Methods f i r Selective Extraction of Chromosomal Nonhistone Proteins JEN-FU CHIU, HIDE0 FUJITANI, LUBOMIR S. HNILICA
Department of Biochemistry. Vanderbilt UniversitySchool of Mediciw, Nashville, Tennessee
I. Introduction During rapid cell proliferation, chromosome puffing, and chromosomal stimulation, there is an increase in the content, synthesis, and metabolic activity of chromosomal nonhistone proteins (1-5). Several studies have implicated chromosomal nonhistone proteins as likely candidates for gene regulatory functions. Fc: example, phosphoproteins, a significant component of the chromosomal nonhistone proteins, are heterogeneous and tissue-specific (6-9), can alter the rate of RNA synthesis in vitro (10-14), and bind specifically to homologous DNA (10,15,16). Changes in the phosphorylation of chromosomal nonhistone proteins were correlated with activation of gene activity in a variety of systems (17-20). Since chromatin nonhistone proteins are very heterogeneous (perhaps over hundreds of individual species), they may have many biological functions. Presently, there are no methods available for a complete separation of all biologically active proteins by a single standard procedure. Techniques taking advantage of particular biological properties of chromosomal nonhistone proteins, such as enzymic activity, transcriptional regulations, immunological specificity, etc., are most useful and probably indicate the future direction of this rapidly moving field. The extent of the tissue specificity of chromosomal nonhistone proteins 283
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was shown immunologically by Chytil and Spelsberg (21). These authors used dehistonized chromatin to elicit tissue-specific antibodies in rabbits. Wakabayashi and Hnilica (22) confirmed these observations and reported that antibodies can be obtained which are specific for various tissues as well as for normal or transformed cells. They also found that the immunospecificity of dehistonized chromatin depended on the presence of tissue-specific complexes between DNA and some nonhistone proteins in chromatin. Only intact chromatin or samples obtained by the reconstitution of nonhistone proteins with the DNA isolated from the same species (homologous) were immunoreactive. Free rat DNA or nonhistone proteins were inactive. Conversely, Wakabayashi et al. (23) digested dehistonized chromatin with DNase I. The immunospecificity of dehistonized chromatin was lost after this treatment. We have recently reported (24) that neoplastic growth changes the immunological specificity of chromatin nonhistone protein complexes with DNA to a new type which is characteristic for the malignant tumor. Complement fixation has shown that the tissue specificity of a fraction of chromatin nonhistone proteins changes gradually with the development of hepatomas in rats fed a carcinogenic diet (24). Nonhistone protein-DNA complexes from fast-growing Morris hepatomas were more immunoreactive than similar proteins from better differentiated, slow-growing tumors (25). We describe here a simple technique for separation of chromosomal proteins into three principal fractions: the bulk of chromosomal nonhistone proteins (UP); histones (HP); and a small, biologically active fraction of nonhistone proteins (NP) with affinity for DNA. The immunological tissue specificityof this latter fraction was used as a marker in developing the described fractionation scheme. In addition to its immunological specificity, the DNAbinding fraction NP was found to contain components active in the in vitro transcriptional regulation of specific genes and in mediating the binding of androgens by target tissue chromatin.
A. Preparation of Nuclei Unless specified otherwise, all work is performed at 2 O-4OC. Novikoff ascites tumor cells are routinely carried in male Sprague-Dawley rats. After collection of the ascites fluid, the cells are diluted with 2.5 volumes of 0.25 M sucrose-5 mM MgC1,-10 mM Tris-acetate (PH 7.2) and washed with
SELECTIVE EXTRACTION OF CHROMOSOMAL NONHISTONE PROTEINS
this buffer several times. Each wash is followed by low-speed centrifugation (100 g, 15 minutes) to separate the cells. About 20-30 ml of the washed, packed cells are homogenized vigorously in 80 ml of 10 mM Tris-acetate (PH 7.4) in a tight-fitting (Potter-Elvehjem type, 0.005 in. clearance) Teflonglass homogenizer. The cells are pelleted (700 g, 5 minutes), resuspended by vigorous homogenization in the same buffer, and passed through a Chaikoff press using a 27-pm pestle. The crude nuclei are pelleted (700 g, 5 minutes) and washed twice by homogenization in 0.25 M sucrose-5 mM MgC1,10 mM Tris-HC1 (PH 7.4). The nuclear pellet is resuspended in 60 ml of 2.2 M sucrose-5 mM MgC1,-10 m M Tris-HC1 (PH 7.4) and centrifuged at 50,000 g for 50 minutes. The pellet contains nuclei free of cytoplasmic tags, while whole or partially broken cells float. The purified nuclei are gently washed once in 40 ml of 0.25 M sucrose-1.5 mM MgC1,-10 mMTris-HC1 (ph 7.4) and finally used for chromatin preparation. Rat livers (1 5 gm) are homogenized in 150 ml of 0.32 M sucrose containing 5 mM MgCl, in a Teflon-glass (Potter-Elvehjem type) homogenizer using eight up and down strokes at 2000 rpm. After filtration through four layers of gauze, the homogenate is centrifuged at 1000gfor 10 minutes. The crude nuclear pellet is then suspended in 150 rnl of 2.2 M sucrose containing 5 mM MgCl, and centrifuged at 75,000 g for 1 hour. The purified pelleted nuclei are suspended in 0.32 M sucrose-5 mM MgCl, and collected by centrifugation at 1000 g for 10 minutes.
B. Preparation of Chromatin Several procedures for the isolation and purification of interphase chromatin have been tried in our laboratory. Best results were achieved by the procedure based on previous studies of several investigators (26-28) with some modifications (29). Nuclei are gently homogenized in 50 volumes of 0.08 M NaC1-0.02 M ethylenediaminetetracetate (EDTA) (PH 6.3) using a Potter-Elvehjem type Teflon-glass homogenizer driven by hand. The suspension is centrifuged at 5000 g for 10 minutes, and the pelleted chromatin is resuspended in the same buffer. The homogenization and centrifugation are repeated twice. The chromatin pellet is then extracted once with 5 volumes of 0.35 M NaCl. Finally, the cromatin is rehydrated by homogenization in 5 volumes of 1.5 mM NaC1-0.15 mM sodium citrate (PH 7.0) and centrifugation at 20,000 g for 10 minutes. This step is repeated once more. The purified chromatin can be stored frozen in 1.5 mM NaC1-O.15mM sodium citrate (PH 7.0) at -20°C. When needed, the chromatin solution is thawed and rehomogenized in a hand-operated Teflon-glass homogenizer to disaggregate the chromatin.
JEN-FU CHIU et al.
C. Fractionation Procedure The fractionation scheme developed in our laboratory (24)is based on the solubility properties of chromosomal proteins in 5 M urea at three different pH values and salt concentrations. The first extraction is performed at pH 7.6 and a relatively low ionic strength. It removes the bulk of proteins that are not firmly associated with DNA in chromatin. The second extraction at high ionic strength and relatively low pH (5.0) takes advantage of the solubility of histones under these conditions. The DNA-binding nonhistone proteins are poorly soluble at this pH. They can be solubilized by increasing the pH to 8.0 in the final extraction step. Isolated rat liver or Novikoff hepatoma chromatin is gently homogenized in 5.0 M urea containing 50 mM sodium phosphate buffer (PH 7.6). After adjusting the DNA concentration to approximately 6 ODunits/ml at 260nm, and stirring for 2 to 3 hours, the mixture is centrifuged at 20,000 g for 30 minutes. The combined supernatants contain 90-95% of chromosomal nonhistone proteins (UP fraction). Histones (HP fraction) are removed from the remaining chromatin pellets (UC) by resuspending them gently in 5.0 M urea -2.5 M NaCl-50 mM sodium succinate (PH 5.0) (final concentration of chromatin is 6 OD/ml at 260 nm) and centrifuging the viscoussolution at 110,000 g for 36 hours. DNA and the associated nonhistone proteins form a pellet (HC), while histones with small amounts of other proteins remain in the supernatant. Finally, the DNA-binding protein fraction (designated as NP fraction) is recovered by dissociation in 5.0 M urea-2.5 MNaC1-50mM Tris-HC1 (pH 8.0) and centrifugation at 110,000 g for 48 hours. The NP fraction in the supernatant represents about 3 4 % of the total chromatin protein content and the pellet (NC) is DNA with a small amount of associated protein. This procedure is summarized in Fig. 1. Because of the time-consuming centrifugations and relatively large volumes, the above fractionation procedure was modified by using hydroxylapatite column chromatography instead of centrifugation to separate histones (HP) and nonhistone proteins (NP) from DNA (30).The isolation of UP proteins follows the same procedure as described above. The residual chromatin pellets are rehomogenized in 1.5 m M NaCl-0.15 mM sodium citrate. After adjusting the DNA concentration of chromatin to approximately 1 mg/ml, the UC suspension is dialyzed against the same buffer overnight with three changes. The dialyzed suspension is then sonicated with a Branson sonifier cell disruptor in 15-second intervals for a total of 2 minutes and adjusted to 2 M NaCl-5 M urea-1 mM sodium phosphate (pH 6.8). It is important to remove urea from the chromatin suspension before its exposure to ultrasound if immunologically active NP proteins are desired. If the sonication is performed in the presence of urea, the immunological activity
SELECTIVE EXTRACTION OF CHROMOSOMAL NONHISTONEPROTEINS
5.0 M urea in 50 mM sodium phosphate buffer (pH 7.6)
20,000 g for 30 minutes
Supernatant (UP) (Nonhistone proteins, 44-47%)
2.5 M NaCI-Si urea in 50 mM sodium succinate buffer (pH 5.0)
Centrifugation at 110,OOOg for 36 hr
M NaCI-5 M urea in 50 mM Tris-HC1 buffer (pH 8.0)
Centrifugation at 110,OOOg for 48 hr I
Supernatant (NP) (DNA-associated Nonhistone proteins 3-573
Pelle: (NC) (DNA + firmly bound proteins about 1%)
FIG.1. Scheme for fractionation of chromatin proteins by sequential extraction method.
is destroyed. After centrifugation at 10,OOO g for 10minutes the supernatant is applied to a hydroxylapatite column (8,31).After elution of the unretained histone fraction (HP) with 2 M NaCl-5 M urea-I mM sodium phosphate (pH 6.8), the nonhistone protein fraction NPis partially fractionated by stepwise elution using 50 mM and 200 mM sodium phosphate (pH 6.8) containing 2 M NaCl and 5 M urea. Finally, DNA can be eluted with 0.5 Msodium phosphate @H 6.8) containing 2 M NaCl and 5 M urea. While the initial steps are performed at 4 "C, the temperature is increased to 25 "Cwhen the phosphate concentration is raised to 200 mM and higher. The elution profile of hydroxylapatite column chromatography is shown in Fig. 2.
111. Properties of the Chromatin Fractions Selectivity of the described chromatin fractionation procedure is shown in Fig. 3. The polyacrylamide gel electrophoresis performed in the presence of sodium dodecylsulfate (SDS) shows that the first step of this fractionation schedule removes most of the nonhistone proteins together with small
JEN-FU CHIU el al.
I I I
4 5 6 7 8 91011 0 .
E f f l u e n t v o l u m e ( m l xlO-')
FIG.2. Hydroxylapatite column chromotography of Novikoff ascites hepatoma chromatin (UC fraction). Isolated chromatin (200 mg DNA) was first extracted with 5 M urea-50 mM sodium phosphate41 mM phenylmethylsulfonyl fluoride (PH7.6)to remove the majority of loosely ,bound nonhistone proteins (UP). The resulting pellet (UC) was hydrated by dialysis against 1.5 mM NaCI-0.15 mM sodium citrate @H 7.0),sonified, and the solution was brought to 5 M urea-2 M NaCI-1 mM sodium phosphate41 mM phenylmethyl sulfonyl fluoride. After removal of insoluble debris by centrifugation (lO,000 g, 10 minutes) the solution was applied to a 5.1 cm x 17 cm (bed volume = 350 ml) hydroxylapatite column and eluted with the same buffer at a flow rate of 1.5 mlhinute. The phosphate concentration was then raised stepwise to 50 mM, 200 mM, and finally to 500 mM.
. ,." c .. .. . . .. .. amounts 01 nistones. I ne secona rractionation step removes essentially all the histones. The NP protein fraction consists of two to three low-molecularweight and several high-molecular-weight components. The efficiencies of the residual chromatin pellets from the individual fractionation steps in serving as a template for the in vitro RNAsynthesis are shown in Table I. The initial extraction of chromatin with buffered 5.0 M urea which removed most of the nonhistone proteins approximately doubled the templating efficiency of the residual chromatin. As can be expected, the removal of histones during the second extraction step derepressed most of the DNA. Finally, the templating efficiency of the NC pellets was only slightly lower than that of the control DNA. The change in templating efficiency of chromatin fractions can result from changes in the total number of available initiation sites or in the rate I
SELECTIVE EXTRACTION OF CHROMOSOMAL NONHISTONE PROTEINS
FIG.3. Polyacrylamide gel electrophoresis of the UP,HP, and NP protein fractions from rat liver chromatin. The electrophoresis was performed in the presence of sodium dodecyl sulfate. The origin of migration is at the top of the gels. From Chiu et al. (33).
of chain elongation. The number of initiation sites available for RNA polymerase on DNA and chromatin can be assayed under conditions that allow only one RNA molecule to be made at each available initiation site (32,33). The experiment is performed by allowing a large excess of enzymes to initiate transcriptions in low salt with only three kinds of nucleotides present. The absence of the fourth nucleotide prevents extensive chain elongation and thus inhibits the formation of multiple initiations at the same site. After 15 minutes, the mixture is brought to 0.4M (NH,),SO,. This prevents further initiations. The fourth nucleotide is then added to permit the elongation of the already initiated nucleotide chains. The number of growing chains is then calculated from sucrose gradient centrifugation of the transcripts. The number (average size) for the RNAs transcribed from pure DNA, NC, HC, and UC pellets, and intact chromatin is between 400 and 600 nucleotides, and the number of RNA molecules is equal to the number of initiation sites (33). From the amount of nucleotides incorporated and the average chain length, the number of initiation sites can be calculated (Table 11). Using 1.5 pg of template DNA (2.2 nmol of base pairs) we observed 1.72 pmol of initiation sites on rat DNA and 0.31, 0.57, 1.51, and 1.61 pmol on rat liver
JEN-FU CHIU et al.
TABLE I TEMPLATING ACTIVITYOF RESIDUAL PELLETS RESULTINGFROM THE SCHEME IN FIG. 1"' Percent [IHIUTP free DNA (pmol/pg of DNA) activity
22.1 50.0 167.5 176.8 195.4
HC NC DNA
11.3 25.6 85.7 90.5 100.0
@FromChiu ef al. (33). bThe results are averages of several preparations of rat liver chromatin. Free DNA was isolated from rat spleen. The reaction mixture (0.25 ml final volume) consisted of 40 mM TrisHC1 buffer (pH 8.0)-120 mM KCl-O.l mM EDTA-2.5 mM MnCl,-1.0 mM dithiothreitol0.08 mM each ATP, GTP, CTP-0.02 mM 'H (sp act 1 Cihol). The concentration of chromatin in each assay was 10-15 pg in respect to DNA, together with 20 units of E. coli RNA polymerase (sp act 600 units/mg of protein). The assay mixtures were incubated at 37°C for 15 minutes, and the reaction was terminated by adding 2 d of 10% trichloroacetic acid-1% sodium pyrophosphate solution.
TABLE I1 DETERMINATION OF THE NUMBER OF GROWING CHAINSBY SUCROSEGRADIENTANALYsIS~"
Template DNA NC HC
Nucleotides incorporated (Pmol)
Chain length (nucleotides)
1015 918 906 308 155
590 570 600 540 500
1.72 1.61 1.51 0.57 0.3 1
From Chiu ef al. (33). 'Assay conditions were as described in Table I. The average chain length was determined from 0.2 ml of each assay tube by sucrose gradient analysis. Each tube contained 1.5 pg of chromatin as indicated. @
SELECTIVE EXTRACTION OF CHROMOSOMAL NONHISTONE PROTEINS
chromatin, UC, HC, and NC fractions, respectively. The removal of the nonhistone proteins UP only slightly increased the number of initiation sites, while the subsequent removal of histones HP increased the initiation considerably. Final removal of the NP proteins did not further increase the initiation. This indicates that histones may function as general repressors and the nonhistone proteins NP may serve as specific regulators of either negative or positive transcriptional control. It was shown by Wakabayashi et al. (16) that the nonhistone protein fraction of chromatin which could be identified by its immunological tissue specificity also contained proteins with affinity for homologous native DNA. To show that the immunospecificity of chromatin nonhistone proteins was not lost during the fractionation schedule, the immunoreactivity of the intact chromatins and of the UC, HC, and NC residual chromatin pellets was assayed. The dehistonized Novikoff hepatoma chromatin was used as antigen for the immunization of rabbits. The removal of urea-soluble proteins UP and histones HP did not change much theextent of complement fixation (Fig. 4). However, the complement-fixing ability of the complex decreased considerably when the DNA-binding proteins NP were removed during the last fractionation step (NC pellet). The residual immunoactivity of the NC pellet was probably caused by traces of NP proteins still present in the NC pellet.
Antigen (ug D N A )
FIG. 4. Complement fixation of chromatin preparations performed in the presence of antiserum against dehistonized Novikoff hepatoma chromatin. All data were corrected for anticomplementarity. (+) Novikoff hepatoma native chromatin; ( A ) Novikoff hepatoma Novikoff hepatoma HC pellet, i.e., UC pellet, i.e., chromatin devoid of UP proteins; (0) chromatin devoid of UP and HP proteins; (A) Novikoff hepatoma NC pellet, i.e., chromatin devoid of UP,HP, and NP proteins; (H) normal rat liver chromatin. From Wang er al. (34).
JEN-FU CHIU et al.
The identity of immunologicallytissue-specific proteins in the NP fraction can be further ascertained by reconstitution experiments. The nonhistone protein fraction NP can be isolated from one tissue type and reconstituted with the DNA isolated from another tissue, e.g., Novikoff hepatoma NP and normal rat liver DNA. As can be seen in Fig. 5 , the immunospecificity of the resulting complexes is determined by the tissue donating the nonhistone proteins NP. To learn if the specific antibodies are directed against the nuclear material and not against some cytoplasmic components that could have associated with chromatin during its isolation, intracellular localization of these antigens can be performed. We use the horseradish peroxidase method to localize antigens of normal rat liver cells. As shown in Fig. 6, the antibodies localize in the nuclei and not in the cytoplasm. Proteins comprising the immunologicallyactive chromosomal nonhistone fraction NP also bind to the DNA. In 10mMNaC1-10 mMTris-HC1 (PH8.0) the binding sites available on homologous DNA (rat spleen) are saturated at the NP protein :DNA ratio of approximately 1.5 :100 (w/w). The reciprocal plot of the saturation curve forms a straight line intercepting the abscissa at the K, value of about 6.7 x 10 -g (34). The binding of NP proteins is species specific. As can be seen in Table 111, both rat spleen and liver DNA bind the liver NP proteins equally well. Calf thymus DNA exhibits a small
AntiNovikoff hepatoma N P C " A
5 u9 DNA
FIG. 5. Complement fixation of native and reconstituted NP-DNA complexes from rat liver and Novikoff hepatoma in the presence of antiserum against Novikoff hepatoma NPDNA. All experimental points were corrected for anticomplernentarity.( A ) Novikoff hepatoma chromatin (native). (A) reconstituted complex of Novikoff hepatoma NP and normal rat liver DNA (NPN-DNAL); (0) normal rat liver chromatin (native); (e)reconstituted complex of rat liver NP and Novikoff hepatoma DNA (NPL-DNA,). From Chiu et ul. (25).
SELECTIVE EXTRACTION OF CHROMOSOMAL NONHISTONEPROTEINS
FIG.6 . Localization of antigens in rat liver by the horseradish peroxidase bridge technique in the presence of rabbit serum against dehistonized rat liver chromatin.
but significant binding while the affinity of rat liver NP proteins to chicken erythrocyte or Escherichiu coli DNA is negligible. To study the association of NPproteins with DNA fractionated according to its renaturation kinetics, sheared rat spleen DNA can be separated by hydroxylapatite chromatography into repetitive, middle repetitive, and slowly renaturing fractions. The 4 Cot values of these fractions in our experi-
JEN-FU CHIU et al.
TABLE I11 INTERACTIONS OF R A T AND
Source of DNA Rat spleen Rat liver Calf thymus Chicken erythrocyte E. coli
LIVERNP FRACTION WITH HOMOLOGOUS
DNA 400 400 400 400
Protein applied (PP)
Protein bound (Pg)
40 40 40 40 40
5.8 5.6 1 .o 0.3 0.1
Protein/DNA binding ratio 0.0145
“From Wang ef al. (34). bThe formation of DNA-protein complexes was assayed by sucrose density gradient centrifugation using 1251-labeledNP protein. The binding ratios represent weight percentages of protein retained by the DNA.
ment were 7.1, 79.4, and about 1000, respectively. The results in Table IV show about 2-fold preference of the NP proteins for the unique sequence DNA. The binding of NP proteins to single- and double-stranded unique sequence DNA is also shown in Table IV.The rat liver NP proteins exhibit a significant preference for native DNA.
IV. Conclusions The fractionation scheme described here is based principally on solubility of the three protein groups in salt and urea at various pH values. Although fraction NP binds selectively to homologous DNA, its separation from histones in sodium succinate buffer (pH 5.0) is principally facilitated by its insolubility at relatively low pH. The NP fraction is heterogeneous. Recent attempts in our laboratory to fractionate the NP proteins resulted in separation of the three principal low-molecular-weightbands from the remaining proteins of higher molecular weight (over 30,000). As determined by complement fixation, the immunologically tissue-specific proteins are in the high-molecular-weight fraction of the NP proteins. Isolated chromatin can be fractionated by a variety of techniques into transcriptionally active and inactive fractions. If the fractionation of chromatin is accomplished by shearing and subsequent sucrose density gradient centrifugation or divalent cation precipitation, the immunologically tissuespecific proteins are selectively accumulated in the extended, transcriptionally active chromatin (34,35). As was mentioned in the introduction,
SELECTIVE EXTRACTION OF CHROMOSOMAL NONHISTONEPROTEINS
TABLE IV RAT LIVERNP PROTEINBINDING TO FRACTIONATED DNA'
DNA fraction Double-stranded repetitive Double-stranded middle repetitive Double-stranded unique sequence Single-stranded unique sequence
Protein applied (pg)
Protein bound (pg)
RoteidDNA binding ratio
'Unless single-strandedDNA is used, fractionated DNAs are renatured to the same percentage of renaturation (80%reassociated). the DNA-binding fraction NP contains macromolecules which can influence the in vitro transcription of chromatin. Using the chicken reticulocyte chromatin system, cDNA probes were prepared by reverse transcription of purified globin mRNA. It was found that the in vitro transcription of globin genes by reticulocyte chromatin depends on the presence of proteins contained in the reticulocyte chromatin fraction NP. Under normal circumstances, chicken liver or brain chromatins do not transcribe in vivo or in vitro RNA sequences complementary to chicken globin cDNA probes. However, when the reticulocyte fraction NP was reconstituted to liver or brain chromatin devoid of its own NP proteins, the final product transcribed the globin genes at a frequency comparable to that of isolated native reticulocyte chromatin (30). Although the detailed mechanism of steroid hormone action is not known, it is anticipated that after its initial association with cytoplasmic receptor, the steroid hormone is transferred into the nucleus where it associates with acceptor sites specific for the target chromatin. It has been shown in several laboratories that chromosomal nonhistone proteins of the target tissue play an important role in the final and selective binding of the steroid-receptor complex. We have found (36) that the NP fraction proteins are principally responsible for the target tissue specific binding of steroid hormone complexes with cytoplasmic receptors. It can be concluded from this brief discussion of the biological properties of proteins comprising the NP fraction that, in addition to its immunological tissue specificity, the NP fraction may be an important source and starting material for the purification and characterization of macromolecules active in the transcriptional regulation of specific genes.
JEN-FU CHIU ef al. ACKNOWLEDGMENT
This work was supported by National Cancer Institute Contract NO1-CP-65730 and USPHS Grant CA- 18389.
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