Relationship between ribonucleoprotein particles containing heterogeneous RNA and ultrastructure and function of chromatin in purified rat hepatocyte nuclei

JOURNAL OF ULTRASTRLICTURE RESEARCH 77, 6 6 4 2 (1981)

Relationship between Ribonucleoprotein Particlea Containing Heterogeneous RNA and Ultrastructure and Function of Chromatin in Purified Rat Hepatocyte Nuclei MASSIMO DERENZINI, ANNALISA PESSION-BRIZZI, AND FRANCESCO NOVELLO

Istituto di Patologia Generale, Via S. Giacomo, 14, 40126 Bologna, Italy Received November 20, 1980, and in revised form June 10, I981 Chromatin of isolated nuclei from regenerating rat hepatocytes, 24 hr after partial hepatectomy, appeared mainly in the dispersed form. Exogenous ribonuclease (RNase) digestion of purified nuclei induced a marked condensation of extranucleolar chromatin, a quantitative reduction of ribonucleoprotein (RNP) perichromatin fibrils, and a solubilization of the heterogeneous nuclear (hn) RNA and of the small-molecular-weight (smw) RNAs contained in the RNP fibrils. Incubation of purified nuclei at 37°C for 30 rain, when endogenous RNase was active, also caused a marked condensation of chromatin, but only a slight solubilization of rapidly labeled hnRNA without affecting the smwRNAs in the RNP fibrils. Both control and enzyme-treated nuclei presented the same quantity of RNA pulymerase II molecules and the same transcriptional activity.

Chromatin in eucaryotic cells has a very dynamic structural arrangement and its changes appear to be closely associated to the modification of gene activity. The relationship between structure and function of chromatin still needs to be clarified: namely we do not know whether the structural-ultrastructural changes of chromatin are a prerequisite or only a consequence of gene activity. In previous studies (Derenzini et al., 1978, 1979) we have demonstrated that the chromatin condensation induced in regenerating rat liver or in cortisol-stimulated rat hepatocytes by o~-amanitin, an inhibitor of RNA polymerase II (Lindel et al., 1970) occurred without any modification of the synthesis of nonhistone nuclear proteins and of their phosphorylation and was not dependent on the quantity of cortisol bound to the chromatin. This chromatin change, i. e., the shift from the dispersed state to the condensed form, was not strictly related to the inhibition of RNA synthesis but it was parallel with the progressive disappearance of the perichromatin fibrils which are the morphological substrate of the newly synthesized heterogeneous nuclear (hn) RNA (Bachellerie et al., 1975; Fakan et

al., 1976). The importance of RNP fibrils in

determining the chromatin ultrastructure has been stressed by the observation that ribonuclease (RNase) treatment induced, in isolated rat hepatocyte nuclei, a condensation of chromatin, a solubilization of hnRNA, and a reduction of the quantity of perichromatin fibrils (Derenzini et al., 1980). These results led us to suggest that the chromatin ultrastructural morphology was determined by the quantity of RNP particles containing hnRNA closely associated to the deoxyribonucleoprotein (DNP) complex. In the present paper we have further investigated chromatin condensation caused by RNase action in purified nuclei from regenerating rat hepatocytes. The aim of this work was, first, to detect which component of the RNP particles containing hnRNA was involved in determining the chromatin ultrastructure bearing in mind that in these RNP particles some small-molecular-weight (stow) RNAs have been demonstrated (Gallinaro and Jacob, 1979; Seifert et al., 1979) to be linked to the nuclear skeleton (Miller et al., 1978a,b). Second, we looked for whether the shift from the dispersed state of chromatin in purified regenerating he66

0022-5320/81/100066-17502.00/0 Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

RNP FIBRILS A N D CHROMATIN U L T R A S T R U C T U R E A N D F U N C T I O N

patocyte nuclei to the condensed form after RNase digestion was associated with a loss of the substances involved in the transcriptional machinery. MATERIALS AND METHODS

Animals. Male Wistar rats, weighing approximately 120 g fed ad libitum on stock laboratory diet, were partially hepatectomized under ether anesthesia according to Higgins and Anderson (1931). Rats were killed 24 hr after partial hepatectomy. Nuclear RNA was labeled by intraperitoneal injection of [6-14C]orotic acid (3/xCi/100 g body wt) 20 rain before killing. Chemicals. [6-14C]Orotic acid (sp act 58 mCi/ mmole), [3H]UTP (15 Ci/mmole) were obtained from the Radiochemical Center Amersham, Buckingamshire. RNase-free sucrose was from Schwarz/Mann or Bio-Rad. Ribonucleoside triphosphates, phenylmethylsulfonyl fluoride (PMSF), DNase-free RNase from bovine pancreas, and RNase-free DNase I from bovine pancreas were obtained from Sigma Company. RNase solution (2 mg/ml in distilled water) was incubated at 70°C for 15 min. Inactivated RNase was prepared according to Hits (1955), DNase was treated with iodoacetic acid (Zimmermann and Sandeen, 1966). ~-Amanitin and O-[3H]methyl-demethyl-3,-amanitin (sp act 2.4 Ci/mmole) were a generous gift of Professor H. Faulstich (Heidelberg). Sodium tetrathionate was purchased from Fluka. Preparation o f purified nuclei and o f subnuclear particles. Livers were homogenized with 9 vol of 0.25 M sucrose containing buffer O (Chevaillier and Philippe, 1973) (25 mM KCI, 0.9 mM MgCI2, 0.9 mM CaCI2,0.14 mM spermidine, 5 mM Tris-HC1, pH 7.5), 0.1 mM PMSF. All subsequent operations were performed at 0°C. After a 10-min centrifugation at 2000 g, the crude nuclear pellets were suspended in 2.2 M sucrose, buffer O, 0.1 mM PMSF. After centrifugation at 33 000 g for 60 rain, the nuclear pellets were resuspended in 0.25 M sucrose, buffer O, 0.1 mM PMSF. After centrifugation at 2000 g for 10 min purified nuclei were suspended in the same medium (1 ml/g of tissue). Samples of purified nuclei (0.4 mg DNA) were incubated in a medium containing in a final volume of 2.8 ml : 0.25 M sucrose buffer O in the presence or in the absence of RNase, 80 tzg/sample. After incubation at 20°C for 10 min, perchloric acid (PCA) was added to 0.7-ml samples to a final concentration of 0.3 N. Untreated samples were precipitated with PCA without incubation at 20°C. In some experiments samples of nuclei were incubated at 37°C for 30 rain in the same medium without added pancreatic RNase but in the presence of 1 mM PMSF and 0.1 mM Na-tetrathionate to inhibit the release of RNA from nuclear skeleton (Berezney, 1979; Miller et al., 1978a).

67

Subnuclear particles were isolated from purified nuclei by sonication and differential centrifugation (Derenzini et al., 1979). Evaluation o f transcription in purified nuclei. From regenerating liver of rats not injected with labeled orotic acid, crude nuclear pellets were prepared in 0.25 M sucrose, buffer O and incubated in the presence or absence of RNase as described above for purified nuclei without 0.1 mM PMSF. After incubation at 20°C, the crude nuclear preparation was diluted with 40 ml of 0.25 M sucrose buffer O and centrifuged at 2500 g for 10 min. The pellet was washed three times with 40 ml of the same medium and nuclei were then purified from 2.2 M sucrose as above reported and resuspended in 0.25 M sucrose, 3 mM MgC12. Nuclear ultrastructure was determined by electron microscopy before incubation. To measure the incorporation of labeled precursor into RNA, samples of nuclei (60 /xg DNA) were incubated for 10 rain at 25°C in the presence of 100 mM Tris-HC1, pH 8.0, 4 mM MnC12, ATP, GTP, and CTP, each 1.8 raM, 0.1 mM UTP, and 1.5 ~Ci of [3HIUTP. Transcription inhibitors were tested for their effects on RNA synthesis without preincubation. Rifampicin derivative AF/O-13 was dissolved in a small quantity of dimethyl sulfoxide (DMSO) before being added to the assay solution. The reaction was stopped with 10% trichloroacetic acid (TCA). The precipitate was collected on Whatman GF/C filters and washed with 5% TCA. After treatment with Soluene 350 (Packard), the radioactivity was measured following the addition of scintillation fluid. The binding of [~H]amanitin to RNA polymerase I1 in purified nuclei was measured according to CochetMeilhac et al. (1974) in the presence of 4 × 10-2 /xg/ 0.5 ml of O-[~H]methyl-demethyl-3,-amanitin. Extraction and analysis o f R N A . From purified nuclei RNA was extracted and fractionated by treatment with phenol at 50 and 85°C (Hadjiolov et al., 1974; Markov and Arion, 1973). The RNA fractions, designated 50 and 85°C RNA, corresponded to nuclear sapnucleolar RNA and to hnRNA, respectively (Hadjiolov et al., 1974). For the isolation of smwRNAs, R N A was extracted from purified nuclei with the phenol-cresol method as described by Miller et al. (1978b) or at 85°C in the presence of 0.5% sodium dodecyl sulfate (Markov and Arion, 1973). Nucleic acids were precipitated with 2.5 vol of ethanol at -20°C and were dissolved in a buffer solution containing 0.01 M Tris-HCl, pH 7.6, and 0.01 M MgC12. DNase I, treated with iodoacetic acid (Zimmermann and Sandeen, 1966), was added to a final concentration of 100/xg/ml and incubated at 30°C for 10 rain. The RNA was extracted with phenol, precipitated with cold ethanol, and then dissolved in distilled water. After the precipitation of high-molecularweight RNAs (Hadjiolov et al., 1974), smwRNAs were analyzed by polyacrylamide gel electrophoresis

68

DERENZINI, PESSION-BRIZZI, AND NOVELLO

u n d e r nondenaturing or denaturing conditions (Hodnett and B u s c h , 1968; Miller et al., 1978b). A b o u t 50/xg of R N A were a d d e d per gel and electrophoresis was performed at 5 m A . In a parallel gel 5 S and 4 S cytoplasmic R N A s were analyzed. Gels were cast in quartz tubes and s c a n n e d at 260 n m in a Gilford spectrophotometer. T h e s m w R N A s were e n u m e r a t e d according to their relative mobilities with respect to 5 S 4 S R N A s (Miller et al., 1978b). Other determinations. R N A was solubilized and m e a s u r e d according to M u n r o and Fleck (1966). The radioactivity of samples and of gel slices was measured as previously reported (Novello et al., 1978). D N A was determined by the m e t h o d of Burton (1956) with calf t h y m u s D N A as standard. Electron microscopy. H e p a t o c y t e nuclei from the s a m e preparations used for biochemical experiments were immediately fixed in a solution of 4% formaldehyde and 2.5% glutaraldehyde in 0.1 M S6rensen buffer, p H 7.2. All the samples were dehydrated in alcohol and e m b e d d e d in an A r a l d i t e - E p o n mixture. Ultrathin sections were obtained with an U l t r a t o m e III L K B m i c r o t o m e , u s i n g a d i a m o n d knife. T w o different staining p r o c e d u r e s were used: (a) double staining with uranyl acetate and lead citrate and (b) preferential staining for ribonucleoproteins with a uranyl a c e t a t e E D T A - l e a d m e t h o d according to B e r n h a r d (1969). The sections were e x a m i n e d with a Siemens Elmiskop 102 electron microscope, at 80 kV, with an objective aperture of 50/zm. RESULTS

B i o c h e m i c a l Evaluation

Purified nuclei from regenerating rat liver were prepared in experimental conditions which preserved all the nuclear structures (Chevaillier and Philippe, 1973); they were

then treated with RNase at the concentrations of 20 /xg/0.1 mg DNA. In the same experiment, from control and RNase-treated nuclei, a subnuclear fraction was prepared enriched in hnRNA particles (Bachellerie et al., 1975) which were equivalent to the so-called perichromatin fibrils in situ (Bachellerie et al., 1975; Fakan et al., 1976). In purified nuclei (Table I) the RNA specific activity of RNase-treated nuclei decreased to 22% of control level and in the isolated subnuclear particles to 15%. The RNA/DNA ratios were 0.46 and 0.33, respectively, for the subnuclear fractions from control and RNase-digested nuclei. The lower specific activity and the decreased RNA/DNA ratio confirmed a large degradation, by added RNase, of rapidly labeled hnRNA, isolated as the ribonuclear particle DNA complex. The smwRNAs were extracted with phenol from nuclei incubated at 20°C, with and without RNase, and analyzed on 10% polyacrylamide gel under denaturing conditions in the presence of 8 M urea (Miller et al., 1978b) (Fig. 1). The pattern of smwRNAs from control nuclei was very similar to that reported by Miller et al. (1978b) for normal liver nuclei, while after RNase digestion none of the different types of smwRNAs could be detected and a large quantity of material absorb-

TABLE I EFFECT OF RNAsE DIGESTION ON R N A CONTENT AND RADIOACTIVITY IN PURIFIED NUCLEI AND SUBNUCLEAR PARTICLES AND ON THE BINDING OF [aH]AMANITIN TO NUCLEI Nuclei

Subnuclear particles

Incubation for 10 min at (°C) 0 20 20 20

RNase

/zg R N A / /~g D N A

Specific activity ~

/zg R N A / /xg D N A

Specific activity a

+ +c

0.31 0.28 0.18 0.27

1271 1165 267 1247

ND 0.46 0.33 ND

ND 8583 1292 ND

[ZH]Amanitin b o u n d to nuclei (cprn//xg DNA) b ND 2.58 _+ 0.23 2.47 _+ 0.32 ND

a The incorporation into R N A is e x p r e s s e d as specific activity [(dpm/mg RNA)//zg DNA]. ND, not determined. b Results are m e a n s of three experiments +_ SEM. ND, not determined. c R N a s e was inactivated by the m e t h o d of Hirs (1955).

RNP FIBRILS AND CHROMATIN ULTRASTRUCTURE AND FUNCTION

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for I0 min without R N a s e ; (b) nuclei were incubated with R N a s e as in (a). R N A was extracted as explained u n d e r Materials and Methods. The samples (50 ~ g R N A ) were denatured (Miller et al., 1978b) and electro-

phoresis was performed in 10% polyacrylamide gels containing 8 M urea for twice the time it took bromophenol blue to r u n off the gel. Gels were s c a n n e d at 260 n m () and radioactivity was counted ( e - e - e ) ; s m w R N A s were e n u m e r a t e d according to Miller et al. (1978b).

ing at 260 nm was recovered toward the end of the gel. Only a small quantity of radioactivity was detectable in gels containing smwRNA from control nuclei while larger quantities of labeled RNA were found in the very low-

molecular-weight fractions after RNase digestion, suggesting that some of these RNAs were produced by the action of RNase on rapidly labeled RNA. Because smwRNAs have the property of being highly resistant to endogenous ribo-

70

DERENZINI, PESSION-BRIZZI, AND NOVELLO 0,6

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FIG. 2. Effect of e n d o g e n o u s ribonuclease on nuclear s m w R N A s . (a) Nuclei were incubated at 0°C for 30 rain; (b) nuclei were incubated at 37°C for 30 min. After the t r e a t m e n t of nuclei with D N a s e (see text), R N A was extracted at 85°C in the p r e s e n c e of 0.5% s o d i u m dodecyl sulfate (Markov and ANon, 1973). Electropboresis was performed u n d e r nondenaturing conditions for 3 hr. Gels were s c a n n e d at 260 n m ( ); s m w R N A s were e n u m e r a t e d according to Miller et al. (1978b).

nuclease digestion at 37°C (Miller et al., 1978b; Seifert et al., 1979) we incubated purified nuclei in this experimental condition. The degradation of rapidly labeled RNA was analyzed: as shown in Table II, a 19% decrease in RNA specific activity was detectable in the acid-precipitated material after the incubation of samples at T A B L E II EFFECT OF ENDOGENOUS RIBONUCLEASE ON THE SPECIFIC RADIOACTIVITY OF TOTAL NUCLEAR R N A AND OF R N A FRACTIONS Incubation for 30 min at (°C) ~

Nuclear RNA

50°C R N A

85°C R N A

b 0 37

266 260 212

ND 208 202

ND 324 247

R N A fractions a

N o t e . Results are e x p r e s s e d as specific radioactivity, dpm//xg R N A . N D , not determined. In another e x p e r i m e n t nuclear R N A was fractionated by t r e a t m e n t with phenol at different temperatures (Hadjiolov et al., 1974); see also Materials and Methods. b Purified nuclei before the addition to the incubation m e d i u m .

37°C for 30 min. Nuclear RNA was fractionated by treatment with phenol at different temperatures; as reported in Table II, after the incubation of purified nuclei at 37°C for 30 min, the specific radioactivity (dpm//~g RNA) of the 50°C RNA was unchanged while that of 85°C RNA was decreased by 21% with respect to the corresponding fraction extracted from nuclei kept at 0°C for 30 min. This result suggested that 85°C RNA, corresponding to hnRNA (Hadjiolov et al., 1974; Markov and Arion, 1973), was the fraction of rapidly labeled nuclear RNA mainly affected by the incubation at 37°C. The smwRNAs were therefore extracted with phenol at 85°C in the presence of 0.5% SDS (see Materials and Methods). To assure better extraction of RNA tightly bound to DNA, nuclei were previously incubated with iodoacetatetreated DNase (250 /xg/ml) for 10 rain at 30°C, so that more than 99% of DNA and RNA were recovered in the aqueous phase after phenol extraction. The s m w R N A s were a n a l y z e d on 10% acrylamide gels. In comparison with nuclei

71

RNP FIBRILS AND CHROMATIN ULTRASTRUCTURE AND FUNCTION T A B L E lII [~H]UMP INCORPORATION INTO PURIFIED LIVER NUCLEI AFTER THE INCUBATION OF CRUDE NUCLEAR PELLET IN THE PRESENCE OR THE ABSENCE OF RNAsE Control nuclei (dprrd/xg DNA)

Addition

0

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0.16 M 0.24 M (NH4)2SO 4 (NH4)2SO 4

None

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34

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42

40

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87

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61

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60

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49

40

68

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Rifampicin AF/O-13 (50/xg/ml)

47

47

68

68

53

36

76

76

Note. For isolation of purified nuclei and experimental conditions see Materials and Methods.

kept at 0°C, all the species of smwRNAs were present after incubation at 37°C although there was an increase of types migrating in the region of 4 S RNAs (Fig. 2). Similar results were obtained when the DNase treatment was omitted before phenol extraction at 85°C in the presence of 0.5% SDS. These data suggested that in nuclei from regenerating rat liver smwRNAs were resistant to endogenous ribonuclease activity as in isolated nuclei and ribonucleoprotein particles from normal liver (Miller et al., 1978b; Seifert et al., 1979). Evaluation of Transcriptional Activity of RNase-Treated Nuclei The number of RNA polymerase II molecules was determined by the binding of 3H-labeled y-amanitin (Cochet-Meilhac et al., 1974): the results (Table I) showed no significant difference in the quantity of the labeled inhibitor bound to purified nuclei after incubation in the presence or absence of RNase. Nevertheless, Cochet-Meilhac et al. (1974) have clearly demonstrated that amanitin and RNA polymerase II bind in 1: l molar ratio and that the binding was not affected by the ionic strength of the medium; this method however cannot discrimi-

nate between functionally active and inactive (unbound) enzyme. We therefore evaluated the functional activity of RNA polymerase II both in control and in RNase-digested nuclei. In a preliminary experiment, data not shown, with the assay system used, the incorporation of labeled precursor into a TCA-insoluble product was inhibited by more than 95% using actinomycin D and RNase. The use of RNase digestion on a crude nuclear pellet was adopted because the repeated washing of the pellet and the purification of nuclei produced a better removal of added RNase while perfectly preserving all the nuclear structures. The results (Table III) showed that no significant difference could be detected in the incorporation of the labeled precursor into TCA-precipitable products at the different ionic strengths used. Some inhibitors of RNA synthesis were also used. Rifampicin derivative AF/O-13 did not inhibit the synthesis of RNA and in the presence of heparin an increased incorporation into RNA was obtained. Both these substances inhibit free but not transcribing RNA p o l y m e r a s e s (Coupar and Chesterton, 1977; Cox, 1973; Meilhac et al., 1972) and

72

DERENZINI, PESSION-BRIZZI, AND NOVELLO

the stimulating effect of polyanions on nuclear and chromatin transcription has been well d o c u m e n t e d (Coupar and Chesterton, 1977; Cox, 1973; Novello and Stirpe, 1969). The latter effect was due to an increase in the p o l y r i b o n u c l e o t i d e e l o n g a t i o n r a t e (Coupar and Chesterton, 1977). c~-Amanitin (0.5 /xg/ml) inhibited the incorporation of the labeled p r e c u r s o r at 0.16 and 0.24 M a m m o n i u m sulfate; at lower salt concentration R N A p o l y m e r a s e II in isolated nuclei has been shown to be barely active (Coupar and Chesterton, 1977; Novello and Stirpe, 1969). The effects of heparin, rifampicin, and ~-amanitin on R N A synthesis were similar in control and R N a s e - t r e a t e d nuclei. The same binding of [~H]amanitin by nuclei and the lack of difference in R N A synthesis in vitro indicated that the total number of R N A p o l y m e r a s e II molecules, the initiated t r a n s c r i p t i o n a l c o m p l e x e s , and factors concerned with R N A synthesis in vitro were unaffected by the R N a s e treatment.

Electron Microscopical Finding We have considered as controls, in the e x p e r i m e n t s p e r f o r m e d with e x o g e n o u s R N a s e , the purified nuclei which have been treated in the same manner as those digested by R N a s e with the exception that inactivated R N a s e replaced the active enz y m e . The inactive e n z y m e did not affect R N A specific activity (Table I) even increasing the quantity of inactive R N a s e up to five times (data not reported). In sections which were stained with uranium and lead nearly all the chromatin of control nuclei appears in a loosened pattern. Condensed chromatin is clearly visi-

ble only closely associated to the inner nuclear m e m b r a n e (Fig. 3). In Fig. 4 purified nuclei after R N a s e digestion are shown: the fairly h o m o g e n e o u s diffusion of the dispersed chromatin in the control nuclei is no longer visible. Large clumps of c o m p a c t chromatin are then present either at the nuclear periphery or scattered throughout the nucleoplasm. In Figs. 5 and 6 control and R N a s e - d i g e s t e d nuclei are, r e s p e c t i v e l y , shown at higher magnification. In the control nucleus also the chromatin lining the inner nuclear m e m b r a n e appears in a loosened form; the dispersed chromatin spreads to almost e v e r y part of the nucleoplasm thus becoming indistinguishable from the nonchromatin structures in the interchromatin spaces. In the large nucleolus the normal~ trabecular structure of the nucleolonema, where granules and fibrils normally intermingle, is perfectly visible. In ribonuclease-digested nuclei, because of m a r k e d chromatin condensation and solubilization of R N P c o m p o n e n t s , the interchromatin spaces a p p e a r completely devoid of any electron-opaque structure (Fig. 6). Also the nucleolar architecture is greatly modified: the nucleolonema can no longer be singled out, the fibrillar c o m p o n e n t appears to be reduced and m a n y vacuoles are then present in the c o m p a c t nucleolus. With the u r a n y l - E D T A - l e a d staining method, a large quantity of perichromatin fibrils is revealed, in the control nuclei; these R N P s t r u c t u r e s are visible either around the areas of unstained chromatin or scattered throughout the nucleoplasm without any detectable relationship with the bleached chromatin (Fig. 7). The dispersed chromatin is in fact hardly recognizable, in

FIG. 3. Isolated nuclei from regenerating rat hepatocytes incubated in the presence of inactive RNase at the concentration of 20/xg/0.1 mg DNA (control nuclei). Chromatin appears to be mainly in the dispersed form and is spread throughout the nucleoplasm. Only little clumps of condensed chromatin are visible. Aldehyde fixation; uranium and lead staining, x 15 600. Fro. 4. Hepatocyte nuclei treated as in Fig. 3 except that active RNase replaced the inactive enzyme in the incubation mixture (RNase-treated nuclei). All the chromatin is condensed in large, very electron-opaque masses. Numerous electron-translucent spaces are now visible in the nucleoplasm. Aldehyde fixation; uranium and lead staining, x 15 600.

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RNP FIBRILS AND CHROMATIN ULTRASTRUCTURE AND FUNCTION contrast to the c o m p a c t chromatin. The large amount of perichromatin fibrils, tog e t h e r with the o t h e r r i b o n u c l e o p r o t e i n c o m p o n e n t s , c o n f e r s a diffuse e l e c t r o n opacity to the nucleoplasm. In the large nucleolus, granules and fibrils are present in the normal structure of nucleolonema. After the R N a s e digestion the quantity of perichromatin fibrils appears to be markedly reduced. Small clusters of these structures are visible closely a s s o c i a t e d with the masses of c o m p a c t bleached chromatin (Fig. 8). Due to the reduction of the quantity of the perichromatin fibrils, nucleoplasmic spaces are now electron translucent. Also, the nucleolar structure appears to be greatly modified; the fibrillar c o m p o n e n t is rarely visualized, whereas granules are frequently o b s e r v e d in small clusters which are separated b y large septa of nucleolus-associated bleached chromatin. As regards the experiments concerned with the action of endogenous R N a s e , we consider, as controls, those nuclei incubated for 30 min in the p r e s e n c e of p r o t e a s e inhibitors at a t e m p e r a t u r e of 0°C instead of a t e m p e r a t u r e of 37°C that has been used for the activation of endogenous R N a s e . The ultrastructural pattern of control nuclei is the same as that described for control nuclei in the experiments with exogenous R N a s e , either using uranium and lead staining or after the regressive E D T A procedure. The incubation of nuclei at 37°C, h o w e v e r , causes a m a r k e d condensation of chromatin (Fig. 9); large clumps of c o m p a c t chromatin are present throughout the nuclear space and along the nuclear m e m -

75

brane (Fig. 10). Agglomerates of electronopaque structures, likely of R N P nature, are visible in the i n t e r c h r o m a t i n spaces where, m o r e o v e r , thin threads are found to c o m p o s e a l o o s e n e d n e t w o r k (Fig. 11). These latter structures are no longer visible in s e c t i o n s s t a i n e d with the u r a n i u m E D T A - l e a d m e t h o d (Fig. 12). On the other h a n d , this staining p r o c e d u r e r e v e a l s a large quantity of ribonucleoprotein particles either associated with the clumps of condensed bleached chromatin or apparently detached f r o m these. In the nucleolus the granular and fibrillar c o m p o n e n t s are o b s e r v e d to f o r m trabecular structures separated from each other by compact, bleached chromatin. DISCUSSION The p r e s e n t results d e m o n s t r a t e d that the solubilization of high-molecular-weight, newly synthesized, hnRNA induced a m a r k e d c o n d e n s a t i o n of e x t r a n u c l e o l a r chromatin in purified nuclei f r o m regenerating rat liver. This chromatin change was not a s s o c i a t e d with modification of the quantity of R N A p o l y m e r a s e I I molecules or with the loss of the substances involved in the transcriptional machinery.

(1) Ribonucleoprotein Particles Containing h n R N A and Extranucleolar Chromatin Ultrastructure The importance of these ribonucleoprotein particles in determining the chromatin ultrastructural pattern has been previously stressed b y ,the following observations: (1) the so called dispersed chromatin a p p e a r e d to be mainly associated with perichromatin

FIG. 5. Control nucleus. Chromatin forms a loosened network in the nucleoplasm. In the nucleolus the trabecular structure of the uucleolonema, in which the granular and the fibrillar components normally intermingle, is perfectly visible. Aldehyde fixation; uranium and lead staining, x 55 000. FIr. 6. RNase-treated nucleus. All the chromatin is condensed in compact clumps. In the nucleoplasm, the interchromatin spaces are devoid of any structure. In the very compact nucleolus, the fibrillar component is not clearly detectable and many vacuoles are present. Aldehyde fixation; uranium and lead staining. × 55 000.

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RNP FIBRILS AND CHROMATIN ULTRASTRUCTURE AND FUNCTION

RNP fibrils (Derenzini et al., 1977); (2) chromatin condensation induced in regenerating rat hepatocytes and in cortisol-stimulated hepatocytes by a-amanitin was not dependent on the synthesis of RNA and of the nonhistone nuclear proteins and their phosphorylation whereas a close relationship between the degree of chromatin condensation and the reduction of perichromatin fibrils was noticed (Derenzini et al., 1978, 1979); (3) RNase treatment of isolated nuclei induced a marked condensation of chromatin with a concurrent reduction of p er ich r o mat i n fibrils (Derenzini et al., 1980). The present experiments have demonstrated that RNase treatment, which induced the condensation of chromatin, solubilized most of the rapidly labeled hnRNA present in the subnuclear fraction containing hnRNA particles (Bachellerie et al., 1975) which are analogous to the perichromatin fibrils, first observed in situ by Monneron and Bernhard (1969). Accordingly, at ultrastructural level, the quantity of perichromatin fibrils was strongly reduced in enzyme-digested nuclei. However, these R NP particles contain smwRNAs and hnRNA (Gallinaro and Jacob, 1979; Seifert et al., 1979); some of these RNAs have been demonstrated to be linked to the nuclear skeleton (Miller et al., 1978b) and their involvement in the ultrastructural organization of chromatin cannot be excluded. However, the observation that chromatin condensation occurred in nuclei incubated at 37°C for 30 min, a condition in which the endogenous RNase digested some hnRNA but not the smwRNAs (Fig. 2), excluded

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that this chromatin change could be due to the solubilization of these latter types of RNA. The same experiment showed that chromatin condensation occurred concurrently with a solubilization of a small amount of hnRNA of high specific radioactivity which corresponded to the newly synthesized molecules. This finding was consistent with the observation that the quantity of RNP fibrils, revealed by uranylEDTA-lead method, was not as greatly reduced as in exogenous RNase-digested nuclei. It is worth noting that in these conditions the uranyl and lead salt staining revealed a fairly electron-opaque fibrillar material in the interchromatin spaces between the clumps of condensed chromatin. These structures were not visualized either in sections preferentially stained for RNP or in sections selectively stained for DNA according to Cogliati and Gautier (1973) (data not shown). Because the nuclei were incubated in the presence of 1 mM PMSF and 0.1 mM Na-tetrathionate, which has been reported to completely prevent the solubilization of the proteins of nuclear matrix (Berezney, 1979), it was very likely that the fibrillar material observed in these preparations might correspond to this latter nuclear structure. (2) Extranucleolar Chrornatin Condensation and Chromatin Transcriptional Machinery We first studied the relationship between the ultrastructural pattern of chromatin and the quantity of RNA polymerase II molecules attached to it. The possibility that the enzyme might induce some morphological

FIG. 7. Control nucleus. A large quantity of ribonucleoprotein fibrils are shown scattered throughout the nucleoplasm. The unstained chromatin appears in the dispersed form. In the large nucleolus both the granular and the fibrillar components are visible. Aldehyde fixation; uranyl-EDTA-lead staining. × 30 000. Fro. 8. RNase-treated nucleus. The quantity of ribonucleoprotein fibrils is very low. The unstained chromatin is condensed in large clumps. Electron-translucent spaces are present in the nucleoplasm. In the nucleolus (nu) only the granular component is visible. Aldehyde fixation; uranyl-EDTA-lead staining. × 30 000.

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RNP FIBRILS AND CHROMATIN U L T R A S T R U C T U R E A N D F U N C T I O N

change in the DNP complexes by becoming linked to chromatin cannot be ruled out considering: (1) that RNA polymerase II has been demonstrated to be associated in polytene chromosomes only with uncondensed and not with condensed chromatin (Jamrich, 1978) and (2) that the number of enzyme-transcribing molecules was increased in regenerating rat hepatocytes with respect to resting hepatocytes (Novello and Stirpe, 1970) which in contrast showed a higher quantity of condensed chromatin (Derenzini et al., 1976). Using [3H]amanitin we demonstrated that the total quantity of both chromatin-bound and nucleoplasmic RNA polymerase II molecules was not modified after ribonuclease digestion (Table I). Moreover in the presence of heparin the transcriptional activity was the same in control and RNase-treated nuclei (Table III). Because heparin affects the transcribing molecules of RNA polymerase II, while blocking the initiation of RNA synthesis by free enzymes (Coupar and Chesterton, 1977), our results demonstrated that the transcriptional activity was due to the enzyme complexed with chromatin and not to the free polymerase adventitiously bound to DNP. Our data therefore clearly showed that the RNase-induced extranucleolar chromatin condensation was not due to the detachment of RNA polymerase II molecules. It has been found that the dispersed chromatin contains a much higher amount of nonhistone proteins and phosphoproteins than the condensed form (Comings et al., 1977; Frenster, 1965). These substances have been postulated to play a role in gene

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activation by shifting the condensed, repressed chromatin to a more dispersed, active form (Allfrey, 1966; Jeter and Cameron, 1974; Paul, 1972). According to this model of control of gene transcription, chromatin condensation would imply a loss and/or an inactivation of the substances involved in the transcriptional activity. However our experiments demonstrated that the shift from a dispersed to a condensed form of chromatin, induced by RNase digestion, was not accompanied by a change in the transcriptional activity of nuclei tested in different experimental conditions for extranucleolar RNA synthesis. These data demonstrated therefore that the substances involved in gene transcription did not influence the chromatin ultrastructure at least as far as the organization level of chromatin considere~t in this work. (3) Proposed Mechanism for Gross Chromatin Dispersal and Condensation As far as the mechanism involved in the RNase-induced chromatin condensation was concerned, two possibilities have been considered. The transcriptionally active chromatin appeared to be mainly associated with perichromatin fibrils (Derenzini et al., 1977); these RNP particles could obviously separate to some extent the DNP fibers, from each other, thus loosening the condensed chromatin. Solubilization of the RNP particles by RNase would recompact the chromatin. Against this very simple explanation one objection could be raised: chromatin in actively transcribing nuclei appeared largely in the dispersed form whereas only a small part of the genome was transcriptionally active (Pederson,

FIG. 9. Isolated nuclei from regenerating rat hepatocytes, incubated at 37°C for 30 min. All the chromatin is in the condensed form. Aldehyde fixation; uranium and lead staining, x 15 000. FIG. 10. Hepatocyte nucleus treated as in Fig. 9. Large masses of condensed chromatin are visible. Arrows indicate nonchromatin, electron-opaque structures with a predominant granular component. Thin threads form a loosened network in the interchromatin spaces, nu, nucleolus. Aldehyde fixation; uranium and lead staining. x 35 000.

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DERENZINI, PESSION-BRIZZI, AND NOVELLO

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FIG, 11. Detail of Fig. 10. Arrow indicates a thread which is b o u n d both to chromatin (ch) and to a granular n o n c h r o m a t i n structure (g). Aldehyde fixation; u r a n i u m and lead staining, x 110 000.

RNP FIBRILS AND CHROMATIN ULTRASTRUCTURE AND FUNCTION 1978). Thus e v e n if a similar m e c h a n i s m plays a part in chromatin dispersal and condensation, this, by itself alone, could not explain the wide chromatin ultrastructural changes occurring either during activation or repression of transcription. It has been d e m o n s t r a t e d that R N P complexes which contained rapidly labeled h n R N A were attached to the nuclear skeleton in rat liver nuclei deprived of chromatin and nucleoplasm (Miller et al., 1978a). Because the R N P fibrils were bound to the chromatin fiber by one end (Puvion-Dutilleul et al., 1978), this resulted in that the transcribing c h r o m a t i n fibers w e r e p r o g r e s s i v e l y anchored to the nuclear skeleton b y means of the other end of the R N P fibrils. The R N P fibrils w e r e s h o w n to migrate f r o m the perichromatin border toward the interchromatin space in isolated rat h e p a t o c y t e s after stimulation of transcription by hydrocortisone (Puvion and Moyne, 1978). Therefore during activation of extranucleolar transcription a new association of R N P fibrils to the nuclear skeleton occurred. This association would progressively stretch the tightly looped chromatin fibers with the multiple anchorage of the D N P fiber to the nuclear skeleton even by means of single i n t e r s p a c e d R N P t r a n s c r i p t s . A similar m e c h a n i s m would be consistent with the morphological peculiarity of extranucleolar transcriptional c o m p l e x e s as visualized in rat liver nuclei with the method of Miller and Beatty (1969). The extranucleolar R N P transcripts were in fact frequently singular on the D N P fiber and when they formed R N P fibril arrays these were n e v e r observed as tandem repeats (Puvion-Dutilleul et al., 1978). The m e c h a n i s m p o s t u l a t e d a b o v e for chromatin ultrastructural changes might explain w h y c h r o m a t i n c o n d e n s a t i o n oc-

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curred after the solubilization of a little quantity of h n R N A by endogenous R N a s e . The observation that m o s t of the R N A solubilized was n e w l y s y n t h e s i z e d d e m o n strated that the e n z y m e digested a portion of R N P fibrils near to the chromatin fiber: the chromatin fibers, thus freed f r o m the c o n n e c t i o n s with the n u c l e a r s k e l e t o n , would collapse giving rise to the clumps of condensed chromatin even if a large quantity of R N P fibrils were still present in the nuclear space. CONCLUSIONS Our results d e m o n s t r a t e d that the gross ultrastructural organization of chromatin must be considered as a c o n s e q u e n c e of the quantity of newly synthesized h n R N A associated with D N P and then as a consequence and not as a cause of gene transcriptional activity. N o u l t r a s t r u c t u r a l functional relationship exists in chromatin other than that chromatin b e c o m e s m o r e dispersed or m o r e condensed b e c a u s e of a major or minor quantity, respectively, of transcriptional products bound to D N P . Our results indicate therefore that, as it has been already suggested by Ris and Kubai (1970), the terms of euchromatin and heterochromatin, must be used only in the morphological sense as s y n o n y m s with disp e r s e d and condensed chromatin. This investigation was supported by grants from CNR (Roma) and by Pallotti's Legacy for Cancer Research. A part of this work was presented in the "Rencontre W. Bernhard" (Onzain, May 12-14, 1980). REFERENCES ALLFREY, V. G. (1966) Cancer Res. 26, 2026-2040. BACHELLERIE, J. P., PUVION, E., AND ZALTA, J. P. (1975) Eur. J. Biochem. 58, 327-337. BEREZNEY, R. (1979) Exp. Cell Res. 123, 411-414.

FIG. 12. Hepatocyte nucleus treated as in Fig. 9. The masses of condensed, bleached chromatin are always bordered by ribonucleoprotein fibrils (--*). Ribonucleoprotein granular clumps are present (I~) in the interchromatin spaces where the thin threads observed in Figs. 9 and 10 are not visible. In the nucleolus, the granular and the fibrillar components normally intermingle. Aldehyde fixation; uranyl-EDTA-lead staining, x 35 000.

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BERNHARD, W. (1969) J. Ultrastruct. Res. 27, 250265. BURTON, B. (1956) Biochem. J. 62, 315-323. CHEVAILLIER, P., AND PHILIPPE, M. (1973) Exp. Cell Res. 82, 1-14. COCHET-MEILHAC, M., NURET, P., COURVALIN, J. C., AND CHAMBON, P. (1974) Biochim. Biophys. Acta 353, 185-192. COGLIATI, R., AND GAUTIER, A. (1973) C. R. Acad. Sci. Ser. D 276, 3041-3044. COMINGS, D. E., HARRIS, D. C., OKADA, R. A., AND HOLMQU1ST, G. (1977) Exp. Cell Res. 105, 349-365. COUPAR, B. E. H., AND CHESTERTON, C. J. (1977) Eur. J. Biochem. 79, 525-533. Cox, R. F. (1973) Eur. J. Bioehem. 39, 49-61. DERENZINI, M., LORENZONI, E., MARINOZZI, V., AND BARSOTTI, P. (1977) J. Ultrastruct. Res. 59, 250-262. DERENZINI, M., MARINOZZI, V., AND NOVELLO, F. (1976) Virchows Arch. B. Cell Pathol. 20, 307-318. DERENZINI, M., NOVELLO, F., AND PESSION-BR1zZI, A. (1978) Exp. Cell Res. 112, 443-454. DERENZINI, M., PESSION-BRIZZI, A., BONETT1, E., AND NOVELEO, F. (1979) J. UItrastruct. Res. 87, 161-179. DERENZINI, M., PESSION-BRIzZI, A., AND NOVELLO, F. (1980) Experientia 36, 181-182. FAKAN, S., PUVION, E., AND SPOHR, G. (1976) Exp. Cell Res. 99, 155-164. FRENSTER, J. H. (1965) Nature (London) 206, 680683. GALLINARO, H., AND JACOB, M. (1979) FEBS Lett. 104, 176-182. HADJIOLOV, A. A., DABEVA, M. D., AND MACKEDONS~I, V. V. (1974) Biochem. J. 138, 321-334. HIGGINS, G. H., AND ANDERSON, R. M. (1931) Arch, Pathol. 12, 186-202. HIRS, C. H. W. (1955) J. Biol. Chem. 219, 611-621. HODNETT, J. L., AND BUSCH, H. (1968) J. Biol. Chem. 243, 6334-6342. JAMRICH, M., GREENLEAF, A. L., BAUTZ, F. A., AND BAUTZ, E. K. F. (1978) Cold Spring Harbor Syrup. Quant. Biol. 42, 389-396.

JETER, J. R., JR., AND CAMERON, J. L. (1974) in CAMERON, J. L., AND JETER, J. R. (Eds.), Acidic Proteins of the Nucleus, p. 213, Academic Press, New York. LINDEL, T. J., WEINBERG, F., MORRIS, P. W., ROEDER, R. G., AND RUTTER, W. J. (1970) Science 170, 447-449. MARKOV, G. G., AND ARION, V. J. (1973) Eur. J. Biochem. 35, 186-200. MEILHAC, M., TYPSER, Z., AND CHAMBON, P. (1972) Eur. J. Biochem. 28, 291-300. MILLER, O. L., AND BEATTY, B. R. (1969) Science 164, 955-957. MILLER, T. E., HUANG, C.-Y., AND POGO, A. O. (1978a) J. Cell Biol. 76, 675-691. MILLER, T. E., HUANG, C.-Y., AND POGO, A. O. (1978b) J. Cell Biol. 76, 692-704. MONYEROY, A., AND BERNHARD, W. (1969) J. Ultrastruct. Res. 27, 266-288. MUNRO, H. N., AND FLECK, A. (1966) Analyst 91, 7888. NOVELLO, F., PESSION-BRIZZI, A., AND DERENZINI, M. (1978) Exp. Cell Res. 112, 219-224. NOVELLO, F., AND STIRPE, F. (1969) Biochem. J. 112, 721-727. NOVELLO, F., AND STIRPE, F. (1970) FEBS Lett. 8, 57-60. PAUL, J. (1972) Nature (London) 238, 444-446. PEDERSON, T. (1978) Int. Rev. Cytol. 55, 1-22. PUVION-DUTILLEUL, F., BERNADAC, A., PUVION, E., AND BERNHARD, W. (1977) J. Ultrastruct. Res. 58, 108-117. PUVION, E., AND MOYNE, G. (1978) Exp. Cell Res. 115, 79-88. PUVION-DUTILLEUL, F., PUVION, E., AND BERNHARD, W. (1978) J. Ultrastruct. Res. 62, 118-131. RIS, H., AND KUBA1, D. F. (1970) Annu. Rev. Genet. 4, 263-294. SEIFERT, n . , SCHEURLEN, M., NORTHEMANN, W., AND HEINRICH, P. C. (1979) Biochim. Biophys. Acta 564, 55-66. Z1MMERMANN, S. B., AND SANDEEN, G. (1966) Anal. Biochem. 14, 269-277.