Experimental
ISOLATION
AND
Cell Research
109 (1977) 151-161
CHARACTERIZATION CHROMATIN
FROM
S. MATSUl
Departmentof Pharmacology,
OF rDNA-CONTAINING
Baylor
NUCLEOLI
and H. BUSCH College
of Medicine,
Houston,
TX 77030,
USA
SUMMARY Fractions corresponding to ‘euchromatin’ and ‘heterochromatin’ were isolated from Novikoff hepatoma nucleoli by a modification of the DNase II method of Gottesfeld et al. [18, 191. Nucleolar chromatin [36] was digested with DNase II, and ‘euchromatin’ was obtained as a soluble fraction following differential precipitation of ‘heterochromatin’ with Mg2+. In this ‘euchromatin’ fraction, the rDNA was enriched approx. 8.5fold over the unfractionated nucleolar chromatin, as determined by DNA-RNA hybridization. On 8-80% glycerol gradients, the ‘euchromatin’ separated into three discrete subfractions as with euchromatin obtained from the bulk chromatin [18, 191. Two subfractions which sedimented at 14s and 2OS, respectively, were consistently found after DNase II digestion (to 15 mitt) which contained all histone fractions. All euchromatin subfractions were enriched in rDNA and had a high protein/DNA ratio, a high RNA-DNA ratio, and a high template activity with exogenous RNA polymerase I. Fraction B with a sedimentation of 14s was most enriched in the parameters related to rRNA synthesis. The Mg*+-precipitable fractions reported to be ‘heterochromatin’ [18, 191 not only contained substantial amounts of ribosomal genes, but also had higher template activities than unsheared chromatin. These results imply (1) nucleolar ‘euchromatin’ has a structural heterogeneity similar to that of the euchromatin containing active single copy sequences; (2) at least a portion of rDNA in ‘euchromatin’ is protected by proteins, including histones, which does not preclude active transcription of rDNA; (3) none of the currently used criteria for ‘euchromatin’ alone defines the chromatin containing rDNA; and (4) an appreciable enrichment of rDNA occurs in isolated euchromatin but not all the rDNA was separated into ‘euchromatin’ by the DNase II method.
Considerable evidence indicates that rDNA is selectively transcribed with high fidelity in isolated nucleoli and the nucleolar chromatin [2, 6, 21, 22, 29, 36, 421. To aid in defining the regulatory mechanism of rDNA (DNA sequences coding for rRNA and transcribed spaces) transcription, various methods have been tested to isolate rDNA-rich nucleolar fractions. Previously, the fibrillar components of nucleoli were isolated by dissociating the nucleoli in solutions containing polyvinyl sulphate (PVS) and DTT followed by purification on sucrose gradients [12]. Although kinetic analyses of
rRNA formation and processing indicated that the librillar components are specific sites of initial assembly of RNP particles, rDNA was not enriched in this fraction (Daskal et al., unpublished). Inasmuch as active genes in eukaryotes are generally believed to be localized in the diffuse ‘euchromatin’ and inactive genes are in the condensed ‘heterochromatin’ [ 16, 30, 311, separation of these fractions has been attempted by differential centrifugation [16], gradient centrifugation [9, 14, 431, differential precipitation [18, 19, 341, thermal elution on hydroxylapatite columns [40], Exp
Cc// Rrs
109 (1977)
152
Matsui
and Busch
gel exclusion chromatography [27] and ionexchange chromatography [48]. Since rDNA represents virtually the only active genes in nucleoli [4,7,8, 20, 21,22,36,42], it seemed reasonable to assume that rDNA exists in nucleoli as ‘euchromatin’. The present study was undertaken to determine tirst whether rDNA is in the form of euchromatin and second whether the rDNA is ‘naked’ or contains fewer proteins so that RNA polymerase may transcribe it efficiently or whether rDNA is associated with most chromatin proteins as reported for single copy sequences of euchromatin [18, 191. MATERIALS
AND
Isolation and fractionation nucleolar chromatin
METHODS
qf
Nucleoli were isolated by the sonic-oscillation method [8] from Novikoff ascites hepatoma cells. Cell suspensions from the peritoneal cavitv were freed of red blood cells by centrifugation in a Sorvall RC-3 centrifuge at 7 000 g for 60 min through 2.0 M sucrose 12 mM MgCl, 10 mM Tris-HCI, pH 7.5. The cells were suspended in 0.88 M sucrose 12 mM MgCI,, 10 mM Tris-HCI, pH 7.5, sonicated until no cells remained intact and centrifuged at 1 100 g for 20 min. After the supernatant was decanted, the pellet was suspended in 0.88 M sucrose 1 mM MgCl,, 10 mM Tris, pH 7.5, and resonicated briefly to disperse the extranucleolar chromatin [36]. The sonicate was centrifuged at I 100 g for 20 min to pellet the nucleoli. Electron microscopic analysis was used for evaluation of nucleolar purity [8]. Nucleoli thus isolated [6] apparently contain intact chromatin structures as evidenced by: (a) high molecular weight DNA (average size of double-stranded molecules>lOx lo6 D [36]) is readily isolated; (b) nucleoli synthesize high molecular weight rRNA including 45s molecules in vitro [21, 22, 361; and (c) nucleoli retain fidelity of rDNA transcribed in vitro [36]. Only 0.046% of nuclear DNA [47] and 0.5 % of nucleolar DNA [47, 581 is rDNA, respectively; isolation of nucleoli produces a IO-fold enrichment of rDNA. Another advantage of isolated nucleoli is that RNA polymerase I is exclusively localized to nucleoli. To obtain nucleolar chromatin, the nucleoli were stirred twice in a Vortex mixer at the highest power setting in 50 vol of 0.075 M NaCl, 25 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), pH 8.0, and then twice in 50 vol of 10 mM Tris-HCl, 0.1 mM PMSF, pH 8.0. Each washing was followed by centrifugation at 7000 g for 10 min. The tinalgelatinous pellet was designated ‘nucleolar chromatin’. 0.1 mM PMSF was used in each wash to prevent degradation of chromatin proteins [ 11. Chromatin thus prepared as E.rr, Cdl
Rcs 109 (1977)
in fig. I was adjusted to 20 AtGo as measured in 1 N NaOH and dialysed overnight against 25 mM Na acetate 0.1 mM EDTA, 0.25 mM PMSF, pH 6.6, in an ice bath [18, 191. A solution of DNase II (26OOO29472 U/mg, Worthington Biochem. Cot-p) was prepared at 1OOOOU/ml in autoclaved distilled water. DNase digestion was carried out in the presence of PMSF at 24°C at an enzyme concentration of 10 U/ OD,,, unit of chromatin solution and terminated by adding 50 mM Tris base to pH 7.6 and rapid chilling in an ice bath. The undigested chromatin was removed by centrifugation at 12000 g for 15 mitt; the supernatant was adjusted to 2 mM MgCI, and stirred for 30 min at 4°C. These samples were centrifuged at 12OOOg for 15 min. The supernatant ‘euchromatin’ fractions (50-75 OD,,, units) were subfractionated bv centrifuaation on 878% glycerol gradients containing 10 mM-Tris, pH 8.0, for 16 h at 85000 g. One ml fractions were collected and monitored using an ISCO fractionator.
Chemical determination Protein was determined by the method of Lowry et al. [32]. For nucleic acid analyses, chromatin samples were precipitated in ice-cold 5% PCA. The precipitates were analysed for DNA and RNA [52, 561.
DNA-RNA
hybridization
For the isolation of DNA, the chromatin solutions were adjusted to 3 M NaCI, 7 M urea by adding slowly solid chemicals, stirred in the presence of 0.1 mM PMSF for 3-5 h at 4°C and centrifuged for 18 h at 165 000 g in a Spinco 50 Tri rotor. The DNA pellet was dissolved in 0.1 N NaOH and kept at 37°C overnight [58]. After neutralizing with 2 M NaH,PO,, the samples were diluted in 2xSSC (
ISOLATION
AND
Cell Research
109 (1977) 151-161
CHARACTERIZATION CHROMATIN
FROM
S. MATSUl
Departmentof Pharmacology,
OF rDNA-CONTAINING
Baylor
NUCLEOLI
and H. BUSCH College
of Medicine,
Houston,
TX 77030,
USA
SUMMARY Fractions corresponding to ‘euchromatin’ and ‘heterochromatin’ were isolated from Novikoff hepatoma nucleoli by a modification of the DNase II method of Gottesfeld et al. [18, 191. Nucleolar chromatin [36] was digested with DNase II, and ‘euchromatin’ was obtained as a soluble fraction following differential precipitation of ‘heterochromatin’ with Mg2+. In this ‘euchromatin’ fraction, the rDNA was enriched approx. 8.5fold over the unfractionated nucleolar chromatin, as determined by DNA-RNA hybridization. On 8-80% glycerol gradients, the ‘euchromatin’ separated into three discrete subfractions as with euchromatin obtained from the bulk chromatin [18, 191. Two subfractions which sedimented at 14s and 2OS, respectively, were consistently found after DNase II digestion (to 15 mitt) which contained all histone fractions. All euchromatin subfractions were enriched in rDNA and had a high protein/DNA ratio, a high RNA-DNA ratio, and a high template activity with exogenous RNA polymerase I. Fraction B with a sedimentation of 14s was most enriched in the parameters related to rRNA synthesis. The Mg*+-precipitable fractions reported to be ‘heterochromatin’ [18, 191 not only contained substantial amounts of ribosomal genes, but also had higher template activities than unsheared chromatin. These results imply (1) nucleolar ‘euchromatin’ has a structural heterogeneity similar to that of the euchromatin containing active single copy sequences; (2) at least a portion of rDNA in ‘euchromatin’ is protected by proteins, including histones, which does not preclude active transcription of rDNA; (3) none of the currently used criteria for ‘euchromatin’ alone defines the chromatin containing rDNA; and (4) an appreciable enrichment of rDNA occurs in isolated euchromatin but not all the rDNA was separated into ‘euchromatin’ by the DNase II method.
Considerable evidence indicates that rDNA is selectively transcribed with high fidelity in isolated nucleoli and the nucleolar chromatin [2, 6, 21, 22, 29, 36, 421. To aid in defining the regulatory mechanism of rDNA (DNA sequences coding for rRNA and transcribed spaces) transcription, various methods have been tested to isolate rDNA-rich nucleolar fractions. Previously, the fibrillar components of nucleoli were isolated by dissociating the nucleoli in solutions containing polyvinyl sulphate (PVS) and DTT followed by purification on sucrose gradients [12]. Although kinetic analyses of
rRNA formation and processing indicated that the librillar components are specific sites of initial assembly of RNP particles, rDNA was not enriched in this fraction (Daskal et al., unpublished). Inasmuch as active genes in eukaryotes are generally believed to be localized in the diffuse ‘euchromatin’ and inactive genes are in the condensed ‘heterochromatin’ [ 16, 30, 311, separation of these fractions has been attempted by differential centrifugation [16], gradient centrifugation [9, 14, 431, differential precipitation [18, 19, 341, thermal elution on hydroxylapatite columns [40], Exp
Cc// Rrs
109 (1977)
152
Matsui
and Busch
gel exclusion chromatography [27] and ionexchange chromatography [48]. Since rDNA represents virtually the only active genes in nucleoli [4,7,8, 20, 21,22,36,42], it seemed reasonable to assume that rDNA exists in nucleoli as ‘euchromatin’. The present study was undertaken to determine tirst whether rDNA is in the form of euchromatin and second whether the rDNA is ‘naked’ or contains fewer proteins so that RNA polymerase may transcribe it efficiently or whether rDNA is associated with most chromatin proteins as reported for single copy sequences of euchromatin [18, 191. MATERIALS
AND
Isolation and fractionation nucleolar chromatin
METHODS
qf
Nucleoli were isolated by the sonic-oscillation method [8] from Novikoff ascites hepatoma cells. Cell suspensions from the peritoneal cavitv were freed of red blood cells by centrifugation in a Sorvall RC-3 centrifuge at 7 000 g for 60 min through 2.0 M sucrose 12 mM MgCl, 10 mM Tris-HCI, pH 7.5. The cells were suspended in 0.88 M sucrose 12 mM MgCI,, 10 mM Tris-HCI, pH 7.5, sonicated until no cells remained intact and centrifuged at 1 100 g for 20 min. After the supernatant was decanted, the pellet was suspended in 0.88 M sucrose 1 mM MgCl,, 10 mM Tris, pH 7.5, and resonicated briefly to disperse the extranucleolar chromatin [36]. The sonicate was centrifuged at I 100 g for 20 min to pellet the nucleoli. Electron microscopic analysis was used for evaluation of nucleolar purity [8]. Nucleoli thus isolated [6] apparently contain intact chromatin structures as evidenced by: (a) high molecular weight DNA (average size of double-stranded molecules>lOx lo6 D [36]) is readily isolated; (b) nucleoli synthesize high molecular weight rRNA including 45s molecules in vitro [21, 22, 361; and (c) nucleoli retain fidelity of rDNA transcribed in vitro [36]. Only 0.046% of nuclear DNA [47] and 0.5 % of nucleolar DNA [47, 581 is rDNA, respectively; isolation of nucleoli produces a IO-fold enrichment of rDNA. Another advantage of isolated nucleoli is that RNA polymerase I is exclusively localized to nucleoli. To obtain nucleolar chromatin, the nucleoli were stirred twice in a Vortex mixer at the highest power setting in 50 vol of 0.075 M NaCl, 25 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), pH 8.0, and then twice in 50 vol of 10 mM Tris-HCl, 0.1 mM PMSF, pH 8.0. Each washing was followed by centrifugation at 7000 g for 10 min. The tinalgelatinous pellet was designated ‘nucleolar chromatin’. 0.1 mM PMSF was used in each wash to prevent degradation of chromatin proteins [ 11. Chromatin thus prepared as E.rr, Cdl
Rcs 109 (1977)
in fig. I was adjusted to 20 AtGo as measured in 1 N NaOH and dialysed overnight against 25 mM Na acetate 0.1 mM EDTA, 0.25 mM PMSF, pH 6.6, in an ice bath [18, 191. A solution of DNase II (26OOO29472 U/mg, Worthington Biochem. Cot-p) was prepared at 1OOOOU/ml in autoclaved distilled water. DNase digestion was carried out in the presence of PMSF at 24°C at an enzyme concentration of 10 U/ OD,,, unit of chromatin solution and terminated by adding 50 mM Tris base to pH 7.6 and rapid chilling in an ice bath. The undigested chromatin was removed by centrifugation at 12000 g for 15 mitt; the supernatant was adjusted to 2 mM MgCI, and stirred for 30 min at 4°C. These samples were centrifuged at 12OOOg for 15 min. The supernatant ‘euchromatin’ fractions (50-75 OD,,, units) were subfractionated bv centrifuaation on 878% glycerol gradients containing 10 mM-Tris, pH 8.0, for 16 h at 85000 g. One ml fractions were collected and monitored using an ISCO fractionator.
Chemical determination Protein was determined by the method of Lowry et al. [32]. For nucleic acid analyses, chromatin samples were precipitated in ice-cold 5% PCA. The precipitates were analysed for DNA and RNA [52, 561.
DNA-RNA
hybridization
For the isolation of DNA, the chromatin solutions were adjusted to 3 M NaCI, 7 M urea by adding slowly solid chemicals, stirred in the presence of 0.1 mM PMSF for 3-5 h at 4°C and centrifuged for 18 h at 165 000 g in a Spinco 50 Tri rotor. The DNA pellet was dissolved in 0.1 N NaOH and kept at 37°C overnight [58]. After neutralizing with 2 M NaH,PO,, the samples were diluted in 2xSSC (
