Eur. 1. Biochern. 58, 315-326 (1975)

Non-histone Chromosomal Proteins Their Isolation and Role in Determining Specificity of Transcription in vitro Horst BLUTHMANN, Stanislav MROZEK, and Alfred GIERER Max-Planck-Institut fur Virusforschung, Tubingen (Received March 27/July 3, 1975)

We describe a method for fractionation of chromatin components by selective dissociation with salt in buffers containing 5 M urea in combination with chromatography on hydroxyapatite at 4 "C. This results in two histone and four non-histone fractions which are recovered in high yield and with minimal proteolytic contamination. Template capacity measurements of the isolated chromatins and pre-saturation competition hybridization experiments support the idea that a group of non-histone proteins activate the transcription of specific DNA sequences which were not transcribed from purified DNA to the same extent. In reconstitution experiments a non-histone protein fraction, NH4, prepared from lymphocyte chromatin by hydroxyapatite chromatography is shown to cause transcription in vitro of lymphocytespecific RNA sequences. A subfraction with a molecular weight of 30000 comprising 40% of the NH4 fraction protein is characteristic for this tissue and not found in liver chromatin. Chromatin, the interphase chromosomal material of eukaryotic cells, is a complex of DNA with histone, non-histone proteins and a small amount of RNA. In differentiated cells, only a portion of the DNA is transcribed. The majority of the genome is repressed. It is generally believed that the non-histone proteins of chromatin play a major role in regulating transcription of DNA (for review see [I]). Previous attempts to characterize this class of proteins have involved strong denaturants [2- 41. More recently, milder isolation conditions have been described in which dissociation of chromatin was achieved either solely with high salt [ 5 ] or high salt in combination with urea or guanidinium chloride [6- 121. Following dissociation, chromatography on hydroxyapatite was used to separate non-histone proteins from histones and DNA [13,14]. The method of fractionation of chromosomal proteins we describe here also employs hydroxyapatite; in addition there is particular emphasis on avoiding proteolytic degradation. Our method consists of a combination of selective dissociation of chromatin with salt in buffered urea and chromatography on hydroxyapatite at 4 "C. With this procedure chromosomal proteins have been resolved into six Abbreviurion. Standard saline citrate, 0.15 M NaCI, 0.015 M sodium citrate, pH 7. Enzyme. RNA polymerase or RNA nucleotidyltransferase (EC 2.7.7.6).

different fractions, two of them being identified as histones and the remaining four as non-histone proteins. The proteins are recovered in high yield, show minimal proteolytic degradation and exhibit reproducible tissue-specific pattern on sodium dodecylsulfate polyacrylamide gels. It has been shown by transcription and hybridization experiments that preparations of chromatin retain a good deal of their native control of gene expression [15- 191. This implies that the structures required for tissue-specific transcription are not lost during the isolation procedure and are recognized by a bacterial polymerase. The ability of non-histone proteins to determine tissue-specific transcription has been shown by reconstitution experiments [I 5,20- 221. In these experiments reconstituted chromatin was transcribed in vitro and the RNA products of various preparations compared in competition hybridization experiments. In interpreting these results it has to be considered that the nucleic acid concentrations and incubation times used in such hybridization experiments allow annealing of the RNA only to repeated sequences. Using stringent conditions of hybridization, which permit less than 5 - 7 % mismatching, we have been able to show that in the case of lymphocyte chromatin a high degree of template specificity is restored in reconstitution experiments using only a small fraction of the non-histone proteins prepared from hydroxyapatite.

Non-histone Chromosomal Proteins

316

MATERIALS AND METHODS Preparation of Nuclei and Chromatin Nuclei from bovine liver and lymphocytes were prepared according to Teng et al. [23] with an additional treatment of the nuclei with 0.5% Triton X-100 [24]. Chromatin was isolated from the nuclei as described [25] and extracted with 0.3 M NaCl as recommended by Johns and Forrester [26] to remove cytoplasmic contamination. Chromatography on Hydroxyapatite Hydroxyapatite was prepared according to the method of Tiselius et al. [27]. The urea used throughout this study was prepared as a 10 M stock solution and deionized on a column of Ionenaustauscher V mixed-bed resin (Merck, Darmstadt). The prepared buffers were stored in the cold and used within two days of preparation. The isolated chromatin was dissolved in 0.5 M NaCI, 5 M urea, 0.001 M sodium phosphate, pH 6.8, to give a final concentration of 0.5 mg/ml DNA and added to a hydroxyapatite suspension in the same buffer. The amount of hydroxyapatite was calculated as follows : 1ml packed hydroxyapatite adsorbs 0.7 mg DNA or 1.O mg non-histone proteins. Adsorption was carried out batchwise for 30 min in the cold. The suspension was poured into a column (ratio of length to diameter about 8) which was run at 4°C with a flow rate of 2-3 ml h-' xcm-2. The first histone fraction (Hl), which was unadsorbed, was washed out with the same buffer. The first non-histone fraction (NH1) was then eluted with 0.45 M NaC1, 5 M urea, 0.05 M sodium phosphate, pH 6.8, and the column washed with the initial buffer (0.5 M NaCI, 5 M urea, 0.001 M sodium phosphate, pH 6.8) to reduce the phosphate concentration to the original level. The NaCl concentration was then raised to 2 M to dissociate the remaining proteins from the DNA. The second histone fraction (H2) appeared with the front of the buffer (2 M NaCl, 5 M urea, 0.001 M sodium phosphate, pH 6.8). Three additional non-histone fractions (NH2, 3 and 4) were eluted stepwise with 0.05 M, 0.10 M and 0.175 M potassium phosphate, pH 7.5, in 2 M KCl, 5 M urea. Fraction NH1 was routinely rechromatographed to remove contaminating histones as follows. After dialysis against 2 M NaCl, 5 M urea, 0.001 M sodium phosphate, pH 6.8, the NH1 fraction was applied to a hydroxyapatite column equilibrated with the same buffer; contaminating histones were eluted with 2 M NaCl, 5 M urea, 0.01 M sodium phosphate, pH 6.8, then fraction NH1 with 2 M KC1, 5 M urea, 0.05 M potassium phosphate, pH 7.5. For isolating the DNA still adsorbed to hydroxyapatite, the column was treated with 40 % guanidinium

hydrochloride, 2.5 M urea, 0.175 M potassium phosphate, pH 7.5. The guanidinium hydrochloride was washed out with 2 M KC1, 5 M urea, 0.175 M potassium phosphate, pH 7.5, and the DNA eluted batchwise with 1 M sodium phosphate, pH 6.8. Virtually all the DNA was recovered from the column. The DNA was passed through a Bio-gel A 1.5 m column; 60 ml of a DNA solution of about 0.6 mg/ml was applied to a 5 x 50-cm column equilibrated with 0.01 M Tris, pH 7.4. Only the DNA excluded by the column was used. The size of native DNA was estimated with a Spinco model E ultracentrifuge employing ultraviolet optics. DNA from both tissues sedimented with szo. values of 15 S, corresponding to a M , = 3.4 x lo6. The sedimentation coefficient of denatured DNA was estimated in alkaline sucrose gradients, according to Studier [28]. Single-stranded DNA from lymphocyte chromatin exhibits an szo,wvalue of 14.4 S , corresponding to M , = 1.2 x loh, whereas single-stranded DNA from liver chromatin sediments with s20.w = 10.5 S, corresponding to M , = 0.56 x lo6. Thus native DNA from lymphocyte has virtually no nicks, whereas native liver DNA contains about two nicks per molecule. Labeling of D N A Bovine lymphocyte cultures were prepared as described [29]. 500 ml of the culture were stimulated with 0.5 ml phytohemagglutinin P (Difco) and labeled with 16 pCi [14C]thymidinefor 68 h. Nuclei, chromatin and DNA were prepared as described above. The purified DNA had a specific activity of 620 counts min pg - .

'

Polyacrylamide Gel Electrophoresis Analytical gel electrophoresis in sodium dodecylsulfate was performed according to the method of Weber and Osborn [30] in 0.56 x 10-cm 10% gels. Approximate molecular weight was calibrated against the relative migrations [31] of bovine serum albumin (67 000), ovalbumin (45 000), chymotrypsinogen (25000) and myoglobin (17 800). Acidic urea polyacrylamide gels were prepared after Panyim and Chalkley [32] using 2.5 M urea and 15% acrylamide. Dissociation and Reconstitution of Chromatin Chromatin was dissociated in 2 M NaC1, 5 M urea, 1 mM MgC12, 0.01 M Tris, pH 8.0, at a concentration of 0.6 mg/ml chromatin DNA. For reconstitution of chromatin from the individual components, 6 mg purified DNA, 8 mg histone and different amounts of individual non-histone frac-

317

H. Bluthmann, S. Mrozek, and A. Gierer

tions as indicated in each experiments were mixed together in 10 m12 M NaCl, 5 M urea, 1 mM MgCl,, 0.01 M Tris, pH 8.0. Gradual removal of salt and then of urea was carried out according to Spelsberg et al. [33], and the reconstituted chromatin purified as described above. Isolation of E . coli RNA Polymerase E. coli RNA polymerase was prepared as described by Burgess [34] up to his fraction 4. The enzyme was further purified by affinity chromatography on a DNA-agarose column [35]and by filtration on Bio-gel A 1.5 m [34].

Synthesis and Isolation of RNA in vitro Reactions mixtures of 10 ml final volume contained 0.02 M Tris, pH 8.0, 0.03 M MgCl,, 0.13 M NH,Cl, 0.1 mM dithiothreitol, 2.5 mM each of unlabeled ATP, GTP and CTP, 0.5mM [3H]UTP (specific activity 360 counts min-' pmol-I), 400- 500 units of E. coli RNA polymerase and 600 to 700 pg primer, either as DNA or as chromatin. After incubation for 150 min at 37 "C, the reaction mixture was centrifuged at 2000 x g for 10 min. The resulting pellet was discarded and the supernatant fraction brought to 1% sodium dodecylsulfate. The solution was applied to a column of Sephadex G-50 fine and eluted with 0.05 M sodium phosphate, pH 6.8. The excluded volume containing the RNA synthesized in vitro was brought to 2 M sodium perchlorate and 5 M urea, and added to 10 ml hydroxyapatite suspended in 0.05 M sodium phosphate, pH 6.8. The suspension was stirred for 30 min at 60"C, then poured into a water-jacketed column, which was heated to 60"C, and washed with 5 vol. of 0.05 M sodium phosphate, pH 6.8. The RNA was eluted with 0.20 M sodium phosphate, pH 6.8. The fraction was lyophilized to reduce the volume to 1/10 and passed through a Sephadex G-50 column equilibrated with 0.01 xstandard saline citrate. The RNA in the excluded volume was concentrated by lyophilization to 1.5 to 2.0 mg/ml. The specific activity of the RNA obtained was between 50 and 200 counts min-' ng-'. RNAs transcribed from native and reconstituted chromatin were also analysed on sucrose gradients in the presence of 0.01 % sodium dodecylsulfate. RNA from both templates exhibited a broad heterogeneous profile from 7 to 18 S .with a peak around 12 S, corresponding to a molecular weight of the order of lo5. DNA-RNA Hybridization

I4C-labeled calf DNA was immobilized on 24-mm nitrocellulose filters as described [36]. 4-mm circles

containing 0.2 pg DNA were punched out of the larger filters. Hybridization was performed by immersing two DNA filters in 20 p1 50% formamide, 2 x standard saline citrate with increasing amounts of [3H]RNA as indicated in the figures for each experiment for 48 h at 41 "C. This is 21 "C below the t , of the DNA (the 1, of calf DNA is 92°C in 2xstandard saline citrate; formamide reduces the t, of DNA by 0.60 "C per 1% [37]). Using these conditions, hybrids were formed with less than 5 - 7 % mismatched bases, as measured by their thermal stability. After washing the filters with 2 x standard saline citrate and treatment with pancreatic RNAase (Sigma) [36] the ratio 3H/14Cwas counted in a Packard Tri-Carb spectrometer. Unspecific adsorption on blank filters was less than 0.005 % of input DNA. For competition hybridization experiments the filters were first pre-saturated with increasing amounts of unlabeled RNA as indicated in the figure for each experiment, processed without RNAase as described [36] and incubated in a second hybridization with a constant, near saturating amount of 3H-labeled RNA. Analyses Histone and non-histone protein content were determined according to Bonner et al. [38]. RNA was separated from DNA by selective hydrolysis as described by Schmidt and Tannhauser [39]. DNA was determined by the method of Keck [40] as modified by Hubbard et al. [41] with calf thymus DNA (Sigma) as standard and RNA by the orcinol method of Dische and Schwarz [42] with E. coli transfer RNA (Boehringer) as standard. RESULTS Separation of Chromatin Constituents on Hydroxyapatite Fig. 1 showsthe chromatography of bovine lymphocyte chromatin on a column of hydroxyapatite. Separation of the chromosomal proteins into six fractions was achieved by a combination of selectivedissociation of the chromatin in salt and chromatography on hydroxyapatite with a stepwise phosphate elution, as described in Materials and Methods. Table 1 and 2 show analyses of hydroxyapatite fractions obtained from lymphocyte and liver chromatin preparations. The first histone fraction, H1, dissociated from the chromatin in 0.5 M NaCI, 5 M urea, 0.001 M phosphate buffer, flow through the column. This fraction contains histone F2a2 and F2b (Fig.2). Raising the phosphate concentration to 0.05 M, but maintaining the ionic strengh of the buffer by reducing the NaCl concentration to 0.45, elutes the first non-histone fraction, NH1 (Fig. 3). Contaminating histones in

Non-histone Chromosomal Proteins

318

Table 1. Analyses of hydroxyapatite column fractions Recovery of protein and RNA related to DNA applied to the column = 100 %. Fractions NH1 are rechromatographed

1.6/

Fraction

Lymphocyte chromatin

Liver chromatin

protein

RNA

protein

RNA

0.2 1.6

65 32 27 1.5 2.5 3.6

0.6 2.0

%

0

20

__3

H1

40

- -

60

80

120 140 Fraction number

NHI

m

NH2

H2

-160

Kx)

H1 H2 NH 1 NH2 NH3 NH4

180 200

61 35 10 3.5 1.2 2.8

NH3 NH4

Fig. 1. Chromatography of’lymphocytr c l i r o m t i n on hydroxyapatite. Lymphocyte chromatin (197 mg chromatin DNA) was dissolved in 0.5 M NaCI, 5 M urea, 0.001 M sodium phosphate (pH 6 4 , adsorbed to 350x111 hydroxyapatite and filled into a column (32 x 3.8 cm). The arrows indicate the buffers with which the column was treated. (1) 0.45 M NaCI, 5 M urea, 0.05 M sodium phosphate (pH 6.8). (2) 0.5 M NaCI, 5 M urea, 0.001 M sodium phosphate (pH 6.8). (3) 2 M NaCI, 5 M urea, 0.001 M sodium phosphate (pH 6.8). (4) 2 M KCI, 5 M urea, 0.05 M potassium phosphate (pH 7.5). (5) 2 M KCI, 5 M urea, 0.10 M potassium phosphate (pH 7.5). (6) 2 M KCI, 5 M urea, 0.175 M potassium phosphate (pH 7.5). Fractions of 10 ml were collected and were pooled as indicatcd by the bars

this fraction, less than 4”/, for both tissues, were removed by rechromatography as described in Methods. With 2 M NaCl in 5 M urea, 0.001 M sodium phosphate, pH 6.8, the remaining proteins dissociate from the DNA. A second histone fraction, H2, containing histone F1, F3 and F2al (Fig.2), appears with the front of the buffer. Three additional non-histone fractions, NH2, NH3 and NH4 were eluted from the column stepwise with 0.05 M, 0.10 M and 0.175 M potassium phosphate in 5 M urea, 2 M KC1. Potassium

Table 2. Amino acid analyses of hydroxyapatite column ,fractions No corrections have been made for losses during hydrolysis Amino acid

Amount in fraction H1

H2

NH1

NH2

NH3

NH4

lympho- liver cyte

lympho- liver cyte

lympho- liver cyte

lympho- liver cyte

lympho- liver cyte

lympho- liver cyte

14.9 2.7 8.7 5.4 4.9 4.5 8.6 4.9 8.8 11.4

8.1 2.3 5.6 9.6 4.9 6.1 13.1 6.5 8.7 7.8 1.8 6.1

8.6 2.2 5.9 9.6 4.4 7.4 14.3 7.4 7.5 7.4 5.5 4.9 9.4 2.2 3.3 23.9 16.1 1.4

10.6 2.1 7.3 9.2 4.4 6.4 14.5 6.2 6.9 7.6

12.7 2.2 5.5 9.8 3.5 5.5 15.3 8.2 6.9 8.2

mo1/100 mol Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Acidic Basic Acidic/basic

13.2 2.5 9.2 5.7 5.1 4.7 8.7 4.6 8.7 11.4 6.5 5.6 10.1 2.5 1.5 14.4 24.9 0.58

8.1 1.9 9.0 7.1 5.1 4.9 9.9 4.9 9.2 11.2 3.0 6.6 -

6.4 8.8 2.1 1.7 17.0 19.0 0.90

-

6.4 5.1 9.8 2.5 1.5 14.0 26.3 0.53

18.3 1.8 12.1 3.2 5.3 3.5 5.8 6.1 7.4 14.7 5.3 4.6 0.4 3.0 5.7 1.4 1.4 9.0 32.0 0.28

-

4.8 8.8 2.4 3.4 22.7 16.0 1.4

6.1 2.0 6.4 8.9 5.0 6.3 13.0 5.9 9.7 1.6 2.9 5.9 1.6 4.6 8.5 2.6 3.6 21.9 14.5 1.5

6.1 2.0 6.1 9.3 4.5 6.1 15.6 6.4 6.4 7.8 3.0 5.7 1.3 4.2 10.2 2.4 3.1 24.9 14.2 1.75

~

5.5 0.9 3.9 8.7 2.3 3.4 23.7 20.0 1.19

6.7 1.9 5.5 9.4 4.3 6.2 14.5 8.5 6.5

7.0 3.8 5.6 1.2 4.1 9.4 2.2 3.1 23.9 14.1 1.7

-

5.1 -

3.1 8.4 1.8 3.9 25.1 20.4 1.23

6.5 2.5 6.3 9.5 4.4 6.4 14.0 6.1 6.9 7.2 4.0 5.9 1.4 4.2 9.1 2.4 3.5 23.5 15.3 1.54

H. Rliithmann. S. Mrozek. and A . Giercr

319

Migration

Fig.2. Electrophoresis of histone fractions HI (-, polyacrylumide gels

-

upper gel) and H2 (----,

was used instead of sodium because of its better solubility. The NH4 fraction from lymphocytes contains a prominent 30000-M band which is characteristic for this tissue and not found in liver chromatin (Fig. 3 D). It represents 40% of the proteins in fraction NH4 and 7 % of the total non-histone proteins from lymphocyte chromatin. Tentative results concerning further fractionation of this band indicate that it consists of a few distinct components. The recovery of the histones in fraction H1 and H2 is 90% or higher for both tissues investigated. The same is true for the non-histone proteins eluted in fractions NH1 to NH4 in the case of lymphocyte chromatin, whereas chromatography of liver chromatin gives a yield of non-histone proteins of about 70 %. The histone fractions and the first two non-histone fractions NHI and NH2 are virtually free of nucleic acids. Most of the chromatin RNA is eluted in fraction NH4 (Table 1). Except for fraction NH2, all fractions are free of proteolytic activities. Added histone F1, which is most susceptible to chromosomal proteases, was not degraded after incubation of the samples for 24 h at room temperature, as revealed by gel electrophoresis in sodium dodecylsulfate. Hybridization Studies of Isolated Chromatin

As in previous studies [43 - 451 both chromatin preparations show only a fraction of the template

lower gel) from lymphocyte chromatin in acidic urea

activity of purified DNA. In saturation hybridization experiments, however, RNA transcribed from liver chromatin and from purified DNA both yield 4%, while RNA from lymphocyte chromatin formed hybrids with 8 % of the denatured DNA (data not shown). Pre-saturation competition experiments [46] were done to compare the RNA families synthesized from both chromatins and from purified DNA. As can be seen in Fig.4A, unlabeled RNA synthesized from liver chromatin competes only partially with labeled RNA synthesized from lymphocyte chromatin. The same is true for the reverse experiment, when unlabeled RNA. from lymphocyte chromatin competes with RNA from liver chromatin, whereas in each case homologous RNA gives essentially complete competition. This implies that both chromatin preparations serve as a template for a specific subset of RNA molecules. A similar conclusion may be drawn from competition experiments between RNA synthesized in vitro using a DNA template and RNA synthesized from both chromatin preparations. Fig. 4B demonstrates that RNA from a DNA template includes some molecules similar to those transcribed from lymphocyte and liver chromatin, but that the majority are distinguishable. In the reverse experiment shown in Fig.4C unlabeled RNA from a DNA template can compete with labeled RNA from lymphocyte and liver chromatin but only to a limited extent, whereas competition with the homologous RNA is complete.

Non-histone Chromosomal Proteins

320

Migration

-

Fig. 3 . Electrophoresis ojhydroxyapatite non-histone fraclionsJrom lymphocyte chromatin >-( upper gel) and liver chromatin (.-~---,lower gel) in sodium dodecylsulfate polyacrylumide gels. (A) Fraction NH1. (B) Fraction NH2. ( C ) Fraction NH3. (D) Fraction NH4

Thus these data show that by the hybridization competition criterion a great part of the DNA sequences available for transcription on lymphocyte and liver chromatin are not transcribed in purified DNA to the same extent. Reconstitution with Different Non-histone Fractions

The ability of the non-histone fractions from lymphocyte chromatin to cause specific transcription

was tested in reconstitution experiments. DNA, histones and the individual non-histone fractions or a mixture of them were added together in ratios found in isolated chromatin and reconstituted by gradient dialysis. RNA transcribed from lymphocyte chromatin reconstituted with a mixture of all non-histone proteins is a good competitor for RNA from a DNA template (Fig.5B) and a weak competitor for RNAfrom lympho-

H. Bliithmann, S. Mrozek. and A . Gierer

cyte chromatin (Fig. 5 A). The same is true for RNA transcribed from chromatin reconstituted with fraction NH1 rather than with a mixture of all non-histones. Thus, these reconstituted chromatin complexes do not exhibit the template specificities of the original lymphocyte chromatin. Part of the template specificity is restored using fraction NH3 for reconstitution; RNA from this template competes well with RNA from lymphocyte

321

chromatin (Fig. 5 A) but is found to be more competitive for RNA from a DNA template than is RNA from lymphocyte chromatin (35 % compared to 20 %, Fig. 5 B). Reconstitution with fraction NH4 gives rise to a complex which resembles the chromatin complex most closely, with respect to transcription. RNA transcribed from this template behaves essentially like RNA from lymphocyte chromatin ; it competes effec-

322

Non-histone Chromosomal Proteins

-

40

-

' 20

OO

2

1

3

4

5

0

0

1

2

3

-

4

5

0

Unlabeled RNA/ [3H]RNA

2

1

0

Unlabeled RNA/ [3H]RNA

3

4

5

Unlabeled RNA/[3H]RNA

Fig. 4. Piv-saturution cony~efitionrxperiments with R N A synlhc,.si;rd from lymphocyte utid liver chromatin and ,fkonz p u r [ j i d D N A . (A) (0-0) Effect of incrcasing amounts of unlabeled RNA from lymphocyte chromatin on the hybridization of [3H]RNA from liver chromatin (2.1 pg); (-0) effect of unlabeled RNA from liver chromatin on the hybridization of [3H]RNA from lymphocyte chromatin (2.9 Fg). In control experiments both the unlabeled and labeled RNA were from the same template: (A-A) from lymphocyte chromatin and (A-A) from liver chromatin. In the absence of competitor hybridization was 1746 and 1834; 1183 and 1064 counts/min per filter, Effect of increasing amounts of unlabeled RNA from lymphocyte chromatin on the hybridization of [3H]RNA respectively. (B) (0-0) from purified DNA (2.1 pg); (0-0) the same experiment with unlabeled RNA from liver chromatin. In the absence of competitor hybridization was 1890 and 1782counts/min per filter. (C) Reverse experiment as in (13). Effect of increasing amounts of unlabeled RNA from purified DNA on the hybridization of rH]RNA from lymphocyte chromatin (H and ) liver chromatin (O-). In a x) control experiment both unlabeled and labeled RNA were from purified DNA ( x ~

A

? ._ +

m 20-

z

0

0

1 2 3 4 Unlabeled RNA / ['HIRNA

0

I

0

I

1

1 2 3 4 Unlabeled RNA/ [3H]RNA

Fig. 5 . Pre-saturation competition wifh unlabeled R N A synthesized from lymphocyte chromatin (M and )lymphocyte chromatin rewith 0.012 mg N H 3 ( x -x ) , with constituted with different non-histone fractions: with 0.10 mg N H l protein per mg DNA (A-A), 0.028 mg NH4 (Mand ), with a mixture of all N H fractions (0.10 mg N H I , 0.035 mg NH2, 0.012 mg NH3 and 0.028 mg NH4) (A----A/. (A) Effect of increasing amounts of unlabeled RNA from the above templates on the hybridization of [3H]RNA from lymphocyte chromatin (2.9 pg) and in (B) on the hybridization of [3H]RNA from purified DNA (2.2 pg). For comparison the effect In ) the .absence of competitor hybridization of unlabeled RNA from native lymphocyte chromatin is shown in (A) and (B): (M was 1306 and 1279; 1448 and 1390 counts/min per filter, respectively

tively with RNA from lymphocyte chromatin (Fig. 5A) and exhibits the same limited competition for either RNA from a DNA template (Fig.6A) or from liver chromatin (Fig.6B) as RNA from lymphocyte chromatin. Furthermore, RNA transcribed from this reconstituted chromatin reaches the same plateau of 8 % in saturation hybridization experiments as RNA from lymphocyte chromatin. The following experiments were done to show that the non-histone fraction is responsible for the observed specificity. When NH4 fraction of lymphocytes and histones and DNA from liver were provided for reconstitution, the specificityof transcription remained

that which was typical of lymphocyte chromatin (Fig. 6A and 6B). Reconstitution of liver chromatin with the equivalent NH4 fraction from liver gives some of the specificity isolated liver chromatin exhibits; RNA from this template is a strong competitor for RNA from a DNA template (Fig.7A) but competes only partially with RNA from liver chromatin (Fig. 7 B). In the case of liver, direct reassociation, i.e. dissociation of the chromatin in 2 M NaCl, 5 M urea. 0.01 M Tris, pH 8.0, followed by gradient dialysis without intervening purification of the components, maintains a great part of the template specificity of

H. Bliithmann, S. Mrozek, and A. Gierer

323

t 1

"

1 2 3 4 Unlabeled RNA/[3H]fWA

0

5

"0

I

1

,

2 3 4 Unlabeled RNA / L3H]RNA

1

5

Fig. 6. Pre-saturution competirion with unlabeled R N A either ,from reconstituted lymphocyte chromatin (fraction N H 4 ) ( 6 0 1 or from reconsrifuredhybrid chromatin ( x x ). Reconstitution was as described in Fig. 5. For reconstitution of hybrid chromatin 1.0 mg liver DNA was mixed with 1.34 mg liver histone and 0.028 mg lymphocyte fraction NH4 protein before gradient dialysis. (A) Effect of increasing amounts of unlabeled RNA from the above templates on the hybridization of fH]RNA from purified DNA (2.2 pg) and in (B) on the hybridization of ['HH]RNA from liver chromatin (2.1 pg). For comparison the effect of unlabeled RNA from native lymphocyte chromatin is shown in (A) and (B): (+o). In the absence of competitor hybridization was 610 and 573; 1310 and 1388 counts/min per filter, respectively ~

0

1

1

I

I

J

1

2

3

4

5

Unlabeled RNAl [ 3 H ] W

I

0

1

2

3 4 Unlabeled RNA / ['HIRNA

5

Fig. 7. Pre-saturation competition with unlabeled RNA either from reassociated liver chromatin (-0) or from reconstituted liver chromafin Ifracrion N H 4 ) ( x --x ). Reconstitution of liver chromatin (fraction NH4) was done with 0.036 mg fraction NH4 protein per mg DNA. Reassociation wds'carried out by dissociation of the chromatin in 2 M NaCl, 5 M urea, 0.01 M Tris, pH 8.0, followed by gradient dialysis without prior isolation of the components. (A) Effect of increasing amounts of unlabeled RNA from the above templates on the hybridization of [3H]RNA from purified DNA (2.2 pg) and in (B) on the hybridization of ['HIRNA from liver chromatin (2.1 pg). For comparison the effect of unlabeled RNA from native liver chromatin is shown in (A) and (B): (M) In the.absence of competitor hybridization was 1170 and 1192; 1520 and 1636 counts/min per filter, respectively

the liver chromatin, as indicated by the competition experiments shown in Fig.7A and 7B. However, in similar experiments with lymphocyte chromatin the template specificity was not recovered (data not shown). Reconstitution with Different Amounts of Fraction NH4

Reconstitution experiments were carried out to estimate the amount of fraction NH4 from lymphocyte chromatin necessary for specific reconstitution with respect to transcription. Fig. 8 A shows that

RNA families transcribed from chromatin reconstituted with 1 %, 2.8 % and 6.4 % of fraction NH4 (w/w protein to DNA) all compete with [3H]RNA from a DNA template to the same limited extent, behaving similarly to RNA from the original lymphocyte chromatin. With larger amounts, i.e. 8 % and 15 % of fraction NH4 in the reconstitution assay, part of the specificity is lost. RNA from these templates competes with RNA from DNA to a greater extent (Fig. 8 B). Omitting fraction NH4 in the reconstitution assay, thus forming a pure DNA . histone complex, results in a complete loss of lymphocyte-specifictranscription.

Non-histone Chromosomal Proteins

324

-

K70

100

A

..“\ :08

O.--o*-%yij

TJ

._ .‘0 5 N

60

-

n

~

E

d

0

x

80 ~

@

r

5

.-U .-

m

A . * 0-.-*-=

! L . -

x

x

c

w

I

I=2

40

-

2 20

-

c 3 0

E,

m

B ._

-

40 x

m z

-

.-

%

20-

d

h I

1

R N A from this template competes fully with that transcribed from DNA (Fig. 8 B).

DISCUSSION Hydroxyapatite chromatography to separate histone and nucleoprotein, partially or totally dissociated by salt, was introduced by Faulhaber and Bernardi [47]. To improve the separation of the chromatin components, MacGillivray et al. [13] used 5 M urea in addition to 2 M NaCl in 1 mM sodium phosphate buffer, pH 6.8, as a dissociating medium. Because of the limited solubility in the salt/urea medium of the sodium phosphate buffers they had to run the column at room temperature. The low yield of histone F 1 they obtained in the unadsorbed fraction together with the appearance of low-molecular-weight components not identified as histones suggest that during chromatography there may have been some proteolytic degradation. In the present study we used potassium phosphate, pH 7.5, and potassium chloride in 5 M urea instead of the sodium salts, because of their better solubility. The hydroxyapatite column could thus be run at 4 “C. Histone fractions from both tissues investigated contain no detectable polypeptides migrating faster than histone F2al on sodium dodecylsulfate polyacrylamide gels. This indicates that proteolytic activities during the whole procedure were minimal. Selective dissociation of chromatin by salt in addition to chromatography on hydroxyapatite allows further separation of chromosomal proteins. The nonhistone protein fractions eluted with 0.05 M sodium phosphate can be resolved into two fractions, NH1 and NH2. Only the NH2 fraction, the smaller one, contains residual proteases isolated together with the chromatin. Furthermore, the histones appear in two fractions, H l and H2. The slightly lysine-rich histones

0

1

1

1

F2b and F2a2 are found in fraction H1, whereas the arginine-rich histones F3 and F2a2 are eluted together with histone F1 in fraction H2. To interpret the results with reconstituted chromatin, it has to be considered that hybridization under the conditions used in this work detects mainly intermediate repetitive sequences. In calf these sequences comprise 38% of the total DNA [48]. They consist of “families” of similar but non-identical sequences with some lo4 members per family. Tissue specificity of RNA transcripts, as seen in competition hybridization experiments, probably reflects differential transcription of the different families. Hybridization does not permit one to distinguish between moderate activation of all the sequences in a family or stronger activation of a few. If a sufficient number of R N A transcripts are made either from few or from many sequences within a family, these will hybridize to all the D N A sequences in the same family, whether or not those are transcribed. Thus, regulation selecting subsets of sequences belonging to the same family would remain undetected. It is experimentally established that repetitive sequences transcribed in different tissues differ quantitatively in composition and that they are similar for native and for reconstituted chromatin [15,16,19-21,241. Furthermore, recent analyses of the Xenopus, sea urchin and rat genomes show intermediate repetitive sequences about 300 nucleotides long, occurring interspersed with unique sequences of the order of 1000 nucleotides [49-511. It is possible that one or a few families of repetitive sequences are represented within the DNA sequence corresponding to a geneI521. A non-random distribution of repetitive sequences in genes could explain why the differential transcription of structural genes leads to a tissue-specific pattern of repetitive sequences. Gene regulation may be combinatorial [53 - 561 and interaction of some regulatory molecules with repetitive sequences could be a neces-

H. Bliithmann, S. Mrozek, and A. Gierer

sary though insufficient condition for transcription. Further regulatory mechanisms undetected by hybridization could lead to the activation of particular structural genes. The pre-saturation competition experiments show that the majority of the repetitive RNA sequences transcribed from either sort of chromatin are distinguishable from those transcribed from purified DNA. Therefore, the RNA sequences from chromatin cannot be subclasses of the RNA synthesized using the corresponding purified DNA as template. The hybridization experiments presented in this study do not exclude derepression mechanisms as proposed by Paul and Gilmour [15]. However, they support the idea that there is also a group of non-histone proteins that activate the transcription of certain sequences in chromatin which are not transcribed from purified DNA to the same extent. The template capacity for RNA is found in the range of 4- 8 % as compared to a total of 38 % of intermediate repetitive sequences in the DNA. Exact evaluation of these numbers depends on assumptions on strand selection and on the spectrum of family sizes of repetitive sequences, but as a rough estimate it appears that 12- 30 % of all families of repetitive sequences are read in a particular tissue; different tissues show an overlap of the order of half of these families, that is 6-15% of the total. While the numbers of genes expressed in a tissue is not known for vertebrates, observations in insects on puffing in giant chromosomes indicate that 15 % of the chromomers are activated [57];of these, only a small fraction (of the order of one tenth) has been shown to be tissue-specific [58]. It is likely that further qualitative and quantitative regulatory selections operate on subsets or individual genes belonging to those families of repetitive sequences which are represented in the RNA transcripts within a tissue. Most reconstitution experiments have been carried out with an unfractionated mixture of non-histone proteins present in the chromatin preparations [15, 19- 22,591. Recently experiments with a non-histone protein fraction of mouse liver obtained by hydroxyapatite chromatography have been reported by MacGillivray et al. [13]. This fraction (which was taken as being representative of the chromatin non-histone proteins) was found to yield reconstituted chromatins in which the template for RNA synthesiswas restricted to the same degree as that found in native chromatin. Our own experiments show that the presence of the lymphocyte NH4 fraction and to a less extent, of the NH3 fraction, permits the reconstitution of a chromatin with template specificitiesresembling native lymphocyte chromatin. A mixture of all lymphocyte non-histone fractions, or the NH1 fraction alone, do not result in lymphocyte-specific templates. It is possible that denatured

325

or inactivated proteins in fraction NH 1, and proteases in unseparated preparations containing fraction NH2, interfere with and prevent proper reconstitution. In our hands, reconstituted liver chromatin produced RNA sequences similar to that transcribed from native chromatin only if reconstitution was performed immediately after dissociation ; after separation of components, only incomplete reconstitution resulted. Again, partially degraded protein may be invoked as an explanation. In lymphocytes, the NH4 fraction is found to be active when present in very small amounts in reconstitution mixtures (1 - 6.4 % wjw of protein to DNA). The amount in native chromatin is 2.8%. It is thus possible that NH4 proteins are partially responsible for activating genes in a tissue-specific manner. It is attractive to suppose that the major protein subfraction detected in the NH4 fraction from lymphocytes is involved in this process. This subfraction has a molecular weight of 30000 and may still be composed of different polypeptides. Since it comprises about 40% of fraction NH4, one can calculate that there are about l o 6 molecules of this component per haploid genome. The results of these experiments suggest that factors comprising only a small proportion of nonhistone components of chromatin are active in tissuespecific regulation of chromatin transcription. We suggest that the tissue specificity of transcription, as detected by repetitive sequence hybridization, reflects only part of the regulatory mechanism within a more complex combinatorial scheme. The possible function of subfractions of the NH4 fraction, in particular of its major protein component in lymphocytes, requires further studies.

REFERENCES 1 . Spelsberg, T. C., Wilhelm, J. A. & Hnilica, L. S. (1972) Subcell. Biochem. 1, 107- 145. L. Shelton, K. R. & Allfrey, V. G. (1970) Nuture (Lond.) 228, 132-134. 3. Benjamin, W. & Gellhorn, A. (1968) Proc. Nut1 Acud. Sci. U.S.A. 59,262-268. 4. Elgin, S. C. R. & Bonner, J. (1970) Biochemistry, 9, 44404441. 5. Van den Broek, H. W. J., Nooden, L. D., Sevall, S. J . & Bonner, J. (1973) Biochemistry, 12, 229-236. 6. Graziano, S. L. & Huang, R. C. C. (1971) Biochemistry, 1 0 , 4110-4177. 7. Shaw, L. M. J. & Huang, R. C. C. (1970) Biuchemisrry, 9, 4530-4542. 8. Arnold, E. A. & Young, K. E. (1972) Biochim. Biophys. A m , 257,482-496. 9. Levy, S., Simpson, R. T.& Sober, H. A. (1912) Biochemistry, Il,I541-1554. 10. Richter, K. H. & Sekeris, C. (1972) Arch. Biochem. Biophys. 148.44-53. 1 1 . Hill, R. J., Poccia, D. L. & Doty, P. (1971) J . Mol. Biol. 61, 445 - 462.

326

H . Bluthmann, S. Mrozek, and A. Gierer : Non-histone Chromosomal Proteins

12. Umanskii, S. R., Tokarskaya, V. I., Zotova, R. N. & Migushind, V. L. (1971) Mol. Biol. 5, 215-222. 13. MacGillivray, A. J., Cameron, A,, Krauze, R. J., Rickwood, D. & Paul, J. (1972) Biochim. Biophys. Actu, 277, 384-402. 14. Rickwood, D. & MacGillivray, A. J. (1975) Eur. J . Biochem. 51,593-601. 15. Paul, J. & Gilmour, R. S. (1968) J . Mol. Biol. 34, 305-316. 16. Smith, K. D., Church, R. B. & McCarthy, B. J. (1969) Biochemistry, 8, 4271 -4277. 17. Axel, R., Cedar, H. & Felsenfeld, G. (1973) Proc. Natl Acad. Sci. U.S.A.70.2029-2032. 18. Gilmour, R. S. & Paul, J. (1973) Proc. Natl Acad. Sci. U.S.A. 70,3440 - 3442. 19. Steggles, A. W., Wilson, G. N., Kantor, J. A., Picciano, D. J., Favley, A. K. & Anderson, W. F. (1974) Proc. Natl Acud. Sci. U.S.A. 71, 1219-1223. 20 Gilmour, R. S. & Paul, J. (1969) J . Mol. Biol. 40, 137-139. 21 Huang, R. C. C. & Huang, P. C. (1969) J . Mol. Biol. 39, 365 378. 22 Bekhor, I., Kung, G. M. & Bonner, J. (1969) J . Mol. Biol. 39,351 364. 23 Teng, C. S., Teng, C. T. & Alfrey, V. G. (1971) J . Biol. Chem. 246, 3597 3609. 24 Blobel, G. & Potter, R. V. (1966) Sciences (Wash. D.C.) 154, 1662-1665. 25. Marushige, K. & Bonner, J . (1966) J. Mol. Biol. IS, 160- 174. 26. Johns, E. W. & Forrcster, S. (1969) Eur. J. Biochem. 8, 547 -551. 27. Tiselius, A,, Hjerttn, S. & Levin, 0. (1956) Arch. Biochem. Biophys. 65, 132-155. 28. Studier, F. W. (1965) J . Mol. Biol. 11, 373-390. 29. Hausen, P., Stein, H. & Peters, H . (1969) Eur. J . Biochem. 9, 542-549. 30. Weber, K. & Osborn, M. (1969) J . Biol. Chem. 244, 44064412. 31. Shapiro, A. L., Viiiuela, E. & Maizel, J . V. (1967) Biochem. Biophys. Res. Commun. 28,815 -820. 32. Panyim, S . & Chalkley, R. (1969) Arch. Biochem. Biophys. 130,337- 346. 33 Spelsberg, T. C., Steggles, A. W., Chytil, F. & O’Malley, B. W. (1972) J . Biol. Chem. 247, 1368-1374. 34 Burgess, R. R. (1969) J . Biol. Chem. 244, 6160-6167. 35 NuRlein, C. & Heyden, B. (1972) Biochem. Biophys. Res. Commun. 47,282-289. ~

~

~

36. Gillespie, S. & Gillespie, D. (1971) Biochem. J. 125, 481 -487. 37. Bliithmann, H., Briick, D., Hiibner, L. & Schoffski, A. (1973) Biochem. Biophys. Res. Commun. 50,91- 97. 38. Bonner, J., Chalkley, G. R., Dahmus, M., Fambrough, D., Fujimura, F., Huang, R. C. C., Huberman, J., Jensen, R., Marushige, K., Ohlenbusch, H., Olivera, B. & Widholm, J. (1968) Methods Enzymol. 12, 3-65. 39. Schmidt, G. & Thannhauser, S. J . (1945) J . Biol. Chem. 161, 83 - 89. 40. Keck, K. (1956) Arch. Biochem. Biophys. 63, 446-451. 41. Hubbard, R. W., Matthew, W. T. & Dubovik, D. A. (1970) Anal. Biochem. 38,190- 201. 42. Dische, Z. & Schwarz, K. (1937) Microchim. Acra, 2, 13-19. 43. Bonner, J., Dahmus, M. E., Fambrough, D., Huang, R. C. C., Marushige, K. & Tuan, D. Y. H. (1968) Science (Wash. D.C.) 159,47-56. 44. Tan, C. H. & Miyagi, M. (1970) J . Mol. Biol. 50, 641 -653. 45. Spelsberg, T. C., Hnilica, L. S. & Ansevin, A. T. (1971) Biochim. Biophys. Acta, 228, 550- 562. 46. Soeiro, R. & Darnell, J. E. (1969) J . Mol. Biol. 44, 551 - 562. 47. Faulhaber, I. & Bernardi, G. (1967) Biochim. Biophys. Actu, 140, 561 - 564. 48. Britten, R. J. (1969) in Problems in Biology: RNA in Development (Hankey, E. W., ed.) pp. 187-216, University of Utah Press, Salt Lake City. 49. Davidson, E. H., Hough, B. R., Amenson, C . S. & Britten, R. J. (1973) J. Mol. Biol. 77, 1-23. 50. Graham, D. E., Neufeld, B. R., Davidson, E. H. & Britten, R. J. (1974) Cell, 1, 127-137. 51. Bonner, J., Garrard, W. T., Gottesfeld, J., Holmes, D. S., Servall, J. S. & Wilkes, M. (1973) Cold Spring Hurbor Symp. Quant. Biol. 38, 303 - 310. 52. Bonner, J. & Wu, J. R . (1973) Proc. Natl Acad. Sci. U.S.A. 70,535- 537. 53. Davidson, E. H. & Britten, R. J. (1973) Q. Rev. Biol. 48, 565 613. 54. Gierer, A. (1967) Naturwissenschaften, 54, 389- 396. 55. Gierer, A. (1973) Cold Spring Harbor Symp. Quant. Biol. 38, 951-961. 56. Kauffman, S. (1973) Science (Wash. D.C.) 181, 310-318. 57. Pelling, C. (1964) Chromosoma, 15, 71 - 122. 58. Berendes, H. D. (1966) J . Exp. Zool. 162, 209-217. 59. Barrett, T., Maryanka, D., Hamlyn, P. H . & Gould, H. J. (1974) Proc. Nut1 Acud. Sci. U . S . A . 71, 5057-5061. ~

H. 1Bliithmann. S. Mrozek, and A. Gierer, Max-Planck-Institut fur Virusforschung, D-7400 Tubingen, SpemannstraBe 35, Federal Republic of Germany