NOTES

Isolation and Tissue Specificity of Chromatin-Associated Proteins in

BARBARA GRAHEK ~ ~ I S C M K E AND OSCAR G . WARD Conlr~littec on Genetics, Tile University of Arizona, T~icson, Arizona 85726

Received May 23, 1974

Mischke, B. G . & Ward, 0. G. (1975) Isolation and Tissue Specificity of Chrornatin- Associated Proteins in Vdciu fabu. Cun. 1. Bisclnem. 53,91-95

A method is described that permits extraction of one class of non-histones in 8 M urea - 0.14 M mercaptoethanol prior to acid extraction of histones and a second class in 0.05 M Tris - 1% sodium dodecyl sulfate following acid extraction of histones. Comparisons of histones and nom-histones extracted by this method with those obtained by other procedures demonstrate two important advantages of the method: ( I ) histones obtained by this method are not contaminated by acid-soluble non-histones, and ( 2 ) non-histones are not subjected to acid or phenol during extraction. Changes in the distributions of ehromatin- associated proteins in different tissues suggest that some species represent regulators of gene action.

Mischke, B. G. & Ward, 0. G. (1975) Isolation and Tissue Specificity of Chromatin- Associated Proteins in Vicir~ fabu. Can. J . Biocl~eraz. 53, 9 1-95

Nous dkcrivons une mkthode qui permet l'extraction d'une premikre cIasse de prot6ines won histoniques dans Be rnklange uree 8 M - rnercaptoCthano1 0.14 A4 avant l'extraction acide des histones et une semnde classe dans le mklange Tris 0.05 M - dodCcyl sulfate de sodium 1% aprZs B'extraction acide des histones. La cornparaison des histones et des non-histones extraites par cette mkthode avec celIes obtenues par d'autres techniques montre deux avantages de cette mkthode: (1 ) les kisaones obtenues ne sont pas contaminkes par les non-histones acidosol~ables, et (2) les non-histones ne subissent pas l'action de 19acide ou du phknol durant l'extraction. Les changements dans la distribution des pratkines associCes h la chromatine dans diffdrents tissus suggkrent que certaines espkces representent des rkgulateurs de I'action gknique. [Traduit par le journal]

Introduetisn During the last few years, much effort has

been expended on non-histone proteins asso- ciated with chromatin, and the accumulated evi- dence indicates their involvement in regulation of gene activity. Characteristics intimating them to be regulatory proteins are the subject of recent reviews (1, 2) . To measure the resdts of differ- ential gene activation, variable features expressed by different tissues of an organism may be ex- amined, since from tissue to tissue, different

Many traditional methods of isolating non- histones rely on removal of histones from chro- matin with acid or salt. The methodology described here isolates non-histones directly from chromatin prior to acid extraction. Originally developed for use with mammalian tissue (31, this method has not yet been widely applied. To date there has been no report of its use with plants.

Materials and Methods portions of the genome are expressed. If chroma- P6at~t Tissue tin-associated proteins represent regulators of seeds of the broad bean, Vick f r t b ~ L. (var. gene activity, changes would be expected in their L o " ~ P o ~ ) ~ were surface-sterilized, in 10% Clorox.

germinated 2 days in running tap water with continu- distribution in different cell types. The ex~er i - ous aeration (41, removed to perlite, and grown 9 days ments described here present an improved eXtrac- in the dark. The 11-day-old etiolated seedlings were tion method for the studv of chromatin-associated harvested and dissected into lateral-root meristem,

proteins in plants, and Grovide evidence support- mature lateral-root tissue, stem tissue, and leaves. Cotyledons were collected at 0, 48, and 120 h of

ing the that these proteins as growth. In addition, epicotyls were dissected from dry regulators of gene activity. V . faba seeds and dark-germinated in glass vials with

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9 2 CAN. J . BIOCHEM. VUL. 53, 1975

5 ml water for periods of 14, 24, 36, or 48 h. These are referred to as "excised epicotyls."

isolndiorr of Chror)aatitm The procedure used was modified from that of

Huang and Bonner (5) for V . faba ( 4 , '7). Plant tissue was homogenized in 0.25 M sucrose - 0.001 ,%4 MgClr! - 0.05 Tris (pH 8.01, and the chromatin pellet was washed at least five times in 0.01 M Tris (pH 8.0) and three times in SSC.' Chromatin was not subjected to sucrose centrifugation, a step included in Muang and Bonner's method (5 1.

Psolcctic~n of CGlaroraanti~1-A~sociabed Proteins The procedure used was modified from that of

Gronow and Griffiths ( 3 ) . In place of N-ethylmale- imide rased by these workers, we have used 2-mercapto- ethanol. The final chromatin pellet was homogenized in 0.5-1.8 ml urea buffer (8 1&f urea - 0.14 1$4 2-mer- captoethanol - 0.05 M sodium phosphate (pH 7.611, and incubated 15 min at rosm temperature. Urea-soluble chromatin-associated proteins (USP) were recovered in the supernatant following centrifugation at 20 080 x g for 20 min. A second supernatant from a se- extraction, performed exactly as above. was pooled with the first, and the USP solution was dialyzed over- night at room temperature against 8 ikf urea - 0- 1 % 2-mercaptsethanol - 0.1 5% SSD - 0.01 M sodium phos- phate (pH 7.15). The remaining chromatin pellet was washed by homogenization in 5-10 ml urea buEer, recovered by centrifugation as before, and washed once in SSC.

TO remove histones, the pellet was homogenized in 1-2 ml of 0.4 N H2S04 and incubated in ice for 15 min. Acid-soluble proteins (HIS) were recovered in the supernatant following centrifugation at 10 000 X g for 15 min. The pellet was washed twice by homogeniza- tion in 5-10 rnl of 0.25 N HCI and recovered by cen- trifugation at 4000 x g for 10 mine The pellet was then homogenized in 1-2 ml of 0.05 M Tris (pH 8.0) containing 1% S S D S , and the suspension was incubated overnight at rosm temperature. Residual proteins (RE§) were recovered in the supernatant following centrifu- gation at 220 000 x g for 20 min. The final pellet, containing DNA and some tightly bound proteins, was saved for extraction of DNA with 5% TCA at 98 "C for 20 min. DNA and protein concentrations were estimated by the diphenylarnine (8 ) and Folin (9 ) methods.

Proteira Elecfroplloresis Electrophoresis of all protein classes was SBS

electrophoresis ( 10, 1 1 ) . Polyacrylan~ide gels were made with 10% Cyanogum 41 (E-C Apparatus Corp.) in 0.1 M sodium phosphate (pH 7.4) containing 0.1 5% SDS. The running buffer was identical with the gel buffer. Electrophoresis was carried out at room

'Abbreviations used: SSC, 0. IS M NaCl - 0.015 A4 trisoditarn citrate (pH 7.0); SDS, sodium dodecyl sul- fate; TCA, trichloroacetic acid; USP, urea-soluble proteins; HIS, presumed histones; RES, residual pro- teins; PSANP, phenol-soluble acidic nuclear proteins; BSP, base-soluble proteins.

temperature for 8 Ba at 5 rnA per tube. Gels were stained for approximately 1 h with 0.255% Coomassie blue in 796 acetic acid and excess stain was removed in '7% acetic acid. Gels were sca~nned at 555 nm and proportions of protein species determined from the integrated area under the peaks.

Results and Discussion

Andysis sf the Extraction Procedure Chromatin prepared by this method probably

contains some cytoplasmic protein. Microscopic examination of Feulgen-stained chromatin preparations showed predominantly Feulgen- positive tissue masses. Numerous starch granules were detected by the iodine test. RNA-positive material was detected in small amounts with a methyl green - pyronin method for tissue homog- enates (1 2). Rigorous over-purification of chro- matin was avoided, however, to minimize the loss of information about regulatory proteins that might be loosely bound to DNA. Repetitive ex- tractions of excised epicotyls demonstrated re- producibility of the isolation methods. A large series of gels was used to establish the presence of each band. The positions sf all USP bands (numbered in Fig. I ) were constant, except for those sf bawds I and 18, which varied slightly. Bands 0, 1, 2, and 4 were not consistently sb- served. Band 7 1 occasionally did not resolve into the two bands 1 1 a and 1 I b. The probable idemti- ficatden of HIS bands (lettered in Fig. I and iden- tified in Fig. 2) was deduced by assuming the same order of migration as that observed by Panyim and Chalkley (13) in their SDS system for histone electropkssssis. Since bands c and d did not always resolve into two bands, data on proportions of HIS subfractions considered pooled values for these bands. Othemiise, HIS bands were not variable. For these experiments, reduction sf the HIS fraction was not routinely done prior to electrophoresis. It has been shown that pea f3-histone dirners exist as artifacts due to oxidation during extraction ( 14). Presumably, the inclrasidsn of rnercaptoethanol during USP iso- lation reduced all histone-f3 dimers to their natural monomeric form since in no HIS gel was a band present at the appropriate position for a histone43 dimer. Much variability was sb- served among RES gels, and the e%eetrophoretic patterns were sometimes similar to USB patterns (Figs. I , 2) . It is possible that RES are USP

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NOTES

FIG. 1. Typical gels obtained with the isolation method. Arrow indicates direction of migration.

that were not completely extracted in urea buffer or that they represent a distinct class of highly variable protein species and that the similarities observed are superficial. The several bandg ob- served in our RES fraction markedly contrast with the lower number (two) of fast-running species obtained by Grsnow and Grifiths ( 3 ) .

Electrophoretic gels sf excised epicotyl Sam- ples, derived by altering the order of isolation of chromatin-associated proteins and by applying various isolation methods, are compared in Fig. 2. These results demonstrate that many non- histsnes are acid-soluble, and direct acid extrac- tion of chromatin results in highly contaminated histones. Measurement of area under the alleged histone peaks of direct acid-extraction gels dem- onstrated that only 66-68 % of all acid-extracted proteins are presumed histones. A slight change in position of bands in urea after acid gels corn- paredtoJJSgels is thought to be due t~ changes in the non-histone protein species during acid extraction. This demonstrates the desirability of direct extraction of non-histones. Some similarity of band patterns for lipoproteins and slow- running USP suggested that a few USP species could be lipoproteins. Lipoproteins were ex- tracted from acid-washed chromatin by solutions of chlorofom :methanol ( 1 : I followed by 2: 1 ) ( 15). Patterns obtained for BSAN tracted with phenol following lipoprotein extrac-

tion ( 15 ) , were similar to USP patterns, and the variability of band position can probably be attributed, at least in part, to the phenol treat- ment to which PSANB were subjected. Base ex- traction following acid extraction resulted in fewer protein species on BSP gels than on either USP or PSANP gels. It was concluded from this series of comparisons that direct urea extraction of chromatin, f s h w e d by acid extraction, repre- sents a significantly improved method for isoIa- tion of chromatin-associated proteins for intensive study sf their properties.

Tissue Specificity Calculation of protein/DNA ratios demon-

strated no statistical differences among different and RES. Although the USP/ not related to any discernible

feature of the tissues examined, the ratios did B Biffermt-tissues -exhibited zhracteris-

Table 1 lists the mean praportions of HIS species for various tissues, and their ranges. The proportions of HIS bands a and b are in line with literature values for histone f 1 for pea (1 61, wheat (17), and V . faha (6) ; however, the pro- portions of HIS c and d are lower than expected for f2a2 $- f2b + f3. This is consistent with problems encountered with quantitative demsi- tometry involving Coomassie-blue stain (I 8).

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94 CAN. J . BIOGKEM. VOL. 53, 1975

4 4 TABLE 1. Proportions of HIS species for various tissuesa 12 b ' h a o s a r e 5 4 3 2 40

UREA EXTRACT

(USPI H I S species

ACID EXTRACT Tissue (fl) (f2b 9 f3' 9 f2a2) (f2a1

Leaf 14 47 3 6 d 7 44-49

UREA AFTER AClD meraseem 18 40 17-1 8 37-43

Mature lateral root 13 49 ACID AFTER UREA 9-1 6 46-5 2

(HIS) Stem 8 41 2-1 4 33-93

Cotyledon, 0 h I2 46 R ES Cotyledon, 48 h 24 40

20-219 30-50 Cotyledon, I20 h 14 46 3 9

L I POPROTEINS 14-19 42-5 1 34-45 aproportions are expressed as percentages of total HIS. Ranges arc

indicated below the mean value. Values for bands c and d are nooled since these bands were not always resolved into two bands.

P SA N P

< FIG. 2. Gel patterns for proteinls obtained from various

iscabtion procedures. USP are obtained from direst extraction of chromatin with urea buffer. Acid extract indicates proteins obtained when chromatin is directly extracted with acid. Urea affer. acid indicates proteins obtained when chromatin is extracted with urea buffer following removal of acid-soluble proteins and lipsprs- teins. HIS indicates proteins obtained when chromatin is extracted with acid following extraction with urea buffer. RES indicates proteins obtained from Tris-SDS extrac- tion following removal sf USP and HIS. La'poprstei~js were extracted from acid-washed chromatin by solutions of chlorsform:methano1. PSANP were obtained by extraction of chomatin with phenol following extractioh of acid-soluble proteins and lipoproteins (15). BSP were obtained from extraction of chromatin with NaBH fs11owing extraction of acid-soluble proteins, Arrow indicates direction of migration.

Leaf and root meristem have a ssmcwhat greater proportion of HIS a and b than obsemed for other tissues. An elevated proportion of lysine- rich histones in meristernatic tissue has been ob- sewed in some experiments (19, 20) but not in sthers (1 6, 1 9 ) . Contrary to the results of former work ( 16, 201, no significant changes are noted in the proportions of HIS e and d. As in the case for pea ( 161, the proportion sf the presumed f 1

11 12 b a 4 0 3 8 7 6 5 4 3

EXCISED EPlCOTYh

f l b 7 6 EXCISED EPICOTYL

2 -48 Rr

13 LEAF l l d o y

ROOT MERISTEM 16 day

MATURE LATERAL ROOT - I? day

STEM 11 day

COTYLEDON 0 - 1 2 8 h r

<

FIG. 3. Tissue-specific USP sf Yicia jaba. The top gel demonstrates complete USP-banding pattern, with the exception of bands 0, 1, and 2. Invariant bands are not pictured in the remaining gel patterns, Arrow indicates direction of migration.

histone was found to change during cotyiedon development.

Figure 3 demonstrates the total USP-banding pattern observed for excised epicotyls and the tissue-specific band patterns in which invariant bands are not pictured. Since bands 0, 1, and 2

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were variable in excised epicotyls, it could not be concluded that these bands were significantly variable among tissues. No differences were dis- cernible at the various stages in cotyledon devel- opment. A striking change, however, is the occurrence of a fast-running species (band 13) in 11-day-old tissues, regardless sf their state of differentiation. This band did not occur in any samples of tissues collected at 120 h or earlier. Band 1 l b appears to be replaced by a slightly faster-running species in cotyledons. Bands 6 and 7 are replaced by one species sf intermediate po- sition for root meristem and stem tissue, and band 7 is absent in leaf tissue. The small amount of tissue specificity observed here is typical of sther experiments that compare electrophoretic- banding patterns. Such tissue specificity has been found in many organisms by a variety of isolation procedures. Extreme species and tissue specificity of USP has been demonstrated for rat and chicken in a two-dimensional electrophoretic system (21 ). No variability in HIS-banding pat- terns was detected in any tissues. RES-banding patterns were not tested due to the great vaka- bility observed, not only among tissues but also between rep%icatisns.

The resaaTts of these experiments provide smp- port for the hypotheses that two classes of non- histones exist (15, 22). The first class includes relatively invariable enzymatic and structural proteins and regulatory proteins concerned with the production of the more ubiquitous WNA's. The second and smaller chss consists of regula- tory proteins that show variability among tissues and species. We believe USP species that we found invariant among tissues to be in the first class, and species that we found variable among tissues to be in the second class.

This investigation was supported in part by Bio- medical Sciences Support grant FW 0780% from the

General Research Support Branch, Division of Re- search Resources, Bureau of Health Manpower Education, National Institutes of Health, and by R

grant from the Arizona Chapter of the American Cancer Society. The research was conducted during the tenure of a National Defense Education Act Title IV predwtaral fellowship to one of 81s (B. G. M.) .

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