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Age-dependent changes in the structure and function of mammalian chromatin—I. Variations in chromatin template activity
Exp. Geront. Vol. 8. pp. 123-133. Pergamon Press 1973. Printed in Great Britain.
AGE-DEPENDENT FUNCTION VARIATIONS
CHANGES OF
IN THE
MAMMALIAN
IN CHROMATIN
STRUCTURE
AND
CHROMATIN--I. TEMPLATE
ACTIVITY
G. S. STEIN*, POLLYL. WANG and R. C. ADELMAN Departments of Pathology and Biochemistry, and the Fels Research Institute, Temple University School of Medicine, Philadelphia, Pa. 19140, U.S.A. (Received 3 M a y 1973)
INTRODUCTION THE PROCESSof biological ageing may be expressed at the molecular level in compositional and transcriptional modifications of the genome, which in eukaryotic cells is a nucleoprotein complex referred to as chromatin, consisting of DNA, RNA, histone and nonhistone protein. In highly differentiated cells the capacity of the genome to act as a template for in vitro and in vivo RNA synthesis is greatly restricted, and the repression of transcription was attributed to the histones, or basic chromosomal proteins (Huang and Bonner, 1962; Allfrey et al., 1963; Marushige and Bonner, 1966). The histones also were shown to impose structural constraints upon DNA (Dory et aL, 1960; Samajima and Yang, 1965; Ohaba, 1966; Ohlenbusch et al., 1967; Tuan and Bonner, 1969). In contrast, the nonhistone chromosomal proteins (Gilmour and Paul, 1970; Spelsberg and Hnilica, 1970; Stellwagen and Cole, 1969; Stein and Baserga, 1972; Stein et al., 1972; Stein and Farber, 1972; Frenster, 1965; Dingman and Sporn, 1964; Kleinsmith, 1966a; Kleinsmith, 1966b; Teng et al., 1971 ; Wang, 1970; Kamiyama and Want, 1971) and chromosomal RNA (Huang and Bonner, 1965; Bonner and Widholm, 1967) were implicated as having a regulatory function and may be responsible for the transcription of specific limited regions of the genome. In recent years several reports indicated modifications in the composition, structure and function of chromatin as animals age. Quantitative as well as qualitative differences were observed in the distribution of nuclear proteins and RNA associated with DNA (Kurtz and Sinex, 1967; Herrman et al., 1969; Pyhtila and Sherman, 1969), structure of chromatin (Herrman et al., 1969; Von Hahn and Fritz, 1966), salt extractability of chromosomal proteins (Hermann et al., 1969; Samis, 1969) and transcriptional capacity (Von Hahn, 1970; Samis and Wulff, 1969; Srivastava, 1968; O'Meara and Herrman, 1972). In contrast, according to Shorey and Sobel (1972), a number of these parameters were found not to be influenced by ageing. Although many of these observations at first appear to be conflicting the contradictions probably can be resolved if the source of material and the biochemical procedures utilized are taken into account. As part of a broad survey of the effects of age on gene regulation in the rat, documented modifications already include delayed initiation of several enzyme inductions in liver (Adelman, 1970; Adelman et al., 1972a), as well as impaired capability for cell proliferation, delayed initiation of DNA synthesis and of inducibility of certain thymidine-metabolizing enzymes and also changes in the time course and degree of synthesis of various species of RNA in the isoproterenol-stimulated submandibular gland (Adelman et al., 1972b). In the *Reprint requests and inquiries should be addressed to Dr. Stein at his present address: Department of Biochemistry, University of Florida, Gainesville, Florida 32601, U.S.A. 123
124
O . S . STEIN, POLLY L, WANG AND R. C. ADELMAN
present paper we report that the template activity in vitro of chromatin extracted from submandibular gland increases as rats age from 2 to at least 12 months, and that this modification probably is attributable to alterations both in the composition of the chromatin and in the association of chromosomal proteins with DNA. We also confirm and extend the recent observations of Shirey and Sobel (1972) that no apparent modifications are detectable in chromatin isolated from certain tissues of young and 12-month-old animals. METHODS AND PROCEDURE
Animals Two- and 12-month-old male Sprague-Dawley rats were purchased from Charles River Breeding Laboratories. All rats were fed a commercial diet, maintained ad lib on Charles River Chow (guaranteed to be of constant dietary composition), and exposed to alternating 12-hr periods of light (6 a.m.-6 p.m.) and dark. The animals were killed by cervical dislocation and the submandibular glands were dissected and freed of lymph nodes and fat tissue. Preparation of chromatin All procedures were carried out at 4°C. Submandibular glands were homogenized in a solution containing 0.25 M sucrose, 0"05 M KC1, 0-005 M MgC12 and 0.05 M Tris, pH 7.4, filtered through 6 layers of cheesecloth and centrifuged at 2000 × g for 4 min. The pellet was washed 3 times in Earle's Balanced Salt Solution and centrifuged at 2000 × g and nuclei were freed of cytoplasmic tags and large segments of the outer layer of the nuclear envelope by 3 washes with 20 volumes of a solution containing 80 mM NaC1, 20 mM EDTA, and 1 per cent Triton X-100 (Hancock, 1969). The nuclei were then washed twice in a solution containing 0"15 M NaCI and 0"01 M Tris pH 8"0, which effectively eliminates ribonuclease activity frequently associated with chromatin preparations (Smart and Bonner, 1971a; Smart and Bonner, 1971b), and lysed in distilled water with several strokes of a Teflon homogenizer. After swelling the chromatin in distilled water for 30 rain, Tris-HC1, pH 8.0, was added to a final concentration of 0.01 M. Five-ml aliquots of the suspension were layered on 25 ml of 1-7 M sucrose in a 1 in. × 3 in. cellulose nitrate tube. The upper twothirds of the contents of the tubes was uniformly mixed and the chromatin was centrifuged at 25,000 rpm for 3 hr in an SW 25.1 Spinco rotor. The chromatin pellet was resuspended in 0.01 M Tris, pH 8.0 and dialyzed for 12 hr against the same buffer. Preparation of E. coli RIgA polymerase RNA polymerase was prepared from early log-phase E. coli (General Biochemicals, Chagrin Falls, Ohio), utilizing the procedure detailed by Bonner et aL (1968). The enzyme was purified to the Fraction-4 stage, which includes chromatography on DEAE cellulose. Assay of chromatin template activity In vitro chromatin template activity was assayed by the method of Bonner et al. (1968), with the addition of 20 mM NaCI (Bekhor and Bavetta, 1971). The incubation mixture, in a final volume of 0"25 ml, contained: 15 ~tg of Fraction-4 E. coli. RNA polymerase; 0.1 llCi of 14C-ATP; 10 I~moles of Tris buffer, ph 8.0; 1 Ilmole of MgC12; 0.25 ~tmole of NnC12; 0.05 I~mole of NaC1; 3 Ilmoles of ~-mercaptoethanol; 0-1 ~tmole each of ATP, CTP, UTP
AGE-DEPENDENT CHROMATIN TEMPLATE ACTIVITY
125
and GTP; chromatin containing up to 40 I~g of DNA. Incubation was for 10 min at 37°C, and the reaction was terminated by the addition of cold 10 per cent (w/v) trichloroacetic acid. The acid-insoluble material was collected on 0.45-1ampore filters, which were washed 4 times with 5 ml of 10 per cent trichloroacetic acid, dissolved in 1 ml of ethylene glycol monoethyl ether and counted in 15 ml of Cellosolve-toluene liquid-scintillation cocktail (Gilman, 1970) in a Packard liquid-scintillation spectrometer. The counting efficiency was 80 per cent.
Preparation of DNA DNA was prepared from submandibular glands by the method of Marmur (1963) and treated with ribonuclease for 30 rain at 37°C (50 Ixg/ml), pronase for 2 hr at 37°C (50 ~tg/ml), and phenol, prior to use (Quincey and Wilson, 1969).
Ribonuclease activity of chromatin Ribonuclease activity of chromatin prepared from submandibular glands was determined by incubating labeled cytoplasmic RNA with chromatin in the reaction mixture for in vitro RNA synthesis described above, for 10 min without 14C-ATP. The samples were precipitated with 10 per cent trichloroacetic acid, collected on 0.45-~tmpore filters, solubilized in ethylene glycol monoethyl ether and counted in Cellosolve-toluene liquid scintillation cocktail. Ribonuclease activity is expressed as acid-insoluble radioactivity recovered. Cytoplasmic RNA (specific activity, 200 dpm/~tg RNA) was prepared from rat liver 4 hr after injection of uridine-5-3 H (1 ~tCi/g body weight, 58 Ci/mmole, New England Nuclear Corporation, Boston, Mass.). The animals were killed by cervical dislocation and the liver was immediately homogenized in a solution containing 0.25 M sucrose, 0"05 M KC1, 0"005 M MgC12, 0"05 M Tris, pH 7.4 and 6 mg/ml Bentonite. The homogenate was filtered through 6 layers of cheesecloth, the nuclei were pelleted by centrifugation at 800 x g for 12 rain, and 0.3 volumes of 10 per cent SDS were added to the cytoplasmic supernatant, which was stirred for 1 man. The cytoplasmic RNA was extracted by the addition of an equal volume of 90 per cent phenol, stirring at 4°C for 30 rain, and centrifugation at 17,000 x g for 10 rain. The clear upper phase containing the RNA was re-extracted with 0.5 volumes of 90 per cent phenol, stirred for 30 man and centrifuged at 17,000 x g for 10 rain. The upper phase was again collected and the RNA was precipitated for 12 hr at -20°C by the addition of 0.1 volume of 1 M NaC1, 0.1 M sodium acetate, pH 5.1 and 2 vol of ethanol. The RNA was then pelleted by centrifugation at 15,000 x g for 20 rain and washed twice in a solution containing 0-05 M NaC1 and 0.002 M sodium acetate.
Proteolytic activity, of chromatin Two- and twelve-month-old rats were injected with L-leucine-3H (5 IxCi/g body weight, 58 Ci/mmole, New England Nuclear Corporation, Boston, Mass.), killed 30 rain later and chromatin was prepared from the submandibular glands as previously described. Proteolytic activity of these labeled chromatin preparations was assayed by incubation for 0, 10 and 30 min in the in vitro RNA-synthesizing system previously described, without 14C-ATP. The samples were precipitated with 10 per cent trichloroacetic acid, collected on 0.45llmpore filters, solubilized in ethylene glycol monoethyl ether, and counted in Cellosolvetoluene liquid-scintillation cocktail. Proteolytic activity is expressed as acid-insoluble radioactivity recovered.
126
G . s . STEIN, POLLY L. WANG AND R. C. ADELMAN
Analysis of nucleic acid and protein composition of chromatin Histones were extracted from chromatin, prepared as previously described, with 3 washings of 0-4 N H2SO ~. Nucleic acid was then extracted with 5 per cent trichloroacetic acid for 15 rain at 90°C, followed by extraction with an equal volume of i N perchloric acid at the same temperature, and the nucleic acid extracts were pooled. The residual pellet (nonhistone chromosomal proteins) was solubilized in 1 N NaOH. The amount of protein present in the histone and nonhistone chromosomal protein fractions was determined by the method of Lowry et al. (1951) and the DNA content of the nucleic acid extract was determined by Burton's modification of the diphenylamine reaction (Burton, 1956). Selective dissociation of histones from chromatin Histones were selectively dissociated from chromatin using the ionic detergent sodium deoxycholate according to the method of Smart and Bonner (1971a and b). Chromatin from the submandibular glands of 2- and 12-month-old rats was diluted with 0.0025 M Tris, pH 8.0, so that a final volume of 10 ml and a final concentration of 10 A~60 nm would be obtained after the addition of sodium deoxycholate. The required amount of 0.25 M sodium deoxycholate--0.0025 M Tris, ph 8.0--was added dropwise while stirring vigorously on a Vortex mixer. Each 10-ml chromatin sample was gently layered onto 2 ml of 1.2 Msucrose-0"0025 M Tris, pH 8.0---and centrifuged in a 50Ti Spinco rotor for 16 hr at 50,000 rpm. The pellet, containing the partially dehistonized chromatin, was then analyzed for DNA, RNA and protein content, or dialyzed extensively against 0.01 M Tris, ph 8.0 and assayed for in vitro chromatin template activity. RESULTS
Template activity of young and old submandibular-gland chromatin In previous studies from this laboratory, age-dependent impairments in the regulation of gene expression in submandibular gland were shown to be expressed progressively and in direct proportionality to chronological rat age, from 2 to at least 24 months. Thus, we feel justified in selecting 12-month-old rats as intermediate points in a progressive senescent process, rather than considerably more expensive, older rats. Figure 1 shows the in vitro template activity of submandibular-gland chromatin from 2and 12-month-old rats. The results are presented as a chromatin concentration curve, using a fixed amount of E. coli RNA polymerase. The transcriptional capacity of chromatin from the old salivary glands is almost threefold greater than that of chromatin from the young salivary glands. A similar age-dependent difference in chromatin template activity of submandibular glands was demonstrated using UTP-ZH (1 ~tCi/250-pl assay) as a precursor (data not shown). Also not shown are data indicating a negligible rate of incorporation of cAMP-14C into chromatin in the absence of E. coli polymerase at both ages. That the age-dependent differences in template activity are not due to variations in ribonuclease activity of the chromatin preparations is supported by two lines of evidence. First, Table 1 indicates that there is no significant loss of acid-insoluble radioactivity from labeled cytoplasmic RNA incubated for 10 rain with young or old chromatin in the reaction mixture for in vitro RNA synthesis. Second, the chromatin preparations were extensively washed with 0.15 M NaC1, 0.01 M Tris, pH 8"3, which presumably results in the removal of ribonuclease activity associated with chromatin (Smart and Bonner, 1971a; Smart and Bonner, 1971b).
127
AGE-DEPENDENT CHROMATIN TEMPLATE ACTIVITY
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DNA AS CHROMATIN (pg)
FIG. 1. Saturation curves for the rate of RNA synthesis as a function of 2- ( 0 - - 0 - - 0 ) and 12-month-old ( O - - © - - © ) rat submandibular gland chromatin concentration. Preparation of chromatin and assay of in vitro template activity are described in the Materials and Methods Section. TABLE 1. RIBONUCLEASE ACTIVITY OF CHROMATIN FROM
YOUNG
AND
ADULT RAT GLANDS*
Sample RNA only Young chromatin (100 lag) Adult chromatin (100 lag)
SUBMANDIBULAR
Radioactivity (dpm) 390 ( ± 9) 401 (±14) 392 (±16)
*Ribonuclease activity is based on the 10 per cent trichloroacetic acid-precipitable radioactivity recovered from uridine-aH-labeled cytoplasmic RNA after incubation for 10 min with 2- and 12-monthold rat submandibular gland chromatin in the reaction mixture for in vitro RNA synthesis. Each value represents a minimum of 3 determinations and the ranges are indicated. Since it was d e m o n s t r a t e d previously that protease activity can be detected in c h r o m a t i n preparations ( P a n y i m e t al., 1968; Bartley a n d Chalkley, 1970), the question arises as to whether differences in the capacity of y o u n g a n d old s u b m a n d i b u l a r - g l a n d c h r o m a t i n to synthesize R N A in vitro can be attributed to a selective loss of c h r o m o s o m a l protein. T a b l e 2 shows that there is n o significant loss of acid-insoluble radioactivity from y o u n g or old TABLE2.
PROTEOLYTIC ACTIVITY OF CHROMATIN FROM YOUNG AND ADULT RAT SUBMAND I B U LA R GLANDS*
Time of incubation Radioactivity (dpm/mg DNA) (min) Young chromatin Old chromatin 0 774 (±19) 1057 (±27) 10 816 ( i 2 3 ) 914 (±17) 30 872 (±18) 1060 (±32) *Proteolytic activity is based on the 10 per cent trichloroacetic acid-precipitable radioactivity recovered from leucine-3H-labeled 2- and 12-month-old rat submandibular gland chromatin after incubation for up to 30 min in the reaction mixture for in vitro RNA synthesis. Each value represents a minimum of 2 determinations, and the ranges are indicated.
128
G . S . STEIN, POLLY L. W A N G AND R. C. ADELMAN
leucine-3H-labeled submandibular-gland chromatin incubated up to 30 rain in the reaction mixture for in vitro RNA synthesis. Thus, under these conditions there is no detectable level of proteolytic activity. The possibility that nucleases and proteases, either nuclear or cytoplasmic, modify chromatin during isolation and thereby result in apparent differences in the template activities of purified chromatin preparations from 2- and 12-month-old submandibular glands may also be discounted. The in vitro transcriptional capacities of chromatin from young or old submandibular glands were compared with that of chromatin prepared from pooled (1 : 1, w/w) young and old tissue. The per cent of recovery of chromatin from 2- and 12-month-old rat submandibular glands was approximately 60. Figure 2 confirms that there is a marked difference in the ability of young and old submandibular gland chromatin to transcribe RNA and since the template activity of the pooled chromatin preparation is intermediate to 2- and 12-month-old levels, it appears that the observed age-dependent effect is not the result of a degradative process. These data also eliminate the possibility that the observed age-dependent differences in chromatin template activity are the consequence of interactions between chromatin and any inhibitory or stimulatory material.
(N
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DNA AS CHROMATiN (pg)
FxG.2. Saturationcurves for the rate of RNA synthesisas a functionof 2- ( 0 - - 0 - - 0 ) and 12-month-old (C)--O--C)) and pooled 2- and 12-month-old(1:1 w/w)([-q--[3--D) rat submandibulargland chromatin concentration.Preparationsof chromatinand assayof in vitrotemplateactivityare describedin the Materials and Methods Section.
Template activity of young and old submandibular-gland DNA Table 3 shows the capacity of DNA from 2- and 12-month-old submandibular glands to transcribe RNA in vitro. Both DNA preparations have a similar template activity, eliminating the possibility that the increased transcriptional capacity of 12-month-old submandibular-gland chromatin results from a modification of the DNA. Rather, it appears that the age-dependent difference in DNA-primed RNA synthesis must be explained by a qualitative or quantitative change in the chromosomal RNA's and proteins or a modification in the association of these macromolecules with the DNA.
AGE-DEPENDENT CHROMATIN TEMPLATE ACTIVITY
129
TABLE 3. In vitro TEMPLATE ACTIVITY OF DNA FROM YOUNG AND ADULT RAT SUBMANDIBULAR GLANDS*
Sample [14C]-AMPincorporated into RNA (50 lag) (pmol) Young DNA 6468 ( -4-135) Adult DNA 6250 (± 162) *The in vitrotemplate activity of DNA from 2- and 12-month-old rat submandibular glands is based on the incorporation of 33C-AMP into 10 per cent trichloroacetic acid-precipitable material during 10 min, as described in the Materials and Methods Section. Each value represents a minimum of 3 determinations and the ranges are indicated.
Composition of young and old submandibular-gland chromatin Table 4 shows that the protein content of rat submandibular gland chromatin decreases as a function of age. Although there is no significant variation in the historic content, the chromatin prepared from old submandibular glands has a reduced level of nonhistone chromosomal protein. This is evident from the actual amounts of protein present, as well as from the histone: nonhistone chromosomal protein ratios (Table 4). However, it should be emphasized that this does not indicate the possibility of differences in the manner in which chromosomal proteins are associated with the D N A . TABLE 4.
COMPOSITION OF CHROMATIN FROM YOUNG AND ADULT RAT SUBMANDIBULAR GLANDS*
Sample Young chromatin Adult chromatin
Histone: DNA 0.80 0.81
Nonhistone protein: DNA 0"52 0.34
Histone: Nonhistone protein 1-54 2.39
*Histone, nucleic acid and nonhistone chromosomal proteins were prepared from 2- and 12-month-old rat submandibular gland chromatin, as described in Materials and Methods. The amount of protein was determined by the method of Lowry et al. (1951), and the amount of DNA by the diphenylamine reaction (Burton, 1956). Each point represents a minimum of 6 determinations and the range of values did not exceed 5 per cent.
Dissociation of histones from young and oM submandibular-gland ehromatin To assess the manner in which histones are associated with D N A in 2- and 12-month-old submandibular gland chromatin, the extractability of these proteins with the ionic detergent sodium deoxycholate was determined. Figure 3 shows that concentrations of sodium deoxycholate which do not remove D N A or nonhistone chromsomal proteins from young and old submandibular gland chromatin are effective in dissociating histories. It is also evident that a lower concentration of the detergent is required to dissociate a given amount of historic from 12-month-old submandibular gland chromatin than that from 2-month-old submandibular gland. These findings suggest that, although the same amount of histone is associated with 2- and 12-month-old submandibular gland chromatin, with increasing age the histories from the old tissue are less tenaciously bound to the D N A . Since histones have been implicated as being responsible for the repression of transcription, one would anticipate that as these proteins become less tenaciously associated with D N A , as in 12-month-old submandibular gland chromatin, an increased template activity
130
G . S . STEIN, POLLY L. WANG AND R. C. ADELMAN
70
60
a
50
~
4o
~.
ao 2o 10
0
.0025
.005
.0075
.01
SODIUM DEOXYCHOLATECONCENTRATION (M)
FIG. 3. Dissociation of historic (young, 0 - - 0 - - 0 ; old, 0 - - ( 3 - - ( 3 ) from 2- and 12-month-old rat submandibular gland chromatin by increasing concentrations of sodium deoxycholate. Each point represents an average of at least 2 determinations and the range of v~tlues for each point did not exceed 4 per cent. The individual values for release of nonhistone chromosomal protein and DNA from 2- and 12-month-old rat submandibulargland chromatin are not indicated, since they show insignificant differences. (Ak--~k--Ak) represents average values of either DNA or nonhistone chromosomal protein released from either 2- or 12-month-old rat submandibular gland chromatin. A minimum of 2 determinations were made for each DNA or nonhistone chromosomal protein value and the range did not exceed 3 per cent.
should be observed. This m i g h t account, at least i n part, for the age-dependent increase in s u b m a n d i b u l a r gland c h r o m a t i n template activity. It is also reasonable to predict that as histones are dissociated from c h r o m a t i n with s o d i u m deoxycholate, a n increased transcriptional capacity should be observed. Table 5 shows that, consistent with a previous report (Smart a n d Bonner, 1971a; Smart a n d Bonner, 1971b), as histones are extracted f r o m 2a n d 12-month-old s u b m a n d i b u l a r gland c h r o m a t i n with s o d i u m deoxycholate, there is an increased capacity for in vitro R N A synthesis. F u r t h e r m o r e , the increased template activity is p r o p o r t i o n a l to the a m o u n t of histone dissociated.
TABLE 5. EFFECT OF SODIUM DEOXYCHOLATE ON in vitro TEMPLATE ACTIVITY OF YOUNG AND ADULT RAT SUBMANDIBULAR GLANDS*
Sodium deoxycholate C o n c e n t r a t i o n s (M)
0 0.0025 0.005 0"01
Young chromatin histone I~C-AMP incorporated released into RNA (pmol) 0 812 9 936 23 1056 50 1586
~ histone released 0 15 38 71
Adult chromatin 14C-AMPincorporated into RNA (pmol) 2280 2898 3202 4568
*The in vitro template activity of chromatin from 2- and 12-month-old rat submandibular glands, after extraction with increasing concentrations of sodium deoxycholate, was determined by incubating 40 nag of DNA as chromatin in 250 lal of assay solution containing 15 I.d of E. eoli RNA polymerase for 10 min. Each value represents a minimum of 2 determinations, and the range of values did not exceed 5 per cent. The figures for the amount of histone released are taken from Fig. 3.
131
AGE-DEPENDENT CHROMATIN TEMPLATE ACTIVITY
Template activity and consumption o f young and old liver chromatin
Although there is a marked age-dependent increase in the template activity of rat submandibular gland chromatin, such a variation in transcriptional capacity is not evident in 2- and 12-month-old rat liver chromatin (Table 4). Another example of absence of an agedependent modification in chromatin template activity is that of Shirey and Sobel for canine cardiac muscle (Shirey and Sobel, 1972). Table 6 also shows that, in contrast to rat submandibular gland chromatin (where there is an age-dependent decrease in the amount of nonhistone chromosomal protein associated with DNA), the nonhistone chromosomal protein content does not vary as a function of age in rat liver chromatin. TABLE 6. In vitro TEMPLATEACrIVr~Y AND COMPOSITIONOF YOUNG AND ADULTRAT LIVERCHROMATIN* Sample Young chromatin Adult chromatin
Histone: DNA
Nonhistone protein: DNA
Histone: Nonhistone protein
x4C-AMP incorporated into R N A (pmol)
0.90 0"88
0.70 0.69
1.29 1.28
1291 1230
*Liver chromatin was prepared from 2- and 12-month-old rats, utilizing the procedure described for rat submandibular gland (see Materials and Methods Section). The composition of the chromatin preparations was determined as described in Table 4. Each figure represents a minimum of 3 determinations and the ranges of values did not exceed 5 per cent. The in vitro template activity of chromatin from 2- and 12-month-old rat liver was determined by incubating 40 lag of DNA as chromatin in 250 ml of assay solution containing 15 lag of E. coli RNA polymerase for 10 min. Saturation curves for the rates of in vitro RNA synthesis in 2- and 12-month-old rat liver chromatin confirmed these results. There was a minimum of 2 determinations for each figure and the range of values did not exceed 5 per cent. DISCUSSION Rat submandibular gland chromatin exhibits an increased age-dependent in vitro template activity using E. coli R N A polymerase. Employment of bacterial R N A polymerase to assess the template function of mammalian chromatin is questionable, because the bacterial enzyme binds to many non-selected sites. However, the data are consistent with an age-dependent increase in the in vivo rate of submandibular gland R N A synthesis, based on the incorporation of uridine-3H into R N A (Roth and Adelman, unpublished). The agedependent difference in DNA-primed RNA synthesis is not due to variations in the activities of nucleases or proteases associated with 2- and 12-month-old chromatin. It has also been shown that the difference in the transcriptional capacity of 2- and 12-month-old submandibular gland chromatin does not result from modifications of chromatin during the isolation and purification procedures. It should be emphasized, however, that these data do not in any way reflect the classes or specific species of informational R N A being synthesized. Since D N A from 2- and 12-month-old submandibular glands has a similar in vitro template activity, it is reasonable to consider the possibility that the age-dependent differences in submandibular gland chromatin template activity might be explained by qualitative or quantitative changes in chromosomal proteins or an alteration in the binding of these proteins to DNA. Although the histone content of 2- and 12-month-old submandibulargland chromatin is similar, the age-dependent increase in extractability of these basic chromosomal proteins with the ionic detergent sodium deoxycholate suggests that with age the histones become less tenaciously bound to the DNA. In as much as histones are believed to be responsible for restricting the transcription of R N A in eukaryotic cells, the less tenacious binding of these proteins to the D N A in chromatin from 12-month-old submandibular gland is in agreement with the observed increased transcriptional capacity.
132
G.S. STEIN,POLLYL. WANGANDR. C. ADELMAN
However, it is not clear whether specific classes of histones are more readily dissociated as a function of age, as well as whether the age-dependent changes in the association of histones with D N A is brought about by a chemical or structural modification of these proteins. Furthermore, the degree to which the histones m a y differ in extent of acetylation, phosphorylation and methylation from animals of increasing age remains to be determined. While histones are responsible for the restriction of template activity (Allfrey et al., 1963; Marushige and Bonner, 1966; Huang and Bonner, 1965), evidence is accumulating which suggests that it is the nonhistone chromosomal proteins which determine the specific genetic information, as well as the amount of template that is available for transcription (Gilmour and Pawl, 1970; Spelsberg and Hnilica, 1970; Stellwagen and Cole, 1969; Stein and Baserga, 1972; Stein et aL, 1972; Stein and Farber, 1972; Frenster, 1965; Dingman and Sporn, 1964). Therefore, one can speculate that the difference in the nonhistone chromosomal protein content of young and old chromatin may be of significance in mediating the age-dependent increased level of DNA-primed R N A synthesis. Experiments are presently in progress to determine whether nonhistone chromosomal proteins, or specific classes of the proteins associated with chromatin at various stages of development, interact with the D N A and thereby modify the transcriptional capacity of the genome. Such interactions m a y occur by means of a modification of the manner in which histones are associated with specific informational sequences. It also remains to evaluate these data against a background of more complete understanding of the complexity of salivary gland tissue with respect to age; i.e. cell turnover, changes in cell population, polyploidy, etc. Acknowledgements--This work was supported by Grants DRG-1138 from the Damon Runyon Memorial
Fund; CA-05222, HD-05874 and HD-04382 from the National Institutes of Health; and P-592 from the American Cancer Society. REFERENCES ADEt~aAN,R. C. (1970) J. biol. Chem. 245, 1032. ADELMAN,R. C., FREEMAN,C. and CO~N, B. S. (1972a) In Advances in Enzyme Regulation (Edited by G. Weber), Vol. 10, p. 365. Pergamon Press, Oxford. AD~LMAN,R. C., STEIN,G., ROTH,G. S. and ENGt.AND~R,D. (1972b) Mech. Age Develop. 1, 49. ALLFI~a~Y,V. G., Ln'a'Au, V. C. and MIRSKY,A. E. (1963) Proc. natn. Acad. Sci. U.S.A. 50, 1026. BARTLEY,J. and C-'a~rdmy, R. (1970) J. biol. Chem. 245, 4286. B~rd-IOR,I. and BAWTrA, L. A. (1971) Biochem. biophys. Res. Commun. 42, 615. BONNER,J., CHALKLEy,G. R., DAnMUS, M., FAMBROUGH,D., FUJIMARA,F., HUANG,R. C., HUaERMAN J., JENSEN,R., MARUSmGE,K., OHLENBUSCH,H., OtZVERA,B. and WIDHOLt~,J. (1968) In Methods in Enzymology (Edited by L. Grossman, and K. Moldave), Vol. 12B, p. 3. Academic Press, New York. BONNER,J. and WIt)HOLM,J. (1967) Proc. natn. Acad. Sci. U.S.A. 57, 1379. BURTON,K. (1956) Biochem. J. 62, 315. DINo~'~, C. W. and SeOR~, M. B. (1964) J. biol. Chem. 239, 3483. DOTY,P., MARMUR,J., EIGNER,J. and SCHILDKRAUT,C. (1960) Proc. natn. Acad. Sci. U.S.A. 46, 461. FRENSTER,J. H. (1965) Nature, Lond. 206, 680. GILMAN,A. G. (1970) Proc. natn. Acad. Sci. U.S.A. 67, 305. GILMOUR,R. S. and PAUL,J. (1970) FEBS Lett. 9, 242. HANCOCK,R. (1969) J. mol. Biol. 40, 457. H E ~ N , R. L., O'MvJ~,A,A. R., Russm.J, A. P. and DOWL~O,L. E. (1969) Proc. 8th Int. Cong. Gerontol., Vol. 1, p. 141. HUANG,R. C. and BONr,mR, J. (1962) Proc. natn. Acad. Sci. U.S.A. 48, 1216. HUANG,R. C. and BONr,mR,J. (1965) Proe. natn. Acad. Sci. U.S.A. 54, 960. ~ A M A , M. and WAN6, T. Y. (1971) Biochim. biophys. Acta 228, 563. KLEINS~ffrn,L. J., ALLFREY,V. G. and MIRSKy,A. E. (1966) Proc. natn. Acad. Sci. U.S.A. 55, 1182. KLEINSVarn,L. J., ALLFREY,V. G. and MIRSKY,A. E. (1966) Science 154, 780. KURTZ,D. and SINEx,F. (1967) Biochim. biophys. Acta 145, 840.
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133
LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L. and RANDALL, R. S. (1951) J. bioL Chem. 193, 265. MARMUR, J. (1963) In Methods in Enzymology (Edited by S. P. Colowick, and N. O. Kaplan), Vol. 6, p. 726. Academic Press, New York. MARUSmGE,K. and BONNER,J. (1966) J. tool Biol. 15, 160. OnAag, Y. (1966) Biochim. biophys. Acta 123, 76. Om.~NBtrSCH, H. H., OLtVERA,B. M., TUAN,D. and DAVlDSON,N. (1967) J. tool. Biol. 25, 299, O'MEARA, A. R. and HERRMAN,R. L. (1972) Biochim. biophys..4cta 269, 419. PANYIM,S., JENSEN,R. H. and CnALg.LEY,R. (1968) Biochim. biophys. Acta 160, 252. PYHTmA, M. J. and SHERMAN,F. G. (1969) Gerontologia 15, 321. QU~CEY, R. and WILSON,S. H. (1969) Proc. natn. Acad. Sci. U.S.A. 64, 981. SAMAJIMA,T. and YANG,J. T. (1965) J. biol. Chem. 240, 2094. SAMtS, H. V. (1969) Proc. 8th int. Cong. Gerontol., Vol. 1, p. 138. SAMIS,H. V. (1969) Proc. 8th Int. Cong. Gerontol., Vol. 1, p. 138. SHIREY, T. L. and SOBEL,H. (1972) Exp. Geront. 7, 15. SMART,J. E. and BONN~R,J. (1971a) J. moL Biol. 58, 651. SMART,J. E. and Bor,u',mR,J. (1971b) J. tool. Biol. 58, 675. SPELSBERG,T. C. and HNILICA,L. (1970) Biochem. J. 120, 435. SRIVASTAVA, B. I. (1968) Biochem. J. 110, 683. STEm, G. S. and BASERGA,R. (1972) Adv. Cancer Res. 15, 287. STEIN, G. S., CnAUt)mJRI,S. and BASERGA, R. (1972) J. biol. Chem. 247, 3918. STEIN, G. S. and FARBER,J. (1972) Proc. natn. Acad. Sci. U.S.A. 69, 2918. STELLWAGEN,R. and COLE,R. (1969) Ann. Rev. Biochem. 38, 951. TENG, C. S., TENG, C. T. and ALLrR~Y, U. G. (1971) J. biol. Chem. 246, 3597. TUAN, D. and BONNER,J. (1969) 3". tool. Biol. 45, 59. VON HAHN, H. P. and FRITZ, E. (1966) Gerontologia 12, 237. VON HAHN, H. (1970) Gerontologia 16, 116. WANG, T. Y. (1970) Expl. Cell Res. 61, 455.
Summary--The in vitro template activity of chromatin isolated from submandibular gland increases threefold as rats age from two to twelve months. The increased capacity for DNAprimed RNA synthesis cannot be attributed to differences in nuclease or protease activity, and there are no variations in the ability of young and older submandibular gland DNA to transcribe RNA under these conditions. The histone content of young and older submandibular gland chromatin is similar. However, these proteins are less tenaciously bound to the DNA with age; consistent with the age-dependent increase in template activity. In contrast, there is an age-dependent decrease in the quantity of nonhistone chromosomal proteins associated with submandibular gland chromatin. R~sum6--L'activit6 matricieUe in vitro de la chromatine isol6e de la glande sous-maxillaire est tripl6e darts le temps que le rat vieillit de deux mois ~t douze mois. La plus grande capacit6 de synth~se d'ARN en d6pendance de I'ADN ne peut ~tre attribu6e h des diff6rences en activit6 de la nucl6ase ou de la prot~ase et il n'y a pas de variations dans raptitude de rADN des glandes sous-maxillaires, jeunes ou plus ~tg6es, h la transcription de I'ARN dans ces conditions. Les teneurs en histone des chromatines des glandes sous-maxillaires, jeunes ou plus ~g~s, sont similaires. Ces prot6ines sont toutefois moins fermement li6es ~t rADN avec l'hge et ceci concorde avec l'augmentation de l'activit6 matricielle en d6pendance de l'Age. On constate, par contre, une diminutionen d6pendance de l'gtgede la quantit6 de prot6ines chromosomiques non histoniques associ6es ~t la chromatine de la glande sous-maxillaire. Zusammenfasstmg--Die in-vitro-Matrizenaktivitiit von Chromatin, welches aus der Submandibulardr~ise der Ratte isoliert wurde, nimmt mit dem Alter der Tiere yon zwei bis zw61f Monaten um das Dreifache zu. Die erh6hte F/ihigkeit der DNS-abhangigen RNS-Synthese kann nicht Unterschieden in der Nuklease- oder Proteaseaktivitfit zugeschrieben werden, und es gibt keine Variationen der F/ihigkeit junger oder ~ilterer Submandibulardr~isen-DNS, RNS unter diesen Bedingungen zu transkribieren. Der Histongehalt von jungem und ~ilterem Submandibulardriasen-chromatin is vergleichbar. Diese Proteine werden jedoch mit zunehmendem Alter weniger fest an DNS gebunden, im Einklang mit dem altersabh/ingigen Anstieg der Matrizenaktivit~it. Im Gegensatz dazu liegt ein altersabh~ingiger Abfall der Menge an Nichthiston-Chromosomenproteinen vor, die mit dem Submandibulardr~isenchromatin verbunden sind.
AGE-DEPENDENT FUNCTION VARIATIONS
CHANGES OF
IN THE
MAMMALIAN
IN CHROMATIN
STRUCTURE
AND
CHROMATIN--I. TEMPLATE
ACTIVITY
G. S. STEIN*, POLLYL. WANG and R. C. ADELMAN Departments of Pathology and Biochemistry, and the Fels Research Institute, Temple University School of Medicine, Philadelphia, Pa. 19140, U.S.A. (Received 3 M a y 1973)
INTRODUCTION THE PROCESSof biological ageing may be expressed at the molecular level in compositional and transcriptional modifications of the genome, which in eukaryotic cells is a nucleoprotein complex referred to as chromatin, consisting of DNA, RNA, histone and nonhistone protein. In highly differentiated cells the capacity of the genome to act as a template for in vitro and in vivo RNA synthesis is greatly restricted, and the repression of transcription was attributed to the histones, or basic chromosomal proteins (Huang and Bonner, 1962; Allfrey et al., 1963; Marushige and Bonner, 1966). The histones also were shown to impose structural constraints upon DNA (Dory et aL, 1960; Samajima and Yang, 1965; Ohaba, 1966; Ohlenbusch et al., 1967; Tuan and Bonner, 1969). In contrast, the nonhistone chromosomal proteins (Gilmour and Paul, 1970; Spelsberg and Hnilica, 1970; Stellwagen and Cole, 1969; Stein and Baserga, 1972; Stein et al., 1972; Stein and Farber, 1972; Frenster, 1965; Dingman and Sporn, 1964; Kleinsmith, 1966a; Kleinsmith, 1966b; Teng et al., 1971 ; Wang, 1970; Kamiyama and Want, 1971) and chromosomal RNA (Huang and Bonner, 1965; Bonner and Widholm, 1967) were implicated as having a regulatory function and may be responsible for the transcription of specific limited regions of the genome. In recent years several reports indicated modifications in the composition, structure and function of chromatin as animals age. Quantitative as well as qualitative differences were observed in the distribution of nuclear proteins and RNA associated with DNA (Kurtz and Sinex, 1967; Herrman et al., 1969; Pyhtila and Sherman, 1969), structure of chromatin (Herrman et al., 1969; Von Hahn and Fritz, 1966), salt extractability of chromosomal proteins (Hermann et al., 1969; Samis, 1969) and transcriptional capacity (Von Hahn, 1970; Samis and Wulff, 1969; Srivastava, 1968; O'Meara and Herrman, 1972). In contrast, according to Shorey and Sobel (1972), a number of these parameters were found not to be influenced by ageing. Although many of these observations at first appear to be conflicting the contradictions probably can be resolved if the source of material and the biochemical procedures utilized are taken into account. As part of a broad survey of the effects of age on gene regulation in the rat, documented modifications already include delayed initiation of several enzyme inductions in liver (Adelman, 1970; Adelman et al., 1972a), as well as impaired capability for cell proliferation, delayed initiation of DNA synthesis and of inducibility of certain thymidine-metabolizing enzymes and also changes in the time course and degree of synthesis of various species of RNA in the isoproterenol-stimulated submandibular gland (Adelman et al., 1972b). In the *Reprint requests and inquiries should be addressed to Dr. Stein at his present address: Department of Biochemistry, University of Florida, Gainesville, Florida 32601, U.S.A. 123
124
O . S . STEIN, POLLY L, WANG AND R. C. ADELMAN
present paper we report that the template activity in vitro of chromatin extracted from submandibular gland increases as rats age from 2 to at least 12 months, and that this modification probably is attributable to alterations both in the composition of the chromatin and in the association of chromosomal proteins with DNA. We also confirm and extend the recent observations of Shirey and Sobel (1972) that no apparent modifications are detectable in chromatin isolated from certain tissues of young and 12-month-old animals. METHODS AND PROCEDURE
Animals Two- and 12-month-old male Sprague-Dawley rats were purchased from Charles River Breeding Laboratories. All rats were fed a commercial diet, maintained ad lib on Charles River Chow (guaranteed to be of constant dietary composition), and exposed to alternating 12-hr periods of light (6 a.m.-6 p.m.) and dark. The animals were killed by cervical dislocation and the submandibular glands were dissected and freed of lymph nodes and fat tissue. Preparation of chromatin All procedures were carried out at 4°C. Submandibular glands were homogenized in a solution containing 0.25 M sucrose, 0"05 M KC1, 0-005 M MgC12 and 0.05 M Tris, pH 7.4, filtered through 6 layers of cheesecloth and centrifuged at 2000 × g for 4 min. The pellet was washed 3 times in Earle's Balanced Salt Solution and centrifuged at 2000 × g and nuclei were freed of cytoplasmic tags and large segments of the outer layer of the nuclear envelope by 3 washes with 20 volumes of a solution containing 80 mM NaC1, 20 mM EDTA, and 1 per cent Triton X-100 (Hancock, 1969). The nuclei were then washed twice in a solution containing 0"15 M NaCI and 0"01 M Tris pH 8"0, which effectively eliminates ribonuclease activity frequently associated with chromatin preparations (Smart and Bonner, 1971a; Smart and Bonner, 1971b), and lysed in distilled water with several strokes of a Teflon homogenizer. After swelling the chromatin in distilled water for 30 rain, Tris-HC1, pH 8.0, was added to a final concentration of 0.01 M. Five-ml aliquots of the suspension were layered on 25 ml of 1-7 M sucrose in a 1 in. × 3 in. cellulose nitrate tube. The upper twothirds of the contents of the tubes was uniformly mixed and the chromatin was centrifuged at 25,000 rpm for 3 hr in an SW 25.1 Spinco rotor. The chromatin pellet was resuspended in 0.01 M Tris, pH 8.0 and dialyzed for 12 hr against the same buffer. Preparation of E. coli RIgA polymerase RNA polymerase was prepared from early log-phase E. coli (General Biochemicals, Chagrin Falls, Ohio), utilizing the procedure detailed by Bonner et aL (1968). The enzyme was purified to the Fraction-4 stage, which includes chromatography on DEAE cellulose. Assay of chromatin template activity In vitro chromatin template activity was assayed by the method of Bonner et al. (1968), with the addition of 20 mM NaCI (Bekhor and Bavetta, 1971). The incubation mixture, in a final volume of 0"25 ml, contained: 15 ~tg of Fraction-4 E. coli. RNA polymerase; 0.1 llCi of 14C-ATP; 10 I~moles of Tris buffer, ph 8.0; 1 Ilmole of MgC12; 0.25 ~tmole of NnC12; 0.05 I~mole of NaC1; 3 Ilmoles of ~-mercaptoethanol; 0-1 ~tmole each of ATP, CTP, UTP
AGE-DEPENDENT CHROMATIN TEMPLATE ACTIVITY
125
and GTP; chromatin containing up to 40 I~g of DNA. Incubation was for 10 min at 37°C, and the reaction was terminated by the addition of cold 10 per cent (w/v) trichloroacetic acid. The acid-insoluble material was collected on 0.45-1ampore filters, which were washed 4 times with 5 ml of 10 per cent trichloroacetic acid, dissolved in 1 ml of ethylene glycol monoethyl ether and counted in 15 ml of Cellosolve-toluene liquid-scintillation cocktail (Gilman, 1970) in a Packard liquid-scintillation spectrometer. The counting efficiency was 80 per cent.
Preparation of DNA DNA was prepared from submandibular glands by the method of Marmur (1963) and treated with ribonuclease for 30 rain at 37°C (50 Ixg/ml), pronase for 2 hr at 37°C (50 ~tg/ml), and phenol, prior to use (Quincey and Wilson, 1969).
Ribonuclease activity of chromatin Ribonuclease activity of chromatin prepared from submandibular glands was determined by incubating labeled cytoplasmic RNA with chromatin in the reaction mixture for in vitro RNA synthesis described above, for 10 min without 14C-ATP. The samples were precipitated with 10 per cent trichloroacetic acid, collected on 0.45-~tmpore filters, solubilized in ethylene glycol monoethyl ether and counted in Cellosolve-toluene liquid scintillation cocktail. Ribonuclease activity is expressed as acid-insoluble radioactivity recovered. Cytoplasmic RNA (specific activity, 200 dpm/~tg RNA) was prepared from rat liver 4 hr after injection of uridine-5-3 H (1 ~tCi/g body weight, 58 Ci/mmole, New England Nuclear Corporation, Boston, Mass.). The animals were killed by cervical dislocation and the liver was immediately homogenized in a solution containing 0.25 M sucrose, 0"05 M KC1, 0"005 M MgC12, 0"05 M Tris, pH 7.4 and 6 mg/ml Bentonite. The homogenate was filtered through 6 layers of cheesecloth, the nuclei were pelleted by centrifugation at 800 x g for 12 rain, and 0.3 volumes of 10 per cent SDS were added to the cytoplasmic supernatant, which was stirred for 1 man. The cytoplasmic RNA was extracted by the addition of an equal volume of 90 per cent phenol, stirring at 4°C for 30 rain, and centrifugation at 17,000 x g for 10 rain. The clear upper phase containing the RNA was re-extracted with 0.5 volumes of 90 per cent phenol, stirred for 30 man and centrifuged at 17,000 x g for 10 rain. The upper phase was again collected and the RNA was precipitated for 12 hr at -20°C by the addition of 0.1 volume of 1 M NaC1, 0.1 M sodium acetate, pH 5.1 and 2 vol of ethanol. The RNA was then pelleted by centrifugation at 15,000 x g for 20 rain and washed twice in a solution containing 0-05 M NaC1 and 0.002 M sodium acetate.
Proteolytic activity, of chromatin Two- and twelve-month-old rats were injected with L-leucine-3H (5 IxCi/g body weight, 58 Ci/mmole, New England Nuclear Corporation, Boston, Mass.), killed 30 rain later and chromatin was prepared from the submandibular glands as previously described. Proteolytic activity of these labeled chromatin preparations was assayed by incubation for 0, 10 and 30 min in the in vitro RNA-synthesizing system previously described, without 14C-ATP. The samples were precipitated with 10 per cent trichloroacetic acid, collected on 0.45llmpore filters, solubilized in ethylene glycol monoethyl ether, and counted in Cellosolvetoluene liquid-scintillation cocktail. Proteolytic activity is expressed as acid-insoluble radioactivity recovered.
126
G . s . STEIN, POLLY L. WANG AND R. C. ADELMAN
Analysis of nucleic acid and protein composition of chromatin Histones were extracted from chromatin, prepared as previously described, with 3 washings of 0-4 N H2SO ~. Nucleic acid was then extracted with 5 per cent trichloroacetic acid for 15 rain at 90°C, followed by extraction with an equal volume of i N perchloric acid at the same temperature, and the nucleic acid extracts were pooled. The residual pellet (nonhistone chromosomal proteins) was solubilized in 1 N NaOH. The amount of protein present in the histone and nonhistone chromosomal protein fractions was determined by the method of Lowry et al. (1951) and the DNA content of the nucleic acid extract was determined by Burton's modification of the diphenylamine reaction (Burton, 1956). Selective dissociation of histones from chromatin Histones were selectively dissociated from chromatin using the ionic detergent sodium deoxycholate according to the method of Smart and Bonner (1971a and b). Chromatin from the submandibular glands of 2- and 12-month-old rats was diluted with 0.0025 M Tris, pH 8.0, so that a final volume of 10 ml and a final concentration of 10 A~60 nm would be obtained after the addition of sodium deoxycholate. The required amount of 0.25 M sodium deoxycholate--0.0025 M Tris, ph 8.0--was added dropwise while stirring vigorously on a Vortex mixer. Each 10-ml chromatin sample was gently layered onto 2 ml of 1.2 Msucrose-0"0025 M Tris, pH 8.0---and centrifuged in a 50Ti Spinco rotor for 16 hr at 50,000 rpm. The pellet, containing the partially dehistonized chromatin, was then analyzed for DNA, RNA and protein content, or dialyzed extensively against 0.01 M Tris, ph 8.0 and assayed for in vitro chromatin template activity. RESULTS
Template activity of young and old submandibular-gland chromatin In previous studies from this laboratory, age-dependent impairments in the regulation of gene expression in submandibular gland were shown to be expressed progressively and in direct proportionality to chronological rat age, from 2 to at least 24 months. Thus, we feel justified in selecting 12-month-old rats as intermediate points in a progressive senescent process, rather than considerably more expensive, older rats. Figure 1 shows the in vitro template activity of submandibular-gland chromatin from 2and 12-month-old rats. The results are presented as a chromatin concentration curve, using a fixed amount of E. coli RNA polymerase. The transcriptional capacity of chromatin from the old salivary glands is almost threefold greater than that of chromatin from the young salivary glands. A similar age-dependent difference in chromatin template activity of submandibular glands was demonstrated using UTP-ZH (1 ~tCi/250-pl assay) as a precursor (data not shown). Also not shown are data indicating a negligible rate of incorporation of cAMP-14C into chromatin in the absence of E. coli polymerase at both ages. That the age-dependent differences in template activity are not due to variations in ribonuclease activity of the chromatin preparations is supported by two lines of evidence. First, Table 1 indicates that there is no significant loss of acid-insoluble radioactivity from labeled cytoplasmic RNA incubated for 10 rain with young or old chromatin in the reaction mixture for in vitro RNA synthesis. Second, the chromatin preparations were extensively washed with 0.15 M NaC1, 0.01 M Tris, pH 8"3, which presumably results in the removal of ribonuclease activity associated with chromatin (Smart and Bonner, 1971a; Smart and Bonner, 1971b).
127
AGE-DEPENDENT CHROMATIN TEMPLATE ACTIVITY
~o 12
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12
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20
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24.
=a
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DNA AS CHROMATIN (pg)
FIG. 1. Saturation curves for the rate of RNA synthesis as a function of 2- ( 0 - - 0 - - 0 ) and 12-month-old ( O - - © - - © ) rat submandibular gland chromatin concentration. Preparation of chromatin and assay of in vitro template activity are described in the Materials and Methods Section. TABLE 1. RIBONUCLEASE ACTIVITY OF CHROMATIN FROM
YOUNG
AND
ADULT RAT GLANDS*
Sample RNA only Young chromatin (100 lag) Adult chromatin (100 lag)
SUBMANDIBULAR
Radioactivity (dpm) 390 ( ± 9) 401 (±14) 392 (±16)
*Ribonuclease activity is based on the 10 per cent trichloroacetic acid-precipitable radioactivity recovered from uridine-aH-labeled cytoplasmic RNA after incubation for 10 min with 2- and 12-monthold rat submandibular gland chromatin in the reaction mixture for in vitro RNA synthesis. Each value represents a minimum of 3 determinations and the ranges are indicated. Since it was d e m o n s t r a t e d previously that protease activity can be detected in c h r o m a t i n preparations ( P a n y i m e t al., 1968; Bartley a n d Chalkley, 1970), the question arises as to whether differences in the capacity of y o u n g a n d old s u b m a n d i b u l a r - g l a n d c h r o m a t i n to synthesize R N A in vitro can be attributed to a selective loss of c h r o m o s o m a l protein. T a b l e 2 shows that there is n o significant loss of acid-insoluble radioactivity from y o u n g or old TABLE2.
PROTEOLYTIC ACTIVITY OF CHROMATIN FROM YOUNG AND ADULT RAT SUBMAND I B U LA R GLANDS*
Time of incubation Radioactivity (dpm/mg DNA) (min) Young chromatin Old chromatin 0 774 (±19) 1057 (±27) 10 816 ( i 2 3 ) 914 (±17) 30 872 (±18) 1060 (±32) *Proteolytic activity is based on the 10 per cent trichloroacetic acid-precipitable radioactivity recovered from leucine-3H-labeled 2- and 12-month-old rat submandibular gland chromatin after incubation for up to 30 min in the reaction mixture for in vitro RNA synthesis. Each value represents a minimum of 2 determinations, and the ranges are indicated.
128
G . S . STEIN, POLLY L. W A N G AND R. C. ADELMAN
leucine-3H-labeled submandibular-gland chromatin incubated up to 30 rain in the reaction mixture for in vitro RNA synthesis. Thus, under these conditions there is no detectable level of proteolytic activity. The possibility that nucleases and proteases, either nuclear or cytoplasmic, modify chromatin during isolation and thereby result in apparent differences in the template activities of purified chromatin preparations from 2- and 12-month-old submandibular glands may also be discounted. The in vitro transcriptional capacities of chromatin from young or old submandibular glands were compared with that of chromatin prepared from pooled (1 : 1, w/w) young and old tissue. The per cent of recovery of chromatin from 2- and 12-month-old rat submandibular glands was approximately 60. Figure 2 confirms that there is a marked difference in the ability of young and old submandibular gland chromatin to transcribe RNA and since the template activity of the pooled chromatin preparation is intermediate to 2- and 12-month-old levels, it appears that the observed age-dependent effect is not the result of a degradative process. These data also eliminate the possibility that the observed age-dependent differences in chromatin template activity are the consequence of interactions between chromatin and any inhibitory or stimulatory material.
(N
g x E o.
0
9
g
3
~
0
!
25 5
,o
2'0
3'0
40
DNA AS CHROMATiN (pg)
FxG.2. Saturationcurves for the rate of RNA synthesisas a functionof 2- ( 0 - - 0 - - 0 ) and 12-month-old (C)--O--C)) and pooled 2- and 12-month-old(1:1 w/w)([-q--[3--D) rat submandibulargland chromatin concentration.Preparationsof chromatinand assayof in vitrotemplateactivityare describedin the Materials and Methods Section.
Template activity of young and old submandibular-gland DNA Table 3 shows the capacity of DNA from 2- and 12-month-old submandibular glands to transcribe RNA in vitro. Both DNA preparations have a similar template activity, eliminating the possibility that the increased transcriptional capacity of 12-month-old submandibular-gland chromatin results from a modification of the DNA. Rather, it appears that the age-dependent difference in DNA-primed RNA synthesis must be explained by a qualitative or quantitative change in the chromosomal RNA's and proteins or a modification in the association of these macromolecules with the DNA.
AGE-DEPENDENT CHROMATIN TEMPLATE ACTIVITY
129
TABLE 3. In vitro TEMPLATE ACTIVITY OF DNA FROM YOUNG AND ADULT RAT SUBMANDIBULAR GLANDS*
Sample [14C]-AMPincorporated into RNA (50 lag) (pmol) Young DNA 6468 ( -4-135) Adult DNA 6250 (± 162) *The in vitrotemplate activity of DNA from 2- and 12-month-old rat submandibular glands is based on the incorporation of 33C-AMP into 10 per cent trichloroacetic acid-precipitable material during 10 min, as described in the Materials and Methods Section. Each value represents a minimum of 3 determinations and the ranges are indicated.
Composition of young and old submandibular-gland chromatin Table 4 shows that the protein content of rat submandibular gland chromatin decreases as a function of age. Although there is no significant variation in the historic content, the chromatin prepared from old submandibular glands has a reduced level of nonhistone chromosomal protein. This is evident from the actual amounts of protein present, as well as from the histone: nonhistone chromosomal protein ratios (Table 4). However, it should be emphasized that this does not indicate the possibility of differences in the manner in which chromosomal proteins are associated with the D N A . TABLE 4.
COMPOSITION OF CHROMATIN FROM YOUNG AND ADULT RAT SUBMANDIBULAR GLANDS*
Sample Young chromatin Adult chromatin
Histone: DNA 0.80 0.81
Nonhistone protein: DNA 0"52 0.34
Histone: Nonhistone protein 1-54 2.39
*Histone, nucleic acid and nonhistone chromosomal proteins were prepared from 2- and 12-month-old rat submandibular gland chromatin, as described in Materials and Methods. The amount of protein was determined by the method of Lowry et al. (1951), and the amount of DNA by the diphenylamine reaction (Burton, 1956). Each point represents a minimum of 6 determinations and the range of values did not exceed 5 per cent.
Dissociation of histones from young and oM submandibular-gland ehromatin To assess the manner in which histones are associated with D N A in 2- and 12-month-old submandibular gland chromatin, the extractability of these proteins with the ionic detergent sodium deoxycholate was determined. Figure 3 shows that concentrations of sodium deoxycholate which do not remove D N A or nonhistone chromsomal proteins from young and old submandibular gland chromatin are effective in dissociating histories. It is also evident that a lower concentration of the detergent is required to dissociate a given amount of historic from 12-month-old submandibular gland chromatin than that from 2-month-old submandibular gland. These findings suggest that, although the same amount of histone is associated with 2- and 12-month-old submandibular gland chromatin, with increasing age the histories from the old tissue are less tenaciously bound to the D N A . Since histones have been implicated as being responsible for the repression of transcription, one would anticipate that as these proteins become less tenaciously associated with D N A , as in 12-month-old submandibular gland chromatin, an increased template activity
130
G . S . STEIN, POLLY L. WANG AND R. C. ADELMAN
70
60
a
50
~
4o
~.
ao 2o 10
0
.0025
.005
.0075
.01
SODIUM DEOXYCHOLATECONCENTRATION (M)
FIG. 3. Dissociation of historic (young, 0 - - 0 - - 0 ; old, 0 - - ( 3 - - ( 3 ) from 2- and 12-month-old rat submandibular gland chromatin by increasing concentrations of sodium deoxycholate. Each point represents an average of at least 2 determinations and the range of v~tlues for each point did not exceed 4 per cent. The individual values for release of nonhistone chromosomal protein and DNA from 2- and 12-month-old rat submandibulargland chromatin are not indicated, since they show insignificant differences. (Ak--~k--Ak) represents average values of either DNA or nonhistone chromosomal protein released from either 2- or 12-month-old rat submandibular gland chromatin. A minimum of 2 determinations were made for each DNA or nonhistone chromosomal protein value and the range did not exceed 3 per cent.
should be observed. This m i g h t account, at least i n part, for the age-dependent increase in s u b m a n d i b u l a r gland c h r o m a t i n template activity. It is also reasonable to predict that as histones are dissociated from c h r o m a t i n with s o d i u m deoxycholate, a n increased transcriptional capacity should be observed. Table 5 shows that, consistent with a previous report (Smart a n d Bonner, 1971a; Smart a n d Bonner, 1971b), as histones are extracted f r o m 2a n d 12-month-old s u b m a n d i b u l a r gland c h r o m a t i n with s o d i u m deoxycholate, there is an increased capacity for in vitro R N A synthesis. F u r t h e r m o r e , the increased template activity is p r o p o r t i o n a l to the a m o u n t of histone dissociated.
TABLE 5. EFFECT OF SODIUM DEOXYCHOLATE ON in vitro TEMPLATE ACTIVITY OF YOUNG AND ADULT RAT SUBMANDIBULAR GLANDS*
Sodium deoxycholate C o n c e n t r a t i o n s (M)
0 0.0025 0.005 0"01
Young chromatin histone I~C-AMP incorporated released into RNA (pmol) 0 812 9 936 23 1056 50 1586
~ histone released 0 15 38 71
Adult chromatin 14C-AMPincorporated into RNA (pmol) 2280 2898 3202 4568
*The in vitro template activity of chromatin from 2- and 12-month-old rat submandibular glands, after extraction with increasing concentrations of sodium deoxycholate, was determined by incubating 40 nag of DNA as chromatin in 250 lal of assay solution containing 15 I.d of E. eoli RNA polymerase for 10 min. Each value represents a minimum of 2 determinations, and the range of values did not exceed 5 per cent. The figures for the amount of histone released are taken from Fig. 3.
131
AGE-DEPENDENT CHROMATIN TEMPLATE ACTIVITY
Template activity and consumption o f young and old liver chromatin
Although there is a marked age-dependent increase in the template activity of rat submandibular gland chromatin, such a variation in transcriptional capacity is not evident in 2- and 12-month-old rat liver chromatin (Table 4). Another example of absence of an agedependent modification in chromatin template activity is that of Shirey and Sobel for canine cardiac muscle (Shirey and Sobel, 1972). Table 6 also shows that, in contrast to rat submandibular gland chromatin (where there is an age-dependent decrease in the amount of nonhistone chromosomal protein associated with DNA), the nonhistone chromosomal protein content does not vary as a function of age in rat liver chromatin. TABLE 6. In vitro TEMPLATEACrIVr~Y AND COMPOSITIONOF YOUNG AND ADULTRAT LIVERCHROMATIN* Sample Young chromatin Adult chromatin
Histone: DNA
Nonhistone protein: DNA
Histone: Nonhistone protein
x4C-AMP incorporated into R N A (pmol)
0.90 0"88
0.70 0.69
1.29 1.28
1291 1230
*Liver chromatin was prepared from 2- and 12-month-old rats, utilizing the procedure described for rat submandibular gland (see Materials and Methods Section). The composition of the chromatin preparations was determined as described in Table 4. Each figure represents a minimum of 3 determinations and the ranges of values did not exceed 5 per cent. The in vitro template activity of chromatin from 2- and 12-month-old rat liver was determined by incubating 40 lag of DNA as chromatin in 250 ml of assay solution containing 15 lag of E. coli RNA polymerase for 10 min. Saturation curves for the rates of in vitro RNA synthesis in 2- and 12-month-old rat liver chromatin confirmed these results. There was a minimum of 2 determinations for each figure and the range of values did not exceed 5 per cent. DISCUSSION Rat submandibular gland chromatin exhibits an increased age-dependent in vitro template activity using E. coli R N A polymerase. Employment of bacterial R N A polymerase to assess the template function of mammalian chromatin is questionable, because the bacterial enzyme binds to many non-selected sites. However, the data are consistent with an age-dependent increase in the in vivo rate of submandibular gland R N A synthesis, based on the incorporation of uridine-3H into R N A (Roth and Adelman, unpublished). The agedependent difference in DNA-primed RNA synthesis is not due to variations in the activities of nucleases or proteases associated with 2- and 12-month-old chromatin. It has also been shown that the difference in the transcriptional capacity of 2- and 12-month-old submandibular gland chromatin does not result from modifications of chromatin during the isolation and purification procedures. It should be emphasized, however, that these data do not in any way reflect the classes or specific species of informational R N A being synthesized. Since D N A from 2- and 12-month-old submandibular glands has a similar in vitro template activity, it is reasonable to consider the possibility that the age-dependent differences in submandibular gland chromatin template activity might be explained by qualitative or quantitative changes in chromosomal proteins or an alteration in the binding of these proteins to DNA. Although the histone content of 2- and 12-month-old submandibulargland chromatin is similar, the age-dependent increase in extractability of these basic chromosomal proteins with the ionic detergent sodium deoxycholate suggests that with age the histones become less tenaciously bound to the DNA. In as much as histones are believed to be responsible for restricting the transcription of R N A in eukaryotic cells, the less tenacious binding of these proteins to the D N A in chromatin from 12-month-old submandibular gland is in agreement with the observed increased transcriptional capacity.
132
G.S. STEIN,POLLYL. WANGANDR. C. ADELMAN
However, it is not clear whether specific classes of histones are more readily dissociated as a function of age, as well as whether the age-dependent changes in the association of histones with D N A is brought about by a chemical or structural modification of these proteins. Furthermore, the degree to which the histones m a y differ in extent of acetylation, phosphorylation and methylation from animals of increasing age remains to be determined. While histones are responsible for the restriction of template activity (Allfrey et al., 1963; Marushige and Bonner, 1966; Huang and Bonner, 1965), evidence is accumulating which suggests that it is the nonhistone chromosomal proteins which determine the specific genetic information, as well as the amount of template that is available for transcription (Gilmour and Pawl, 1970; Spelsberg and Hnilica, 1970; Stellwagen and Cole, 1969; Stein and Baserga, 1972; Stein et aL, 1972; Stein and Farber, 1972; Frenster, 1965; Dingman and Sporn, 1964). Therefore, one can speculate that the difference in the nonhistone chromosomal protein content of young and old chromatin may be of significance in mediating the age-dependent increased level of DNA-primed R N A synthesis. Experiments are presently in progress to determine whether nonhistone chromosomal proteins, or specific classes of the proteins associated with chromatin at various stages of development, interact with the D N A and thereby modify the transcriptional capacity of the genome. Such interactions m a y occur by means of a modification of the manner in which histones are associated with specific informational sequences. It also remains to evaluate these data against a background of more complete understanding of the complexity of salivary gland tissue with respect to age; i.e. cell turnover, changes in cell population, polyploidy, etc. Acknowledgements--This work was supported by Grants DRG-1138 from the Damon Runyon Memorial
Fund; CA-05222, HD-05874 and HD-04382 from the National Institutes of Health; and P-592 from the American Cancer Society. REFERENCES ADEt~aAN,R. C. (1970) J. biol. Chem. 245, 1032. ADELMAN,R. C., FREEMAN,C. and CO~N, B. S. (1972a) In Advances in Enzyme Regulation (Edited by G. Weber), Vol. 10, p. 365. Pergamon Press, Oxford. AD~LMAN,R. C., STEIN,G., ROTH,G. S. and ENGt.AND~R,D. (1972b) Mech. Age Develop. 1, 49. ALLFI~a~Y,V. G., Ln'a'Au, V. C. and MIRSKY,A. E. (1963) Proc. natn. Acad. Sci. U.S.A. 50, 1026. BARTLEY,J. and C-'a~rdmy, R. (1970) J. biol. Chem. 245, 4286. B~rd-IOR,I. and BAWTrA, L. A. (1971) Biochem. biophys. Res. Commun. 42, 615. BONNER,J., CHALKLEy,G. R., DAnMUS, M., FAMBROUGH,D., FUJIMARA,F., HUANG,R. C., HUaERMAN J., JENSEN,R., MARUSmGE,K., OHLENBUSCH,H., OtZVERA,B. and WIDHOLt~,J. (1968) In Methods in Enzymology (Edited by L. Grossman, and K. Moldave), Vol. 12B, p. 3. Academic Press, New York. BONNER,J. and WIt)HOLM,J. (1967) Proc. natn. Acad. Sci. U.S.A. 57, 1379. BURTON,K. (1956) Biochem. J. 62, 315. DINo~'~, C. W. and SeOR~, M. B. (1964) J. biol. Chem. 239, 3483. DOTY,P., MARMUR,J., EIGNER,J. and SCHILDKRAUT,C. (1960) Proc. natn. Acad. Sci. U.S.A. 46, 461. FRENSTER,J. H. (1965) Nature, Lond. 206, 680. GILMAN,A. G. (1970) Proc. natn. Acad. Sci. U.S.A. 67, 305. GILMOUR,R. S. and PAUL,J. (1970) FEBS Lett. 9, 242. HANCOCK,R. (1969) J. mol. Biol. 40, 457. H E ~ N , R. L., O'MvJ~,A,A. R., Russm.J, A. P. and DOWL~O,L. E. (1969) Proc. 8th Int. Cong. Gerontol., Vol. 1, p. 141. HUANG,R. C. and BONr,mR, J. (1962) Proc. natn. Acad. Sci. U.S.A. 48, 1216. HUANG,R. C. and BONr,mR,J. (1965) Proe. natn. Acad. Sci. U.S.A. 54, 960. ~ A M A , M. and WAN6, T. Y. (1971) Biochim. biophys. Acta 228, 563. KLEINS~ffrn,L. J., ALLFREY,V. G. and MIRSKy,A. E. (1966) Proc. natn. Acad. Sci. U.S.A. 55, 1182. KLEINSVarn,L. J., ALLFREY,V. G. and MIRSKY,A. E. (1966) Science 154, 780. KURTZ,D. and SINEx,F. (1967) Biochim. biophys. Acta 145, 840.
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LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L. and RANDALL, R. S. (1951) J. bioL Chem. 193, 265. MARMUR, J. (1963) In Methods in Enzymology (Edited by S. P. Colowick, and N. O. Kaplan), Vol. 6, p. 726. Academic Press, New York. MARUSmGE,K. and BONNER,J. (1966) J. tool Biol. 15, 160. OnAag, Y. (1966) Biochim. biophys. Acta 123, 76. Om.~NBtrSCH, H. H., OLtVERA,B. M., TUAN,D. and DAVlDSON,N. (1967) J. tool. Biol. 25, 299, O'MEARA, A. R. and HERRMAN,R. L. (1972) Biochim. biophys..4cta 269, 419. PANYIM,S., JENSEN,R. H. and CnALg.LEY,R. (1968) Biochim. biophys. Acta 160, 252. PYHTmA, M. J. and SHERMAN,F. G. (1969) Gerontologia 15, 321. QU~CEY, R. and WILSON,S. H. (1969) Proc. natn. Acad. Sci. U.S.A. 64, 981. SAMAJIMA,T. and YANG,J. T. (1965) J. biol. Chem. 240, 2094. SAMtS, H. V. (1969) Proc. 8th int. Cong. Gerontol., Vol. 1, p. 138. SAMIS,H. V. (1969) Proc. 8th Int. Cong. Gerontol., Vol. 1, p. 138. SHIREY, T. L. and SOBEL,H. (1972) Exp. Geront. 7, 15. SMART,J. E. and BONN~R,J. (1971a) J. moL Biol. 58, 651. SMART,J. E. and Bor,u',mR,J. (1971b) J. tool. Biol. 58, 675. SPELSBERG,T. C. and HNILICA,L. (1970) Biochem. J. 120, 435. SRIVASTAVA, B. I. (1968) Biochem. J. 110, 683. STEm, G. S. and BASERGA,R. (1972) Adv. Cancer Res. 15, 287. STEIN, G. S., CnAUt)mJRI,S. and BASERGA, R. (1972) J. biol. Chem. 247, 3918. STEIN, G. S. and FARBER,J. (1972) Proc. natn. Acad. Sci. U.S.A. 69, 2918. STELLWAGEN,R. and COLE,R. (1969) Ann. Rev. Biochem. 38, 951. TENG, C. S., TENG, C. T. and ALLrR~Y, U. G. (1971) J. biol. Chem. 246, 3597. TUAN, D. and BONNER,J. (1969) 3". tool. Biol. 45, 59. VON HAHN, H. P. and FRITZ, E. (1966) Gerontologia 12, 237. VON HAHN, H. (1970) Gerontologia 16, 116. WANG, T. Y. (1970) Expl. Cell Res. 61, 455.
Summary--The in vitro template activity of chromatin isolated from submandibular gland increases threefold as rats age from two to twelve months. The increased capacity for DNAprimed RNA synthesis cannot be attributed to differences in nuclease or protease activity, and there are no variations in the ability of young and older submandibular gland DNA to transcribe RNA under these conditions. The histone content of young and older submandibular gland chromatin is similar. However, these proteins are less tenaciously bound to the DNA with age; consistent with the age-dependent increase in template activity. In contrast, there is an age-dependent decrease in the quantity of nonhistone chromosomal proteins associated with submandibular gland chromatin. R~sum6--L'activit6 matricieUe in vitro de la chromatine isol6e de la glande sous-maxillaire est tripl6e darts le temps que le rat vieillit de deux mois ~t douze mois. La plus grande capacit6 de synth~se d'ARN en d6pendance de I'ADN ne peut ~tre attribu6e h des diff6rences en activit6 de la nucl6ase ou de la prot~ase et il n'y a pas de variations dans raptitude de rADN des glandes sous-maxillaires, jeunes ou plus ~tg6es, h la transcription de I'ARN dans ces conditions. Les teneurs en histone des chromatines des glandes sous-maxillaires, jeunes ou plus ~g~s, sont similaires. Ces prot6ines sont toutefois moins fermement li6es ~t rADN avec l'hge et ceci concorde avec l'augmentation de l'activit6 matricielle en d6pendance de l'Age. On constate, par contre, une diminutionen d6pendance de l'gtgede la quantit6 de prot6ines chromosomiques non histoniques associ6es ~t la chromatine de la glande sous-maxillaire. Zusammenfasstmg--Die in-vitro-Matrizenaktivitiit von Chromatin, welches aus der Submandibulardr~ise der Ratte isoliert wurde, nimmt mit dem Alter der Tiere yon zwei bis zw61f Monaten um das Dreifache zu. Die erh6hte F/ihigkeit der DNS-abhangigen RNS-Synthese kann nicht Unterschieden in der Nuklease- oder Proteaseaktivitfit zugeschrieben werden, und es gibt keine Variationen der F/ihigkeit junger oder ~ilterer Submandibulardr~isen-DNS, RNS unter diesen Bedingungen zu transkribieren. Der Histongehalt von jungem und ~ilterem Submandibulardriasen-chromatin is vergleichbar. Diese Proteine werden jedoch mit zunehmendem Alter weniger fest an DNS gebunden, im Einklang mit dem altersabh/ingigen Anstieg der Matrizenaktivit~it. Im Gegensatz dazu liegt ein altersabh~ingiger Abfall der Menge an Nichthiston-Chromosomenproteinen vor, die mit dem Submandibulardr~isenchromatin verbunden sind.