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Covalent modifications of chromosomal proteins during aging
Arch. GerontoL Geriat~, 2 (1983) 1-10
1
Elsevier
Review
Covalent modifications of chromosomal proteins during aging M.K. Thakur Biochemistry Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221005, India
(Received 9 December 1982; accepted 20 January 1983)
Summa~ Covalent modifications of proteins introduce negative or positive charges into the molecules and thereby cause alterations in the ionic interactions of protein-protein or DNA-protein complexes. Whereas modifications of histones largely affect the organization of chromatin, those of non-histone proteins are believed to be involved in the expression of genes. These modifications during aging have been reviewed here. The available data suggest that the extent of covalent modificationsof histones and non-histone chromosomal (NHC) proteins change during aging and such modifications may have an important role in the differential expression of genes at different phases of life span of an organism. covalent modifications; histones and non-histone chromosomalproteins; aging
Introduction The process of biological aging may be explained at the molecular level by understanding the compositional and transcriptional modifications of the chromatin. Numerous biochemical parameters, which vary as a function of age, indicate that the expression of information encoded in D N A alters with age (Orgel, 1963; Comfort, 1974; Sinex, 1977; Kanungo, 1980a). Different regions of the total chromosomal D N A are transcribed into R N A in different cell types at different periods and in the same cell at different phases of development. An important mechanism by which such manifestations may be brought about is an alteration in the proteins associated with the chromatin. Histones and non-histone chromosomal (NHC) proteins have been shown to be intimately involved in dictating the structural organization of the genetic material and in regulating the expression of defined genetic sequences. Regulation of gene expression is thought to play a key role during development and normal functioning of cells of higher organisms (Kanungo, 1975; 1980b; Medvedev, 1981). The most important characteristic of chromosomal proteins is that they undergo post-translational covalent modifications like phosphorylation, acetylation, methyla0167-4943/83/$03.00 © 1983 Elsevier Science Publishers B.V.
tion, (ADP)-ribosylation, thiolation, etc. Since only the first three modifications have been studied in relation to aging, the latter will not be discussed here, Post-synthetic modifications of DNA-associated proteins is one of the main means of regulation for modulating the outflow of genetic information (Allfrey, 1971; Stein et al., 1974). These chemical modifications occur in the side chains of amino acid residues of proteins during or after their synthesis in the cytoplasm (Uy and Wold, 1977). Then the modified proteins are translocated to the nucleus where they bind to DNA (Ruiz-Carrillo et al., 1975). Some modifications have also been reported to occur in the nucleus while the proteins are bound to DNA. The modifications which decrease the net positive charge on histones - such as phosphorylation and acetylation - have been correlated with a diffused state of chromatin and with enhanced template activity of DNA. On the other hand, the modification which increases the net positive charge on histones - such as methylation - has been correlated with condensation of chromatin and a loss of RNA synthetic activity (Allfrey, 1971). Thus, the covalent modifications of chromosomal proteins may be an important regulatory mechanism for the differential expression of genes at different phases of the life span of an organism. It is surprising that despite the large number of reports on modifications of chromosomal proteins, relatively little is known about the function of these structural changes. Whether and how post-translational modifications of chromosomal proteins modulate the structure and function of chromatin is a central problem of molecular biology. These modifications have been studied mostly in relation to functional activity of chromatin, cell cycle and development. Histones are better studied than N H C proteins. This is because of the difficulty in the experimental procedure of isolation and fractionation of N H C proteins. Our knowledge concerning the age-associated alterations in the covalent modifications of chromosomal proteins is very poor. This article does not describe the biochemical aspects of modifications of chromosomal proteins because its main aim is to review the changes in covalent modifications of histones and N H C proteins which occur as a function of age.
Phosphorylation Phosphorylation occurs on serine (Kleinsmith et al., 1966) and threonine (Langan and Hohmann, 1974) residues of histones and N H C proteins. It shows high turn over. Rapid phosphorylation is seen not only in dividing cells, but also in non-dividing cells after their stimulation by various effectors. Phosphorylation of histones is known to be essential for mitosis (Gurley et al., 1974). It occurs during S phase of the cell cycle. This may accelerate uncoiling of chromosomes and induce the expression of specific genes. Unlike phosphorylated histones, which have effect on the structure of chromatin, phosphorylation of N H C proteins is believed to be involved in modifying their structural and functional interaction with D N A in a way so as to bring about the regulation of gene function (Stein et al., 1974; Park et al., 1977). This is substantiated by the fact that phosphorylated N H C proteins are highly
heterogeneous, phosphorylation patterns are tissue-specific, changes in their phosphorylation correlate with changes in chromatin structure as well as gene activity, and phosphorylated NHC proteins bind specifically to DNA (Kleinsmith, 1975). Liew and Gornall (1975) reported higher phosphorylation of nucleohistones in the liver of old mice. Ermini et al. (1977) have found an age-dependent incorporation of phosphate into individual histones of chromatin of the skeletal muscle of the dog and neuronal and glial cells of humans. Phosphorylation of histone H 1 is high in the developing rat liver but is negligible in the adult liver (Balhorn et al., 1972). However, it increases greatly when the liver cells divide after partial hepatectomy (Balhorn et al., 1971). In immature erythroid cells histone H 5 is phosphorylated to a considerably higher degree than in mature erythrocytes. Lower degree of phosphorylation causes total inactivation of nuclear transcription in mature erythrocytes (Adams et al., 1970). Thomas and Hempel (1976) made an indirect study of the age-dependence of phosphorylation of histones. They observed that the phosphorylation of H 1 ceased sharply and that of H 2A diminished in the culture of old resting cells as compared to that of young proliferating cells. Kanungo and Thakur (1977) measured the level of phosphorylation of individual histones using slices of cerebral hemisphere of rats of various ages. Phosphorylation of H 1 and H 4 was higher than that of others. The degree of 32p incorporation into these two histone species decreased significantly with increasing age of the rat. Kanungo and Thakur (1979a) also analysed the phosphorylation of individual N H C proteins after separating them on SDS-polyacrylamide gels. Individual bands of NHC proteins of cerebral cortex of young (2-wk-old) rats showed a higher level of phosphorylation than that of 84-wk-old rats. The phosphorylation pattern of NHC protein fractions is specific for each age. Different fractions are phosphorylated in young and adult rats. However, such differential phosphorylation of specific NHC protein fractions is not seen in old age. Das and Kanungo (1980) also found a progressive decline in the in vitro phosphorylation of NHC proteins of the brain with increasing age of the rat. Alterations in the levels of phosphorylation of individual histones and NHC proteins may result from differences in (a) rates of phosphorylation and dephosphorylation, (b) activity of specific phosphokinases and phosphatases for each protein species, (c) availability of serine and threonine residues which serve as the sites for phosphorylation, and (d) metabolic activity of cells.
Acetylation Histones are modified by acetylation in two ways. Acetylation of the NH2-terminal serine of H l, H 2A and H 4 histones occurs during their synthesis (Phillips, 1968) and this blocks the amino terminus of histones. This terminal acetate may remain permanently or may be lost slowly. Secondly, acetylation occurs at internal lysine residues of histones H 2A, H 2B, H 3 and H 4 post-translationally (Gershey et al., 1968). This internal acetate turns over fast with a I l l 2 of about 3 min. Such modifications are important for the dynamics of chromatin structure. Acetylation
may alter histone-histone, histone-DNA or h i s t o n e - N H C protein interactions. Consequently, the conformation of nucleosomes may be converted to a state which is more conducive to transcription of the associated DNA. Thus, acetylation of histones plays a direct role in the activation of genes. It has been suggested that both acetylation and phosphorylation may open the DNA for transcription or replication by reducing the affinity of histones and N H C proteins to DNA. Although different degrees of histone acetylation have been observed in various states of the cell, and suggestions have been made to correlate histone acetylation with transcriptional activity (Allfrey, 1971; Wangh et al., 1972; Covault and Chalkley, 1980), the true biological role of histone acetylation is still not known. Furthermore, little is known about the acetylation of N H C proteins except for the report of Sterner et al. (1979) that high mobility group N H C proteins of calf thymus and duck erythrocyte nuclei are acetylated. The first attempt to study the level of acetylation of histones of thymus and liver was made by Oh and Conard (1972). They measured the incorporation of acetate into histones of normal and regenerating livers of rats of 1 to 24 ruth of age. Acetylation of liver histones was generally higher in young growing rats. In normal livers, acetylation declined to minimal levels by 6 mth of age. Regenerating livers showed higher acetylation. This declined steadily after 9 mth of age to low levels comparable to those of normal livers of 20- to 24-mth-old rats. Such decline in acetylation of histones is believed to be related to reduced metabolic capacity of the liver associated with the aging process. In order to determine the relative degree of acetylation of different histone species, histones were fractionated on a CM-cellulose column. The incorporation was particularly high in H 3 and H 4 histones. However, no significant difference was found in these histone fractions of rats of different ages. Acetylation of H 2A and H 2B was slightly higher in young rats than in the old (10-24 mth). Ryan and Cristofalo (1972) studied human diploid cells in culture and reported an age-dependent decrease in the rate of histone acetylation with increasing passage number. It was suggested that this reduction in acetylation rate was due to the older cultured cells reaching a stage of the cell cycle not compatible with histone acetylation. In contrast to this, using the tissue culture technique, Liew and Gornall (1975) have reported a higher rate of acetylation of nuclear proteins of the liver of mice with increasing age. These studies further showed that H 3 histone was acetylated at a rate of 129% and H 4 histone to 112% of the value found for young mice. Petricevic et al. (1978) also studied acetylation of histones of liver and thymus of rats of various ages. The acetylation of liver histones decreased from 1- to 16-mth stabilizing thereafter until 24 mth of age. The 16- to 17-mth limit of the age for the decrease of acetylation in liver was suggested as related to age-associated increase in polyploidization of liver cells (Epstein and Gatens, 1967; Brasch, 1982). Thymus histones showed lower rate of acetylation than liver histones. Acetylation of thymus historic increases initially up to the third month, decreases thereafter up to 8 mth and this is followed by a rise in the rate of acetylation up to 24 mth of age of the rat. The difference between the liver and thymus acetylation may be due to a difference in the rate of organ maturation. The increase in acetylation of thymus histones of old rats may be related to infiltration of older cell types, such as connective tissue
cells and fat cells. Sarkander and Knoll-ki3hler (1978) studied in vitro acetylation of cerebral histones of rats of different ages. Whereas H 1 was not acetylated, nucleosomal histones showed an increase in acetylation between 1 and 12 mth of age. These findings contradict reports suggesting that acetylation of histones decreases with age (Ryan and Cristofalo, 1972; Kanungo and Thakur, 1979b). The discrepancy may be due to the use of different tissues with different mitotic a n d / o r due to age-related changes in acetate pool size. Kanungo and Thakur (1979b) followed acetylation of chromosomal proteins up to the 110-wk age of rats. They found a steady fall of acetylation of histones of the brain with increasing age of the rat. Acetylation in old rats was only about 50% of that in young animals. Thakur et al. (1978) further analysed the incorporation of [3H] acetate into individual histones and observed that acetylation of nucleosomal histones H 4, H 3 and H 2B was high in immature rats and later decreased with age. The significant decrease in the acetylation of histones, particularly those of the nucleosome, after early development may be due to cessation of neuronal division (Johnson and Erner, 1972). Also, a decrease in the activity of histone acetyltransferase a n d / o r the availability of acetylation sites, particularly of lysyl residues, due to conformational changes in the histone-DNA complex after the cells stop dividing, may account for the decrease in acetylation of histones. O'Meara and Pochron (1979) also observed an age-dependent decline in the incorporation of acetate into liver histones of rats up to 24 mth of age. H 4 was predominantly labelled at 2 mth, whereas in 12-, 16- and 24-mth-old animals H 3 was more highly labelled; at 27 mth the two fractions were labelled equally. Assay of histone acetylase and deacetylase activities indicates that deacetylase activity increases with age (Pochron et al., 1978). Liew and Gornall (1975) reported a higher level of acetylation of nuclear acidic proteins in the liver of old mice. Acetylation of phenol-soluble nuclear acidic proteins was also found to increase to 250% and phosphorylation to 138%, as compared to young mice, as a function of age. Kanungo and Thakur (1979c) found an age-dependent decline in the level of acetylation of NHC proteins of brain cells. They further electrophoresed these proteins on SDS-polyacrylamide gels, but failed to find any significant incorporation of labelled acetate into a particular NHC protein fraction. The decrease in acetylation of NHC proteins in the adult appears to be mainly due to a decrease in the acetylation of high mol. wt. fractions. In the old, all the NHC protein fractions are acetylated to a lesser degree.
Methylation Methylation occurs largely on Lys 9 and 27 in H 3, and Lys 10 in H 4 histone (Isenberg, 1979). It is irreversible; once a histone is methylated, it remains methylated (Byvoet et al., 1972). Methylated histones may have several functions: (a) Methylation of lysyl residues, particularly the trimethyl residues, may raise the pK of the c-NH 2 group of lysine and increase the basicity of histones (Gershey et al., 1969). This may strengthen the binding of histones to DNA. Thus, methylation of histones
may play a role in the condensation of chromatin leading to the tightly coiled state of the premitotic chromosome. (b) Methylation of H 3 and H 4 histones may play an important role in the structure of nucleosome. (c) Methylated histones may lock in with DNA irreversibly and prevent replication. This may account for the cells becoming post-mitotic. (d) They may inhibit transcription. Duerre and Lee (1974) first observed that only trace amounts of [laC]methyl groups were incorporated into histones of the brain cells after maturity. During brain development (up to 50 days) the arginine-rich histones have a half life of 32 days while no turn over is observed after maturity as measured by either 3H or ~aC decay. Lee and Duerre (1974) found that the level of histone methylases and the ability of brain histones to accept methyl groups decrease during aging. Methylation of brain histones was high during the first few days after birth. After approximately 11 days the activity decreased progressively throughout the life of the animal. The extent of methylation of histones of brain nuclei was three to four times greater than that of liver nuclei. The age-dependent decrease in the level of methylation was more pronounced in brain nuclei. After fractionation on Bio-Gel P-10, H 3 and H 4 histones of the liver and brain nuclei were found to be highly methylated. The amount of labelled methyl groups incorporated into H 3 and H 4 histones decreased during aging in both liver and brain. However, the degree of methylation of H 3 was always greater than that of H 4. The apparent decrease in methylation of brain and liver histones with age may be attributed to a decrease in the activity of histone methylases a n d / o r the availability of sites on the histones to accept methyl groups. To examine these two possibilities, histone methylases were assayed in young and old animals. The activity of histone methylase was considerably higher in brain and liver nuclei of young animals. There was no significant difference in the ability of histones prepared from liver nuclei of young or old animals to accept methyl groups. However, brain histones of young rats appeared to be somewhat better methyl group acceptors than those from old brains. These data agree with those of Paik and Kim (1973) who also found that the activity of histone methylase III of rat brain increased after birth until the 10th day and decreased significantly thereafter. The levels of methyl donor compound (S-adenosylmethionine) also show a remarkable decrease in the liver and brain of senescent (30-mth-old) rats (Stramentinoli et al., 1977). Duerre et al. (1977) observed that the molar ratio of mono-:di: trimethyl lysine in H 3 of 10-day-old rats is 0.55:1.0:0.35. The molar ratio of mono-: dimethyl lysine in H4 of the same age is 0.1:0.9. Thus, there is a gradual shift towards more methylated forms of lysine residues. Kanungo and Thakur (1979c) found lower methylation of histones of the adult brain than that of the immature. Surprisingly, methylation in the old is greater than that of the adult. Repeated experiments showed the same pattern of data. Higher activity of methylase III in the young than in the adult may account for greater methylation of histones in the former (Paik and Kim, 1973), but how methylation increases in the old cannot be accounted for from these experiments. The availability of specific sites on histones to accept methyl groups may also differ with age due to conformational changes in the chromatin. Even the degree of methylation of the same active site on a histone may
depend on the functional status of the chromatin. Methylation of H 3 and H 4 which are known to be the most dominant forms of methylated histones is highest in the immature and decreases significantly with age (Kanungo and Thakur, 1979c). Methylation of other histones is lower than that of H 3 and H 4, and also does not change markedly with age. This is of much significance since (H 3)2 (H 4)2 tetramer defines the length and basic fold of the core DNA (Kornberg, 1974; Camerini-Otero et al., 1976). Stronger binding of H 3 and H 4 to DNA in old age may be responsible for the unavailability of methylation sites. Methylation of certain histones has been implicated in the process of development (Borun and Stein, 1972). Likewise, differential incorporation of methyl groups into different histones species may play a significant role in the process of senescence. Klimenko and Malyshev (1976) reported an age-dependent increase in the methylation of histones and NHC proteins of the liver of rats. However, Kanungo and Thakur (1979c) observed a progressive decrease in the methylation of brain NHC proteins with increasing age of the rat. They also observed the incorporation of [ 14 C]methyl groups into individual NHC protein fraction and found that it varies in NHC proteins of the brain of rats of different ages (Thakur and Kanungo, 1981). Almost all NHC protein fractions showed lower methylation in adult and old rats as compared to those of the immature. Such a general pattern of change may be due to a decrease in the level of methylase responsible for methylation of NHC proteins.
Conclusion It is surprising that with so many indirect indications about the possible involvement of covalent modifications of chromosomal proteins in the differential expression of genes occurring during aging, no definite function has yet been assigned to these modifications. There still remains a great deal of work to be done to understand fully the role of covalent modifications of chromosomal proteins, and to understand as well, the mechanism by which these modifications influence the structure and function of chromatin at different phases of the life span.
Acknowledgements I express my gratitude to Professor M.S. Kanungo for his encouragement and critical review of the article. The part of the work cited above was supported partially by University Grants Commission, New Delhi, India.
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1
Elsevier
Review
Covalent modifications of chromosomal proteins during aging M.K. Thakur Biochemistry Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221005, India
(Received 9 December 1982; accepted 20 January 1983)
Summa~ Covalent modifications of proteins introduce negative or positive charges into the molecules and thereby cause alterations in the ionic interactions of protein-protein or DNA-protein complexes. Whereas modifications of histones largely affect the organization of chromatin, those of non-histone proteins are believed to be involved in the expression of genes. These modifications during aging have been reviewed here. The available data suggest that the extent of covalent modificationsof histones and non-histone chromosomal (NHC) proteins change during aging and such modifications may have an important role in the differential expression of genes at different phases of life span of an organism. covalent modifications; histones and non-histone chromosomalproteins; aging
Introduction The process of biological aging may be explained at the molecular level by understanding the compositional and transcriptional modifications of the chromatin. Numerous biochemical parameters, which vary as a function of age, indicate that the expression of information encoded in D N A alters with age (Orgel, 1963; Comfort, 1974; Sinex, 1977; Kanungo, 1980a). Different regions of the total chromosomal D N A are transcribed into R N A in different cell types at different periods and in the same cell at different phases of development. An important mechanism by which such manifestations may be brought about is an alteration in the proteins associated with the chromatin. Histones and non-histone chromosomal (NHC) proteins have been shown to be intimately involved in dictating the structural organization of the genetic material and in regulating the expression of defined genetic sequences. Regulation of gene expression is thought to play a key role during development and normal functioning of cells of higher organisms (Kanungo, 1975; 1980b; Medvedev, 1981). The most important characteristic of chromosomal proteins is that they undergo post-translational covalent modifications like phosphorylation, acetylation, methyla0167-4943/83/$03.00 © 1983 Elsevier Science Publishers B.V.
tion, (ADP)-ribosylation, thiolation, etc. Since only the first three modifications have been studied in relation to aging, the latter will not be discussed here, Post-synthetic modifications of DNA-associated proteins is one of the main means of regulation for modulating the outflow of genetic information (Allfrey, 1971; Stein et al., 1974). These chemical modifications occur in the side chains of amino acid residues of proteins during or after their synthesis in the cytoplasm (Uy and Wold, 1977). Then the modified proteins are translocated to the nucleus where they bind to DNA (Ruiz-Carrillo et al., 1975). Some modifications have also been reported to occur in the nucleus while the proteins are bound to DNA. The modifications which decrease the net positive charge on histones - such as phosphorylation and acetylation - have been correlated with a diffused state of chromatin and with enhanced template activity of DNA. On the other hand, the modification which increases the net positive charge on histones - such as methylation - has been correlated with condensation of chromatin and a loss of RNA synthetic activity (Allfrey, 1971). Thus, the covalent modifications of chromosomal proteins may be an important regulatory mechanism for the differential expression of genes at different phases of the life span of an organism. It is surprising that despite the large number of reports on modifications of chromosomal proteins, relatively little is known about the function of these structural changes. Whether and how post-translational modifications of chromosomal proteins modulate the structure and function of chromatin is a central problem of molecular biology. These modifications have been studied mostly in relation to functional activity of chromatin, cell cycle and development. Histones are better studied than N H C proteins. This is because of the difficulty in the experimental procedure of isolation and fractionation of N H C proteins. Our knowledge concerning the age-associated alterations in the covalent modifications of chromosomal proteins is very poor. This article does not describe the biochemical aspects of modifications of chromosomal proteins because its main aim is to review the changes in covalent modifications of histones and N H C proteins which occur as a function of age.
Phosphorylation Phosphorylation occurs on serine (Kleinsmith et al., 1966) and threonine (Langan and Hohmann, 1974) residues of histones and N H C proteins. It shows high turn over. Rapid phosphorylation is seen not only in dividing cells, but also in non-dividing cells after their stimulation by various effectors. Phosphorylation of histones is known to be essential for mitosis (Gurley et al., 1974). It occurs during S phase of the cell cycle. This may accelerate uncoiling of chromosomes and induce the expression of specific genes. Unlike phosphorylated histones, which have effect on the structure of chromatin, phosphorylation of N H C proteins is believed to be involved in modifying their structural and functional interaction with D N A in a way so as to bring about the regulation of gene function (Stein et al., 1974; Park et al., 1977). This is substantiated by the fact that phosphorylated N H C proteins are highly
heterogeneous, phosphorylation patterns are tissue-specific, changes in their phosphorylation correlate with changes in chromatin structure as well as gene activity, and phosphorylated NHC proteins bind specifically to DNA (Kleinsmith, 1975). Liew and Gornall (1975) reported higher phosphorylation of nucleohistones in the liver of old mice. Ermini et al. (1977) have found an age-dependent incorporation of phosphate into individual histones of chromatin of the skeletal muscle of the dog and neuronal and glial cells of humans. Phosphorylation of histone H 1 is high in the developing rat liver but is negligible in the adult liver (Balhorn et al., 1972). However, it increases greatly when the liver cells divide after partial hepatectomy (Balhorn et al., 1971). In immature erythroid cells histone H 5 is phosphorylated to a considerably higher degree than in mature erythrocytes. Lower degree of phosphorylation causes total inactivation of nuclear transcription in mature erythrocytes (Adams et al., 1970). Thomas and Hempel (1976) made an indirect study of the age-dependence of phosphorylation of histones. They observed that the phosphorylation of H 1 ceased sharply and that of H 2A diminished in the culture of old resting cells as compared to that of young proliferating cells. Kanungo and Thakur (1977) measured the level of phosphorylation of individual histones using slices of cerebral hemisphere of rats of various ages. Phosphorylation of H 1 and H 4 was higher than that of others. The degree of 32p incorporation into these two histone species decreased significantly with increasing age of the rat. Kanungo and Thakur (1979a) also analysed the phosphorylation of individual N H C proteins after separating them on SDS-polyacrylamide gels. Individual bands of NHC proteins of cerebral cortex of young (2-wk-old) rats showed a higher level of phosphorylation than that of 84-wk-old rats. The phosphorylation pattern of NHC protein fractions is specific for each age. Different fractions are phosphorylated in young and adult rats. However, such differential phosphorylation of specific NHC protein fractions is not seen in old age. Das and Kanungo (1980) also found a progressive decline in the in vitro phosphorylation of NHC proteins of the brain with increasing age of the rat. Alterations in the levels of phosphorylation of individual histones and NHC proteins may result from differences in (a) rates of phosphorylation and dephosphorylation, (b) activity of specific phosphokinases and phosphatases for each protein species, (c) availability of serine and threonine residues which serve as the sites for phosphorylation, and (d) metabolic activity of cells.
Acetylation Histones are modified by acetylation in two ways. Acetylation of the NH2-terminal serine of H l, H 2A and H 4 histones occurs during their synthesis (Phillips, 1968) and this blocks the amino terminus of histones. This terminal acetate may remain permanently or may be lost slowly. Secondly, acetylation occurs at internal lysine residues of histones H 2A, H 2B, H 3 and H 4 post-translationally (Gershey et al., 1968). This internal acetate turns over fast with a I l l 2 of about 3 min. Such modifications are important for the dynamics of chromatin structure. Acetylation
may alter histone-histone, histone-DNA or h i s t o n e - N H C protein interactions. Consequently, the conformation of nucleosomes may be converted to a state which is more conducive to transcription of the associated DNA. Thus, acetylation of histones plays a direct role in the activation of genes. It has been suggested that both acetylation and phosphorylation may open the DNA for transcription or replication by reducing the affinity of histones and N H C proteins to DNA. Although different degrees of histone acetylation have been observed in various states of the cell, and suggestions have been made to correlate histone acetylation with transcriptional activity (Allfrey, 1971; Wangh et al., 1972; Covault and Chalkley, 1980), the true biological role of histone acetylation is still not known. Furthermore, little is known about the acetylation of N H C proteins except for the report of Sterner et al. (1979) that high mobility group N H C proteins of calf thymus and duck erythrocyte nuclei are acetylated. The first attempt to study the level of acetylation of histones of thymus and liver was made by Oh and Conard (1972). They measured the incorporation of acetate into histones of normal and regenerating livers of rats of 1 to 24 ruth of age. Acetylation of liver histones was generally higher in young growing rats. In normal livers, acetylation declined to minimal levels by 6 mth of age. Regenerating livers showed higher acetylation. This declined steadily after 9 mth of age to low levels comparable to those of normal livers of 20- to 24-mth-old rats. Such decline in acetylation of histones is believed to be related to reduced metabolic capacity of the liver associated with the aging process. In order to determine the relative degree of acetylation of different histone species, histones were fractionated on a CM-cellulose column. The incorporation was particularly high in H 3 and H 4 histones. However, no significant difference was found in these histone fractions of rats of different ages. Acetylation of H 2A and H 2B was slightly higher in young rats than in the old (10-24 mth). Ryan and Cristofalo (1972) studied human diploid cells in culture and reported an age-dependent decrease in the rate of histone acetylation with increasing passage number. It was suggested that this reduction in acetylation rate was due to the older cultured cells reaching a stage of the cell cycle not compatible with histone acetylation. In contrast to this, using the tissue culture technique, Liew and Gornall (1975) have reported a higher rate of acetylation of nuclear proteins of the liver of mice with increasing age. These studies further showed that H 3 histone was acetylated at a rate of 129% and H 4 histone to 112% of the value found for young mice. Petricevic et al. (1978) also studied acetylation of histones of liver and thymus of rats of various ages. The acetylation of liver histones decreased from 1- to 16-mth stabilizing thereafter until 24 mth of age. The 16- to 17-mth limit of the age for the decrease of acetylation in liver was suggested as related to age-associated increase in polyploidization of liver cells (Epstein and Gatens, 1967; Brasch, 1982). Thymus histones showed lower rate of acetylation than liver histones. Acetylation of thymus historic increases initially up to the third month, decreases thereafter up to 8 mth and this is followed by a rise in the rate of acetylation up to 24 mth of age of the rat. The difference between the liver and thymus acetylation may be due to a difference in the rate of organ maturation. The increase in acetylation of thymus histones of old rats may be related to infiltration of older cell types, such as connective tissue
cells and fat cells. Sarkander and Knoll-ki3hler (1978) studied in vitro acetylation of cerebral histones of rats of different ages. Whereas H 1 was not acetylated, nucleosomal histones showed an increase in acetylation between 1 and 12 mth of age. These findings contradict reports suggesting that acetylation of histones decreases with age (Ryan and Cristofalo, 1972; Kanungo and Thakur, 1979b). The discrepancy may be due to the use of different tissues with different mitotic a n d / o r due to age-related changes in acetate pool size. Kanungo and Thakur (1979b) followed acetylation of chromosomal proteins up to the 110-wk age of rats. They found a steady fall of acetylation of histones of the brain with increasing age of the rat. Acetylation in old rats was only about 50% of that in young animals. Thakur et al. (1978) further analysed the incorporation of [3H] acetate into individual histones and observed that acetylation of nucleosomal histones H 4, H 3 and H 2B was high in immature rats and later decreased with age. The significant decrease in the acetylation of histones, particularly those of the nucleosome, after early development may be due to cessation of neuronal division (Johnson and Erner, 1972). Also, a decrease in the activity of histone acetyltransferase a n d / o r the availability of acetylation sites, particularly of lysyl residues, due to conformational changes in the histone-DNA complex after the cells stop dividing, may account for the decrease in acetylation of histones. O'Meara and Pochron (1979) also observed an age-dependent decline in the incorporation of acetate into liver histones of rats up to 24 mth of age. H 4 was predominantly labelled at 2 mth, whereas in 12-, 16- and 24-mth-old animals H 3 was more highly labelled; at 27 mth the two fractions were labelled equally. Assay of histone acetylase and deacetylase activities indicates that deacetylase activity increases with age (Pochron et al., 1978). Liew and Gornall (1975) reported a higher level of acetylation of nuclear acidic proteins in the liver of old mice. Acetylation of phenol-soluble nuclear acidic proteins was also found to increase to 250% and phosphorylation to 138%, as compared to young mice, as a function of age. Kanungo and Thakur (1979c) found an age-dependent decline in the level of acetylation of NHC proteins of brain cells. They further electrophoresed these proteins on SDS-polyacrylamide gels, but failed to find any significant incorporation of labelled acetate into a particular NHC protein fraction. The decrease in acetylation of NHC proteins in the adult appears to be mainly due to a decrease in the acetylation of high mol. wt. fractions. In the old, all the NHC protein fractions are acetylated to a lesser degree.
Methylation Methylation occurs largely on Lys 9 and 27 in H 3, and Lys 10 in H 4 histone (Isenberg, 1979). It is irreversible; once a histone is methylated, it remains methylated (Byvoet et al., 1972). Methylated histones may have several functions: (a) Methylation of lysyl residues, particularly the trimethyl residues, may raise the pK of the c-NH 2 group of lysine and increase the basicity of histones (Gershey et al., 1969). This may strengthen the binding of histones to DNA. Thus, methylation of histones
may play a role in the condensation of chromatin leading to the tightly coiled state of the premitotic chromosome. (b) Methylation of H 3 and H 4 histones may play an important role in the structure of nucleosome. (c) Methylated histones may lock in with DNA irreversibly and prevent replication. This may account for the cells becoming post-mitotic. (d) They may inhibit transcription. Duerre and Lee (1974) first observed that only trace amounts of [laC]methyl groups were incorporated into histones of the brain cells after maturity. During brain development (up to 50 days) the arginine-rich histones have a half life of 32 days while no turn over is observed after maturity as measured by either 3H or ~aC decay. Lee and Duerre (1974) found that the level of histone methylases and the ability of brain histones to accept methyl groups decrease during aging. Methylation of brain histones was high during the first few days after birth. After approximately 11 days the activity decreased progressively throughout the life of the animal. The extent of methylation of histones of brain nuclei was three to four times greater than that of liver nuclei. The age-dependent decrease in the level of methylation was more pronounced in brain nuclei. After fractionation on Bio-Gel P-10, H 3 and H 4 histones of the liver and brain nuclei were found to be highly methylated. The amount of labelled methyl groups incorporated into H 3 and H 4 histones decreased during aging in both liver and brain. However, the degree of methylation of H 3 was always greater than that of H 4. The apparent decrease in methylation of brain and liver histones with age may be attributed to a decrease in the activity of histone methylases a n d / o r the availability of sites on the histones to accept methyl groups. To examine these two possibilities, histone methylases were assayed in young and old animals. The activity of histone methylase was considerably higher in brain and liver nuclei of young animals. There was no significant difference in the ability of histones prepared from liver nuclei of young or old animals to accept methyl groups. However, brain histones of young rats appeared to be somewhat better methyl group acceptors than those from old brains. These data agree with those of Paik and Kim (1973) who also found that the activity of histone methylase III of rat brain increased after birth until the 10th day and decreased significantly thereafter. The levels of methyl donor compound (S-adenosylmethionine) also show a remarkable decrease in the liver and brain of senescent (30-mth-old) rats (Stramentinoli et al., 1977). Duerre et al. (1977) observed that the molar ratio of mono-:di: trimethyl lysine in H 3 of 10-day-old rats is 0.55:1.0:0.35. The molar ratio of mono-: dimethyl lysine in H4 of the same age is 0.1:0.9. Thus, there is a gradual shift towards more methylated forms of lysine residues. Kanungo and Thakur (1979c) found lower methylation of histones of the adult brain than that of the immature. Surprisingly, methylation in the old is greater than that of the adult. Repeated experiments showed the same pattern of data. Higher activity of methylase III in the young than in the adult may account for greater methylation of histones in the former (Paik and Kim, 1973), but how methylation increases in the old cannot be accounted for from these experiments. The availability of specific sites on histones to accept methyl groups may also differ with age due to conformational changes in the chromatin. Even the degree of methylation of the same active site on a histone may
depend on the functional status of the chromatin. Methylation of H 3 and H 4 which are known to be the most dominant forms of methylated histones is highest in the immature and decreases significantly with age (Kanungo and Thakur, 1979c). Methylation of other histones is lower than that of H 3 and H 4, and also does not change markedly with age. This is of much significance since (H 3)2 (H 4)2 tetramer defines the length and basic fold of the core DNA (Kornberg, 1974; Camerini-Otero et al., 1976). Stronger binding of H 3 and H 4 to DNA in old age may be responsible for the unavailability of methylation sites. Methylation of certain histones has been implicated in the process of development (Borun and Stein, 1972). Likewise, differential incorporation of methyl groups into different histones species may play a significant role in the process of senescence. Klimenko and Malyshev (1976) reported an age-dependent increase in the methylation of histones and NHC proteins of the liver of rats. However, Kanungo and Thakur (1979c) observed a progressive decrease in the methylation of brain NHC proteins with increasing age of the rat. They also observed the incorporation of [ 14 C]methyl groups into individual NHC protein fraction and found that it varies in NHC proteins of the brain of rats of different ages (Thakur and Kanungo, 1981). Almost all NHC protein fractions showed lower methylation in adult and old rats as compared to those of the immature. Such a general pattern of change may be due to a decrease in the level of methylase responsible for methylation of NHC proteins.
Conclusion It is surprising that with so many indirect indications about the possible involvement of covalent modifications of chromosomal proteins in the differential expression of genes occurring during aging, no definite function has yet been assigned to these modifications. There still remains a great deal of work to be done to understand fully the role of covalent modifications of chromosomal proteins, and to understand as well, the mechanism by which these modifications influence the structure and function of chromatin at different phases of the life span.
Acknowledgements I express my gratitude to Professor M.S. Kanungo for his encouragement and critical review of the article. The part of the work cited above was supported partially by University Grants Commission, New Delhi, India.
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