Nuclear chromatin changes during post-natal myocardial development

387

Biochimica et Biophysica Acta, 521 (1978) 387--396

© Elsevier/North-HollandBiomedicalPress

BBA 99281 NUCLEAR CHROMATIN CHANGES DURING POST-NATAL MYOCARDIAL DEVELOPMENT

CONSTANTINOS

J. L I M A S and CHRISTINE C H A N -S T I E R

Department of Medicine (CardiovascularDivision), University of Minnesota School of Medicine, Minneapolis, Minn. 55455 (U.S.A.) (Received January 3rd, 1978) (Revised manuscript received March 24th, 1978)

Summary The proliferative capacity of rat myocardium declines rapidly during the first few weeks of post-natal life. In order to gain insight into the mechanisms involved in this decline, we studied the structure and function of nuclear chromatin from isolated rat myocardial cells during post-natal growth. Chromatin template activity decreased progressively (7.5 _+0.3 pmol [SH]UTP/~g DNA per min at age 5 days compared to 2.2 _+0.1 pmol [3H]UTP//~g DNA per rain at age 6 months) and was associated with a decrease in the number of transcription initiation sites. This decline was accompanied by changes in chromatin structure as evidenced by: (a) decreased susceptibility to DNAase I digestion with advancing age, (b) decreased poly-L-lysine binding (60% decrease between day 5 and six months of age), (c) progressive decline in positive ellipticity of circular dichroism spectra between 250--300 nm, and (d) derivative melting profiles showing a decrease in DNA regions bound by non-histone proteins and concomitant increase in histone-bound regions. The protein composition of myocardial chromatin also changed during post-natal development, chiefly due to a progressive increase in the histone/DNA ratio. These results indicate substantial changes in the organization and functional capacity of myocardial chromatin during early post-natal growth. These changes accompany, and may contribute to, the restriction in the proliferative capacity of myocardial cells.

The proliferative capacity of myocardial cells decreases rapidly during postnatal development and reaches almost negligible levels within three weeks in the rat [1--3]. This restriction in proliferative capacity poses severe limitations on cardiac adaptations to chronic overload or injury. The adult myocardium can only respond to increased functional demands by hypertrophy, i.e., an increase in cell size with no change in total cell number. This form of adapta-

388 tion is associated with structural and functional changes that eventually lead to cardiac decompensation [4]. In contrast, interstitial cells retain their capacity to proliferate through adulthood, as reflected in the substantial increase in collagen content of hypertrophied myocardium [5] ; this increase may further impair the mechanical performance of the heart [6]. It is n o t known what regulatory factors underlie the progressive restriction in myocardial proliferative capacity. Studies in numerous other cell systems have indicated that transition from quiescence to cell proliferation in response to appropriate stimuli is associated with, and may be dependent on, changes in the structural organization and functional capabilities of nuclear chromatin [7-11]. We, therefore, u n d e r t o o k to examine the possibility that the "de-activat i o n " of myocardial chromatin accompanies and, perhaps, determines the progressive loss of mitotic potential during postnatal development. Materials and Methods Experiments were conducted on Holtzman rats aged 5--240 days. Myocardial cell isolation. Myocardial cells were isolated from rat hearts by a modification of the m e t h o d described b y Glick et al. [12]. Cardiac ventricles from rats of different ages were diced into 3 mm slices and incubated at 37°C in a 50 ml conical flask containing 3--5 ml of a phosphate-buffered medium (pH 7.4) with 1 mg/ml collagenase. The flasks were shaken at 100 strokes/min. The composition of the medium (g/l) was: 8.8 NaC1, 0.4 KC1, 0.21 Na2HPO4 and 0.9 glucose. Cells were decanted from the tissue pieces at 20-min intervals (harvests 1--4) and the tissue resuspended in fresh buffer. Harvests 1 and 2 were mostly erythrocytes and fragmented cells and were discarded. Cells from harvests 3 and 4 were collected by centrifugation (60 × g for 2.5 rain), washed three times and counted with a h e m o c y t o m e t e r . Approximately 93--95% of the cells were m y o c y t e s and 65% of the latter excluded Trypan blue. The cell yield varied from 0.3 • 107--1.4 • 10-7/heart, depending on the age of the animals. Isolated m y o c y t e s were used in all the experiments of the present study. Isolation o f nuclei. Cardiac tissue from pooled rat hearts was chilled and finely minced with scissors. All steps in the isolation procedure were performed in the cold. The minced tissue was homogenized in 5 vols. of 0.32 M sucrose/ 3.3 mM CaC12 with a Polytron PT-20 homogenizer. Phenylmethylsulfonyl fluoride {final concentration, 0.1 mM) was present in all the buffers during isolation of nuclei and chromatin. The homogenate was filtered through a double gauze and cold deionized water was added to the filtrate to reduce the total sucrose concentration to 0.25 M. Aliquots of the homogenate were taken for determination of total protein and DNA content. The remainder was transferred to centrifuge tubes and 0.25 ml of 0.32 M sucrose/3.3 mM CaC12 were pipetted into each tube to form a layer beneath the broken cell suspension. The tubes were centrifuged at 1000 × g for 10 min. The crude nuclear pellet was resuspended in 10 vols. of 2.4 M sucrose/1 mM MgC12 and the nuclei were pelleted by centrifugation at 1 0 0 0 0 0 × g for I h. The nuclear preparations were routinely checked by phase microscopy and were shown to be free of intact cells, myofibrils or cytoplasmic contaminants; the nuclear membrane was shown to be intact by electron microscopy.

389

Isolation and dissociation of chromatin. Chromatin was prepared by lysing nuclei in 80 vols. of triple glass
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390 Roeder and R u t t e r procedure [16]. The enzyme had a specific activity of 53 units per mg protein; one unit incorporates 1 nmol [3H]UTP/10 min into acidinsoluble precipitate. The assay mixture contained, in a total volume of 0.25 ml, 10 ~mol Tris • HC1 (pH 7.9), 0.75 ~ m o l MnCl2, 1.15 ~mol MgC12, 12.5 ~mol (NH4)2SO4, 0.17 ~ m o l EDTA, 1.0 ~mol ~-mercaptoethanol, 62.4 nmol each of ATP, CTP and GTP, 10 nmol [3H]UTP, 0.5 unit R N A polymerase, and appropriate amounts of chromatin. The reaction was carried o u t at 37°C for 30 rain and was stopped with 3 ml of cold 5% trichloroacetic acid. After 15 min, the precipitate was collected on a Millipore filter (0.65 ~m, pore size), washed four times with 3 ml of 2% trichloroacetic acid, dried and counted. The number of transcription initiation sites was determined according to Cedar and Felsenfeld [17]. R N A polymerase (5 units) and DNA (0--20 ~g) were incubated at 37°C in 0.5 ml containing 10 mM Tris • HC1 (pH 7.0), 1.0 mM MnC12, 0.08 mM each of ATP and GTP, and 0.02 mM [3H]UTP. After 20 rain, the initiation reaction was stopped b y the addition of 0.16 ml 1.6 M (NH4)2SO4. Propagation in high salt was then started b y the addition of CTP (final concentration 0.063 mM) and MgC12 (final concentration, 5 mM). Incorporation into R N A was measured at various times during the assay, as described above. In this procedure, chromatin template is titrated against a fixed amount of R N A polymerase. The a m o u n t of template required to achieve maximum enzymatic activity indicates the number of available initiation sites. As the a m o u n t of template is increased, the number of R N A chains synthesized is correspondingly increased until a plateau is reached. Thermal denaturation. Myocardial chromatin was dialyzed against 2 . 5 . 10 -4 M EDTA (pH 8.0) and adjusted to a concentration of A26oam = 1.0. Thermal denaturation was performed on a Gilford Model 2400 spectrophotometer equipped with a Haake constant temperature regulator. The derivative of each melting profile, dh/dT, where h is the hyperchromicity at 260 nm and T the temperature, was calculated according to Li and Bonnet [ 18]. Circular dichroism spectra. Circular dichroism spectra were obtained with a Jasco Model J-41C spectropolarimeter measured at ambient temperature. The molar extinction coefficient of 6500 M -1 • C ~ at 260 nm was used for estimating chromatin concentration. The mean ellipticity is expressed in degree • cm 2 per dmol of nucleotide residue, assuming a mean molecular weight of a nucleotide as 330. All readings were taken in 10 mM Tris • HC1. Polylysine titration. Poly(1-1ysine) hydrochloride (degree of polymerization = 147) was obtained from Schwartz/Mann, Orangeburg, N.Y. Both polylysine and chromatin were dissolved in 2 . 5 - 1 0 - 4 M EDTA and adjusted to pH 8.0. Polylysine was added slowly to the chromatin solution with vigorous shaking [19]. At .each point in the titration, the polylysine • chromatin complex was centrifuged at 1 0 0 0 0 × g for 10 min. The supernatant was collected and its absorbance at 260 nm recorded. Deoxyribonuclease digestion [20]. Chromatin samples were dialyzed overnight against 0.01 M NaC1/1.5 mM MgC12/0.01 M Tris. HCI (pH 7.0) and adjusted to contain 15--50 ~g of DNA/ml; in a given set of experiments all the chromatin samples contained equal amounts of D N A per ml. To 1.0 ml of chromatin was added 0.1 ml of buffer containing 11 times the desired concen-

391 tration of DNAase (pencreatic deoxyribonuclease I, electrophoretically purified, RNAase-free); the tubes were stoppered and incubated at 37°C for 1 h. Digestion was stopped by the addition of cold perchloric acid to 0.25 M and the samples were centrifuged at 3 7 0 0 0 X g for 15 min to deposit the acidprecipitated, undigested chromatin. The supernatants were then aspirated off and the amount of DNA hydrolyzed was determined by measuring the absorbance at 260 nm. Results

The transcriptive capacity of chromatin using homologous RNA polymerase II decreased progressively with advancing age (Fig. 2); most of the decline occurred within the first three weeks of age and was also apparent when re-initiation was prevented {Fig. 3) indicating a decrease in the number of transcription initiation sites on the chromatin. Using the Cedar-Felsenfeld procedure [ 17], the amount of chromatin DNA required to achieve maximum polymerase activity increased from 5 ug at day 5 to 10 pg at 6 months of age, indicating a 100% decrease in the number of transcription initiation sites. The possibility of changes in chromatin-associated proteins as an explanation for the altered transcriptive capacity was then examined. As shown in Table I, the protein/ DNA ratio increased with advancing age as a consequence of enrichment with histones. The non-histone proteins as a whole did not show any significant quantitative changes. It was of interest that, although the molar percentages of the four histones H2A, H2B, H3 and H4 were retained throughout development, that of histone HI, the very lysine-rich histone, increased progressively with age (Fig. 4). Polylysine titration. The restriction in chromatin template activity with age suggested an alteration resulting in fewer DNA sequences available for transcription. This conclusion was re-inforced by the polylysine titration data of

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Fig. 5 which show that chromatin from later ages bound less polylysine. Based on the midpoint of chromatin precipitation curves, myocardial chromatin bound 60% more polylysine at age 5 days compared to 6 months. These results indicate progressive loss of open DNA regions in chromatin which would be available for transcription. The deoxyribonuclease digestion results (Fig. 6) also support a progressive unavailability o f DNA for gene expression. The dose-response curve for DNAase I is shifted to the right as age advances indicating increasing resistance to endonuclease attack. These data, in turn, may be interpreted as showing alterations in protein-DNA interactions in myocardial chromatin during postnatal development. This is reflected in the thermal denaturation profiles {Fig. 7). The derivative plots of the melting profiles can be separated into three bands which,

TABLE I CHROMATIN PROTEIN AND DNA CONTENT CARDIAL DEVELOPEMENT

AT VARIOUS

AGES DURING POST-NATAL MYO-

S e p a r a t i o n o f c h r o m a t i n - a s s o c i a t e d p r o t e i n s w a s carried o u t using B i o - R e x 7 0 c h r o m a t o g r a p h y , as d e s c r i b e d in t h e t e x t . T h e a m o u n t o f p r o t e i n w a s a s s a y e d b y t h e m e t h o d o f L o w r y et al. [ 4 4 ] a n d D N A b y t h e d i p h e n y l a m i n e r e a c t i o n [ 4 5 ] . R e s u l t s r e p r e s e n t averages for s e v e n d i f f e r e n t d e t e r m i n a t i o n s in e a c h age g r o u p . * P < 0 . 0 5 , ** P < 0 . 0 1 , c o m p a r e d t o age 5 days. Age (days)

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according to Li and Bonner [18] can be assigned as follows: melting band I at approximately 60°C corresponds to DNA regions bound by non-histone proreins and/or very short DNA gaps between histone-bound regions, while bands II (at 75°C) and III (at 90°C) represent regions bound only by histones. The data in Fig. 7 show a decrease in the area of melting band I with concomitant increase in the areas of melting bands II and III as age advances. Apparently, a shift occurs in a small amount of DNA originally bound to non-histone proteins towards that bound by histones; this shift could partially account for the changes in template activity. Circular dichroism spectra of myocardial chromatin at different ages (Fig. 8) showed a progressive decrease in positive ellipticity between 250--300 nm as age advanced indicating conformational changes in DNA-bound proteins. These conformational results and those obtained from thermal denaturation studies

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394 are evidence of structural modification of myocardial chromatin during post° natal development. Discussion Two processes take place in the developing myocardium during.the first few weeks of postnatal life. One is the decrease in the number of cells undergoing mitosis and is reflected in the gradual decrease in [3H]thymidine incorporation [2,3]. This is probably a process c o m m o n to all organs as they achieve their full complement of cells (which may be genetically determined). The second process concerns the rapid loss by myocardial cells of their potential for mitosis in response to appropriate stimuli. This is a change with profound functional implications for the heart and has not been adequately studied. Although the activities of key enzymes concerned with DNA synthesis, such as DNA polymerases and thymidine kinase, decline during postnatal cardiac development [21--24] this is n o t adequate explanation for the loss of myocardial proliferative capacity. For example, at 3--4 weeks of age, rat myocardial cells still have the ability to proliferate in response to cardiac overload [25-27]. In contrast, adult myocardial cells subjected to the same stimuli cannot undergo mitosis despite no interim change in DNA polymerase activity [24] to account for this difference. Clearly, other mechanisms must account for the age-dependent loss of myocardial mitotic potential. We have sought to gain more information about these mechanisms by exploring the possibility of chromatin changes during development. Studies in other systems have indicated that chromatin structure and function are exceedingly sensitive to changes in the proliferative state of the cell [7--11]. Chromatin from resting cells stimulated to proliferate has increased template activity in the pre-replicative phase, indicative of adaptations preparatory to mitosis [8,28,29]. DNA synthesis and subsequent progression to mitosis are dependent on RNA and protein synthesis in the prereplicative phase [8,28] and this may explain the increased chromatin template activity. The latter is associated with structural changes in chromatin, namely, an increase in positive eUipticity in the 250--300 n m region of circular dichroism spectra [9] and an increase in ethidium bromide or polylysine binding capacity [30,31]. There is a close correlation between capacity to bind intercalating dyes and enhanced transcriptive ability of "activated" chromatin [28] which may indicate a higher number of binding sites for RNA polymerase. The decrease in thermal stability, another chromatin change regularly observed in proliferating or stimulated cells [10], points to an alteration in the interaction between DNA and chromatinassociated proteins. Since unmodified histones are generally repressors of gene transcription [34], the high histone/DNA ratio in the adult myocardium may provide a partial explanation for the decreased transcriptive capacity of maturing ~myocardial chromatin. The data in Fig. 4 demonstrate that four of the histones (H2A, H2B, H3 and H4) remained in equimolar proportions throughout development although their total amount per unit DNA increased. This suggests that they remain together in a nucleosome-like structure. On the other hand, HI behaved independently; as the myocardium reached adulthood, the relative proportion

395 of H1 increased progressively. Since H1 has been proposed as modifier of DNAhistones interactions [35], this behavior may be more than coincidental. It should be noted, however, that our results do not give any information about functional modifications of H1 (such as phosphorylation) which may control its effect on gene expression. A direct implication of these observations is that the accessibility of DNA in chromatin to endonucleases should decrease during post-natal development, and this was indeed noted. This was indicated both by the results of the DNAase I digestion and the binding to polylysine. Histone H1 is assumed to help in condensing chromatin [35] and its increase during maturation may contribute largely to the functional inactivation of the myocardial genome. The increase in the histone/DNA ratio in the developing myocardium and decrease in DNA accessibility in chromatin can best be interpreted as an increase in nucleosome number along the DNA fiber. This suggests that although nucleosomes are clearly associated with actively transcribed genes [7,32], nucleosome density along the DNA fiber may be a crucial parameter in controlling gene expression. Such a dramatic change can probably be demonstrated only when cell division is required for differentiation and when a large number of genes have to be switched off and on. For example, recent data indicate that the differentiation of the avian red blood cell is accompanied by a progressive packaging of more and more histones along the DNA until it is almost inactivated [8,9]. The reverse situation prevails during the activation of a repressed genome in the germinating pea embryo [36]. These histone changes are very likely associated with modifications in nuclear nonhistone proteins. The onset' of DNA synthesis and subsequent mitosis in a variety of systems activated by appropriate stimuli are usually preceded by increased synthesis and/or phosphorylation of nuclear nonhistone proteins [37--41]. Changes in the proliferative capacity during differentiation and development are also associated with similar changes in NHPs [42]. Although the total amount of myocardial nonhistone proteins did not substantially change during development, variations in individual key nonhistone proteins would be undetected and require resolution of this nuclear fraction into individual proteins. This type of analysis is now in progress in our laboratory. That nonhistone protein modifications do occur is indicated by changes in the derivative melting profiles of maturing chromatin which indicate a shift from nonhistone to histone-associated components and by our recent finding of decreased nonhistone protein-associated nuclear protein kinases during postnatal development (unpublished observations). Our results suggest that significant changes occur in the structure and template activity of myocardial chromatin during a period of restriction in proliferative capacity. Despite this temporal correlation, we have no direct evidence that the chromatin changes are causally related to the decline in mitotic potential; this possibility is only made likely by the fact that the reorganization of myocardial chromatin results in progressive repression of genome expression with aging and may, thus, contribute to the loss of proliferative capacity. Alternatively, the chromatin changes described in this report may simply refect the transition from "active" to "inactive" state as an adaptation secondary to mitotic quiescence. Definitive choice between these alternatives will require further studies.

396

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