AU:SDS gel system (Fig. 1A). In summary, the order of migration of Achlya histones on the first-dimension TAU gels was H2A = H3.1~ H3.2 < H4 < (Y (Fig. 2A). The order of migration of rabbit kidney histones on the first-dimension TAU gels was H2A < H3 < H4 < Hl < H2B (Fig. 2B). No major Achlyu nuclear protein was observed to migrate to a position similar to that of rabbit histone Hl in the TAU:SDS gel system. Peptide mapping of Achlya histones H3.1, H3.2 and rabbit kidney histones H3 and H2B isolated from TAU: SDS gels. To confirm the identity of the two Achlya H3 proteins seen on the TAU:SDS gels, Achlya histones H3.1 and H3.2 and rabbit histones H3 and H2B were isolated from TAU:SDS gels and treated with protease V8. The peptide maps of Achlya H3.1 and H3.2 and of rabbit H3 and H2B are shown in Fig. 3, lanes d, e, f, and g, respectively. Achlya Histones H3.2 and H3.1 and rabbit histones H3 and H2B which had not been treated with V8 are shown in Fig. 3, lanes a, b, i, and j, respectively, The autodigestion products of protease V8 alone, which are visible with the sensitive silver-staining pro-

AND

SILVER

cedure used, are shown in Fig. 3, lanes c, h, and k, so that these may be easily compared with the digestion products of the Achlya histones. The peptide maps of H3.1 and H3.2 were similar to one another and to that of rabbit H3 but were different from that of rabbit H2B (Fig. 3) and also different from the peptide maps of any of the other rabbit kidney histones (compare Fig. 4). These results confirmed the presence of two major subtypes of histone H3 in Achlya nuclei. Comparison of Achlya histone a with rabbit kidney histones by peptide mapping. Achlya histone o has not been reported in other organisms but is observed in Achlya and in the closely related Oomycete Saprolegnia [l, 31. In order to determine if Achlya histone (Yshowed similarity to a known mammalian histone, histone (Y was excised from TAU:SDS gels and digested with protease V8. The peptide products were compared with the peptide maps of the five rabbit kidney histones Hl, H3, H2B, H2A, and H4 which had been excised from TAU: SDS gels and treated similarly (Fig. 4, lanes a to e, respectively). The autodigestion pattern of protease V8 alone is shown in Fig. 4, lane g. Unlike the other histones tested, rabbit histone H4 was not appreciably digested by protease V8 under the conditions employed. As shown in Fig. 4, lane f, the peptide map of histone CYwas bcdefg

a

HI

H3

Ii28 rabbat

1

H2A

H4,,

c-c

j

V8

Achlya

VE-treated

FIG. 4. Comparison of Achlya histone (Y with rabbit kidney histones by peptide mapping. Achlya acid-soluble nuclear proteins were electrophoresed in TAU:SDS two-dimensional gels. Specific hiEtOne8 were excised from the second-dimension gel and treated with Staphylococcus aureu.s V8 protease. V8-treated Achlya histone o and rabbit kidney histones which had been treated similarly were electrophoresed in a 20 to 25% acrylamide gradient SDS gel. The peptides were located by silver staining. (a) V8-treated rabbit Hl; (b) V8-treated rabbit H3; (c) VS-treated rabbit H2B; (d) V8-treated rabbit H2A; (e) V8treated rabbit H4; (f) Vb-treated Achlya a; (g) V8 only.

FUNGAL

“3: 2N

1N

492 3N

369

2N

246

1N

A

NUCLEOSOMAL

123

B

FIG. 5. Analysis of Achlyu nucleosomal DNA. Achlya nuclei were treated with micrococcal nuclease and the resulting mono- and oligonucleosomes of different sizes were separated by electrophoresis on 2.5% polyacrylamide-0.5% agarose gels. The fragment length of the DNA associated with each nucleosome band was determined by electrophoresing extracted DNA on a 2.5% polyacrylamide-0.5% agarose gel with the DNA fragments of a 123-bp ladder as size markers. (A) Mono- and oligonucleosome nucleoprotein particles. (B) DNA associated with nucleosomes. The DNA size markers are shown at the right. The accompanying numbers indicate the size in base pairs of each marker. The mononucleosome band is indicated by lN, while 2N, 3N, and 4N indicate multimers.

found to be relatively distinct from that of any of the five rabbit kidney histones (Fig. 4, lanes a to e).

Nucleosomal Location of Achlya Histones As reported previously by Silver [l, 31 and as suggested in the studies herein, Achlya chromatin appears to lack a nucleosome linker histone analogous to histone Hl of higher eucaryotes. A unique Achlya histone designated a was however observed. To determine if Achlya histone a would show functional similarity to histone Hl, that is, would be associated with nucleosomal linker regions rather than with nucleosome cores, Achlya chromatin was digested with micrococcal nuclease to isolate nucleosomes. The resulting nucleosome monomers and multimers of different sizes were separated on 2.5% polyacrylamide-0.5% agarose gels and visualized using ethidium bromide (Fig. 5A). To determine the exact fragment length of the DNA associated with each of the deoxynucleoprotein (DNP) bands, gel pieces containing DNP particles of monomer, dimer, and trimer size were cut from unstained sample tracks adjacent to the stained tracks and the DNA was extracted. The extracted DNA was electrophoresed on 2.5% acrylamide-0.5% agarose gels alongside a 123-bp DNA ladder as marker (Fig. 5B). In three replicate experiments the average fragment length of the DNA associated with the monomers so produced (indicated as 1N in Fig. 7) was 144 + 4 bp. The nucleosomal DNA repeat length was calculated by determining the slope of a plot of DNA fragment length in base pairs versus band number [ 161. The average nucleosomal repeat length of Achlya chromatin was calculated to be 160 t- 2 bp, which is in close agreement with previous reports [2, 31. The

CORE

21

HISTONES

DNA size determinations allowed the monomer band to be unequivocally identified. The length of DNA of 144 + 4 bp associated with the monomers generated, was consistent with the identification of the monomers as “stripped” core particles, i.e., those free of linker DNA, The proteins associated with mononucleosomes and oligonucleosomes were identified using 2D gel electrophoresis as described under Materials and Methods. The first-dimension gel separated monomers and multimers of DNP particles and the second-dimension SDS gel separated the proteins associated with these particles. Achlya histones H3, H2A, a, and H4 were all present both in multimers (2N, 3N) and in stripped mononucleosome core particles (IN) containing DNA of 144 bp in length (Fig. 6). These four Achlya histones appeared to be present in the same proportions in monomers and multimers. With the exception of a protein doublet which appeared to be associated only with dimers (Fig. 6, arrow), no protein present in the multimers was specifically released as digestion proceeded to stripped mononucleosome cores. These results support the conclusion that Achlya histones H3, H2A, CY,and H4 are nucleosomal core histones and that Achlya nucleosomes lack an identifiable linker protein analogous to histone Hl of higher eucaryotes. The nature of the protein doublet which appeared to be associated specifically with nucleosome dimers is not known, but a similar protein 1N 2N 3N

-H3 -H2A %I

FIG. 6. Nucleosomal location of Achlya histones. Achlyu nucleosomes were isolated and mononucleosomes and multimers of different sizes were separated by electrophoresis in a 2.5% polyacrylamide0.5% agarose first-dimension gel. The associated histones and DNA were resolved by electrophoresis in a 15% acrylamide SDS seconddimension gel. The second-dimension gel was silver-stained. Achlyu histone markers electrophoresed in the second dimension only are shown at the left. The positions of mononucleosomes (1N) and multimers (2N and 3N) are indicated at the top. The arrow indicates the position of a high-molecular-weight band discussed in the text. The arrowheads indicate the position of DNA which migrated into the second-dimension gel.

22

PEKKALA

AND

SILVER

Achlya histone (Y was found to be a nucleosomal core histone. However, both the partial peptide map as well as the electrophoretic behavior of Achlya histone (Y differed from that of any rabbit or Achlya histones (Fig. 4). Thus, although (Y is clearly a nucleosomal core histone, it appears to be a novel histone. As noted above, although not seen in other fungal chromatins studied to date, histone (Y is found in another Oomycete, S. ferax [3] and may therefore be an oomycete-specific histone. The results of this and previous studies [l, 31 suggest that Achlya chromatin does not appear to contain a nucleosomal linker-associated protein analogous to the histone Hl of higher eucaryotes. In animal cells, histone Hl is the only major nuclear protein soluble in 5% PCA, and histone Hl-like proteins from the slime mould Dictyostelium [19] and the zygomycete fungus Entomophaga [20] are also soluble in 5% PCA. However, no Achlya nuclear protein present in the stoichiometry expected for a histone Hl was selectively soluble in 5% PCA (data not shown). More significantly, analysis of DISCUSSION Achlya nucleosomes revealed no major nuclear protein associated with nucleosomal linker regions (Figs. 5 and Histone proteins have been extensively characterized in higher eucaryotes, in which five main types of his6). Taken together the results suggest that Achlya chromatin does not contain a major nuclear protein exhibittones exist. The histones of fungi are less well characterized, in part due to the difficulty in isolating nuclei and ing similarity to the histone Hl seen in most eucaryotes. histone proteins from these organisms. In previous work The yeast S. cereuisiae, like Achlya, also appears to lack employing 1D gels [ 1,3], Achlya chromatin was found to a histone Hl protein [21] and an Hl gene sequence has yet to be identified [ 221. Both Achlya and yeast chromacontain a somewhat unusual histone complement marked by the presence of a novel histone, designated CY, tins have unusually short nucleosomal DNA repeat and by the apparent absence of histone Hl [ 11. A histone lengths, that of Achlya being approximately 160-bp ([ 1, complement and chromatin structure nearly identical to 31 and this report) and that of yeast being 160-165 bp that of Achlya was seen also in the closely related oomy[23, 241. It has been noted that there may be a correlation between repeat size and levels of heterochromatin cete fungus S. ferax [3] but is not reported for other [20]. Relatively little or no heterochromatin is evident fungi. To further investigate the Achlya histones and to de- in nuclei of the fungi Sacchuromyces, Neurospora, and termine their relationship to one another and to the Aspergillus or in mammalian neurons [25-301, all of which have short nucleosomal repeats in the range of well-characterized histones of higher eucaryotes, Achlya 154-170 bp. Similarly, interphase or mitotic nuclei of the histones were analyzed in two different 2D gel systems, Oomycetes Achlya and Saprolegnia contain no discernpartial peptide maps were generated, and the nucleosoma1 location (core vs linker) of each Achlya histone was ible condensed chromatin [4, 311. These characteristics are not a feature of all fungal chromatins since the fundetermined. In these studies, Achlya nuclei were shown gus Entomophaga exhibits extensive amounts of conto contain two histone H3 subtypes, designated H3.1 densed chromatin in interphase nuclei. Furthermore, and H3.2. It is not known if H3.1 and H3.2 represent the same protein which has been differently modified by Entomophaga chromatin has a long nucleosomal repeat (197 f 1.2 bp) typical of that of higher eucaryotes and post-translational modification or whether these H3 has stoichiometric amounts of an Hl-like histone, albeit subtypes exhibit differences in amino acid sequence. less basic and smaller (18.9 kDa) [ 201. Preliminary analyses of Achlya histone genes indicate Histone Hl with a stoichiometry of one molecule per that there appear to be at least five putative H3 genes in the Achlya genome which exhibit homology to H3 genes nucleosome [32] is believed to be essential for the folding from S. cerevisiae (Allen and Silver, unpublished). It of the lo-nm fiber into 30-nm solenoids [33,34]. It is of would be of interest to determine the sequences of the particular interest therefore to find that Achlya chromatin, despite the apparent absence of a linker-associated Achlya histone H3 genes and proteins. Such analyses Hl-like protein, becomes markedly condensed and remight elucidate the basis for the electrophoretic differfractory to DNase I digestion during heat shock [4]. This ences in the H3 subtypes and perhaps suggest possible observation indicates that, at least in Achlya, chromatin functional differences, e.g., in potential phosphorylation condensation may possibly occur in a manner not medisites, between the H3 subtypes. has been reported previously [ 171 for the yeast Saccharomyces cereviseae. Nucleosome-associated proteins migrating faster than histone H4 in lanes lN, 2N, and 3N were not seen in the histone marker preparations (Fig. 6, left side). The histone marker preparations consisted of only acid-soluble nuclear proteins while the nucleosome samples contained both acidic and basic proteins as well as DNA. The nature of these additional nucleosome-associated proteins is unknown. The large silver-stained areas which migrated on a diagonal above the core histone bands (Fig. 6, arrowheads) consisted of the DNA which had been associated with the nucleosome particles and which migrated onto the second-dimension SDS gel. DNA is stained with the silver-staining procedure [X3] and similar diagonal DNA patterns can be obtained when the gels are stained with ethidium bromide but not with Coomassie blue.

FUNGAL

NUCLEOSOMAL

ated by histone Hl. Although in most eucaryotes Hl appears to mediate chromatin packaging, the core histones may also be important. The physical and biochemical properties of the amino-terminal regions of the nucleosomal core histones suggest that they may help to stabilize chromatin structure through interactions with the DNA on the outside of adjacent nucleosomes [35-371. Furthermore, proteolytic removal of the amino-terminal regions of the core histones has been shown to result in disruption of the 30-nm solenoid and in relaxation of chromatin structure [38, 391. The amino-terminal regions of the core histones contain the sites at which posttranslational modifications such as acetylation and phosphorylation occur. Together, these observations suggest that changes in the composition, abundance, or post-translational modification of nucleosomal core histones can be involved in changes in the extent of chromatin condensation. It is therefore of interest that the chromatin condensation which occurs in Achlya during heat shock is accompanied by the markedly increased phosphorylation of the Achlya nucleosomal core histone CYand one of the Achlya histone H3 subtypes, H3.1, and by the decreased phosphorylation of the other Achlya H3 subtype, H3.2 [4]. This observation suggests that, at least in Achlya, phosphorylation of these core histones may be more essential than the presence of an Hl histone in chromatin condensation. In summary, to date the histones of relatively few fungi have been characterized and relatively few fungal histone genes have been analyzed [40,41]. However, it is apparent that certain fungal histones, such as Achlya histone (Y, are somewhat different from the canonical histones of higher eucaryotes. These observations suggest that considerable evolutionary divergence may exist with respect to certain histones and therefore in the packaging of chromatin into nucleosomes and higher-order structures. Further analysis of Achlya histones and chromatin could therefore enhance our understanding of strategies of chromatin organization and packaging. The nucleosomal linker-associated histone Hl is widely believed to mediate, and to be required for, condensation of chromatin. However, despite the apparent absence of an Hl-like histone, under heat-shock conditions, Achlya chromatin becomes markedly condensed and refractory to DNase I digestion [4]. Concomitant with these changes, marked increases in the phosphorylation of Achlya histones H3.1 and CYare observed. These results suggest that in Achlya, the increased phosphorylation of the two core histones H3.1 and (Y, rather than the presence of histone Hl, may be responsible for, or play a significant role in, chromatin condensation. These observations may reflect the existence of a mechanism of chromatin condensation which has not as yet been widely considered but which may also occur in other cell types.

CORE

23

HISTONES

This study was supported by NSERC Canada Grants to J.C.S. and by Ontario Graduate Scholarships to D.H.P.

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