A general method for purification of H1 histones that are active for repression of basal RNA polymerase II transcription

PROTEIN EXPRESSION AND PURIFICATION 2, 162-169 (1991)

A General Method for Purification of HI Histones That Are Active for Repression of Basal RNA Polymerase II Transcription

Glenn E. Croston, Lucy M. Lira, and James T. Kadonagal Department of Biology, 0322, and Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093

Received February 13,1991, and in revised form May 21, 1991

Hl histones were purified from extracts of salt- treated nuclei as a co-product of RNA polymerase II transcription factors from both Drosophila embryos and HeLa cells by a simple and general method. This procedure was also used to purify Hl as co-product of the core histones from calf thymus. The key steps in this purification exploit the solubility of Hl in 2.26 M ammonium sulfate and the chromatographic properties of the highly charged Hl molecules on a phenyl-Sepharose resin, Hl that is prepared by this procedure is active for in vitro repression of basal RNA polymerase II transcription. This method provides a new means of purifying Hl by a mild procedure that is likely to be generally useful for studies of transcription and chromatin structure. 0 1991 Academic Press, Inc.

Histone Hl is a central component of chromatin (for reviews, see Refs. (1,2)). Hl and its variants, H1° and H5, are often referred to as the linker histones because of their association with the linker DNA between nucleosomal cores, and there is roughly 1 molecule of Hl per nucleosome repeat (3). Hl is often viewed as com- prising a globular domain, which can be prepared by partial digestion with trypsin (4), that is flanked by N- and C-terminal “tails.” It is generally believed that the lysine-rich tails bind to the linker DNA while the globular domain interacts with the nucleosomal dyad (4,s). In addition, Hl is required for condensation of the lo-nm chromatin filament into the 30-nm filament (6-9). Hence, Hl has an integral role in the structure of chromatin.

Hl, along with nucleosomal cores, also appears to be involved in repression of gene expression (for reviews,

’ To whom correspondence should be addressed.

162

see Refs. (1,2)). Biochemical studies of RNA polymerase III transcription in Xenopus have directly demon- strated repression of transcription by Hl (10-12). Pro- tein-DNA cross-linking experiments have shown that Hl and core histones are either depleted or reconfigured at the promoter regions of active genes in vivo (13,14). In addition, nonmethylated CpG islands, which appear to represent active chromatin, were found to be depleted of Hl and core histones (15). Moreover, Hl-dependent higher order structures were found to have a more “open” conformation in active genes (16). These data collectively suggest that depletion or reconfiguration of Hl and nucleosomes at promoter regions is a prerequi- site for gene activation.

We have recently found that purified Hl, in the absence of nucleosomes, is capable of repression of basal RNA polymerase II transcription in vitro (17). Further- more, sequence-specific transcription factors, such as Spl, GAL4-VP16, and GAGA factor, were able to coun- teract Hl-mediated repression of transcription. These findings suggest that promoter- and enhancer-binding factors function, at least in part, to relieve chromatin- mediated repression of basal transcription. Since these studies were carried out in the absence of nucleosomes, however, the interpretation of the experiments is some- what limited. Nevertheless, the biochemical data com- bined with the in vivo observations that Hl is depleted at active promoter regions are consistent with a model for gene activation that involves displacement or reconfiguration of Hl at promoters by sequence-specific transcription factors.

In the course of our studies of Hl, we have developed an efficient method for purification of the protein from standard in vitro transcription extracts. Standard nuclear extracts for in vitro transcription by RNA polymerase II (for instance, see Refs. (l&19)) typically involve salt extraction of transcription factors from nuclei at salt concentrations at which Hl is also efficiently ex-

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PURIFICATION OF Hl HISTONES 163

tracted (20). We have devised a general procedure for purification of Hl from such nuclear extracts to >95% homogeneity. Two key properties of Hl that are ex- ploited in this purification method are its solubility in 2.26 M ammonium sulfate and the chromatographic properties of the highly charged protein on a phenyl-Se- pharose resin. Moreover, this mild, two-column procedure yields Hl that is active for repression of basal RNA polymerase II transcription.

EXPERIMENTAL PROCEDURES

Materials and Methods

Column chromatography was carried out with a Pharmacia-LKB fast-protein liquid chromatography (FPLC) system. Phenyl-Sepharose CL-4B andS-Sepha- rose Fast Flow resins were obtained from Pharmacia- LKB. Nonidet P-40 was purchased from United States Biochemicals. Biochemicals and reagents were obtained from Sigma Chemical Co., Aldrich Chemical Co., and Fisher Scientific. Protein concentrations were determined with the bicinchoninic acid protein assay (Pierce) by using bovine serum albumin as a protein standard. The salt concentrations in protein fractions were determined by using a Radiometer CDMSO conductivity meter with the appropriate buffer standards. All buffers and solutions were prepared with deionized, glass-dis- tilled water. HEMG buffer is 25 mM Hepes (K+), pH 7.6, 0.1 mM EDTA, 12.5 mM MgCl,, 10% (v/v) glycerol, 1 mM dithiothreitol, and the indicated concentration of KCl. HEG buffer is identical to HEMG, except that it does not contain MgCl,. Buffer AB is 15 mM Hepes (K+), pH 7.6,llO mM KCl, 5 mM MgCl,, 0.1 mM EDTA, 2 mM dithiothreitol, 1 mM sodium bisulfite, 1 mM ben- zamidine, and 0.2 mM phenylmethylsulfonyl fluoride. The latter three components of buffer AB were added to inhibit the action of proteases.

In Vitro Transcription Assays

In vitro transcription reactions and primer extension analysis of the RNA were carried out as described previ- ously (21-23). Drosophila nuclear extracts were prepared by various methods (19,22,24), and the transcription factors were subjected to two successive cycles of precipitation with 2 M ammonium sulfate to deplete the preparations of histone Hl (17,25), which is ineffi- ciently precipitated with ammonium sulfate. Alterna- tively, transcription reactions could be carried out with a soluble nuclear fraction (23) that is deficient in Hl. The presence of Hl or any other nonspecific DNA binding inhibitor of transcription can be determined easily by carrying out in vitro transcription reactions with relatively low levels (~25 ng) of template DNA in the presence or absence of 100 to 200 ng of a nonspecific competitor DNA, such as pUC. If the addition of the nonspe-

cific competitor DNA yields an increase in the level of transcription, it is likely that a DNA-binding inhibitor of RNA synthesis is present in the preparation of transcription factors. The only DNA-binding transcriptional inhibitor that we have found in Drosophila nuclear extracts is Hl.

Hl-mediated repression of RNA polymerase II transcription was quantitatively measured as a nonspecific DNA-binding activity that represses RNA synthesis in vitro. One unit of transcription repression activity was defined to be the amount of protein required to repress >95% of the transcription from 100 ng of covalently closed, circular pKr template DNA, which contains the promoter region of the Drosophila Kriippel gene (21). Purified Drosophila Hl possesses a specific activity of 7140 units/mg. In these assays, two DNA templates that yield distinguishable transcripts were used. Hl (typically, 50 to 140 ng) was incubated with the first template DNA (100 ng) for 20 min at 4°C. Next, the second template DNA (100 ng), nuclear extract (150 pg), and ribonucleoside triphosphates (to a final concentration of 0.5 mM in each of the four ribonucleoside triphosphates) were added at 4”C, and then the transcription reaction was performed at 21°C for 30 min. Transcripts were detected by primer extension analysis. The medium for the incubation and the transcription reactions was 0.5~ HEMG buffer (12.5 mM Hepes (K+), pH 7.6, 50 PM EDTA, 6.25 mM MgCl,, 0.5 mM dithiothreitol) containing 50 mM KCl, 1% (w/v) polyethylene glycol compound (average molecular weight, 15,000-20,000), and 1% (w/ v) polyvinyl alcohol (average molecular weight, 10,000). The relative amounts of RNA synthesized were determined by using an LKB Ultroscan scanning densitome- ter. When 0.5 to 1 unit of Hl was added to the first template, there was moderate (50%) to complete (>95%) inhibition of transcription from the first template DNA, whereas there was either no detectable decrease or only a slight (~10%) decrease in transcription from the second template DNA. In this manner, it was possible to distinguish transcription repression by Hl, which selectively represses the first template DNA, from an overall decrease in the level of transcripts caused by soluble agents such as RNases, DNases, and phosphatases, which would nonspecifically reduce the amount of intact RNA derived from both the first and the second template DNAs.

Purification of Hl from Drosophila Embryos

Preparation of the crude nuclear fraction. All opera- tions were performed at 4’C. Drosophila embryos (50 to 150 g) were collected between 0 and 12 h after fertiliza- tion, and nuclei were prepared as described by Wampler et al. (22). The nuclei were suspended in buffer AB (1 ml buffer/g embryos) with a 40-ml Dounce homogenizer with a B pestle. To extract Hl (as well as the general

164 CROSTON, LIRA, AND KADONAGA

RNA polymerase II transcription factors) from the nuclei, 4 M ammonium sulfate (Na+), pH 7.0 (stored at room temperature), was added (l:lO, v/v, 4 M ammonium sulfate:nuclei suspension) to give a final concentration of 0.36 M ammonium sulfate. The viscous mixture was placed on a rotating wheel for 20 min and then subjected to centrifugation in a Beckman 45 Ti rotor at 35,000 rpm (142,000g) for 1 h. The supernatant was collected, and the general RNA polymerase II transcription factors were precipitated by the addition of solid, pulverized ammonium sulfate (0.3 g/ml of supernatant) to a final concentration of 2.26 M. This mixture was magnetically stirred for 15 min and then subjected to centrifugation in a Sorvall SS-34 rotor at 15,000 rpm (26,900g) for 20 min. Histone Hl was present in the supernatant. When desired, the general transcription factors were isolated from the pellet as described by Wampler et al. (22). Glycerol was added to the supernatant to a final concentration of 10% (v/v), and, if desired, the mixture was frozen in liquid nitrogen and stored at -100°C. In a typical preparation, 80 g of embryos yielded 100 ml of supernatant possessing a protein concentration of 2.5 mg total protein/ml.

Phenyl-Sepharose CL-4B chromatography. The supernatant from the 2.26 M ammonium sulfate precipitation (100 ml) was thawed in cold (4°C) water and clari- fied by centrifugation in a Sorvall SS-34 rotor at 10,000 rpm (12,OOOg) for 10 min. The liquid containing Hl was separated from the insoluble material at both the bot- tom and the top of the centrifuge tubes. The protein was then subjected to chromatography with phenyl-sepharose CL-4B resin: column volume = 30 ml; column di- mensions (diameter X height) = 1.6 X 15 cm; flow rate = 0.5 ml/min; fraction size = 4.0 ml; full scale absorbance range at 280 nm = 2 absorbance units. The sample (in buffer AB containing 2.26 M ammonium sulfate and 10% glycerol) was applied to the column equilibrated with HEMG buffer containing 2.1 M ammonium sulfate, and the resin was then washed with 4 column volumes (120 ml) of the same buffer. Protein was eluted with a linear gradient of 2.1 to 0.1 M ammonium sulfate over 6 column volumes (180 ml), and the column was washed with an additional 4 column volumes (120 ml) of HEMG buffer containing 0.1 M ammonium sulfate. (Due to materials such as lipids in the ammonium sulfate supernatant, the resin became discolored and slightly compacted during chromatography-we dis- card the used resin.) The fractions from the phenyl-sepharose column, except for small aliquots (100 ~1) to be used for assays, were frozen in liquid nitrogen and stored at -100°C. The fractions containing Hl were determined by assaying for Hl-mediated repression of transcription and by subjecting the protein fractions to SDS-polyacrylamide gel electrophoresis. The majority (>80%) of the Hl elutes from the phenyl-Sepharose re-

sin from 1.5 to 1.1 M ammonium sulfate in a volume of 24 ml (3.2 mg total protein).

S-Sepharose chromatography. The peak phenyl- Sepharose fractions were thawed in cold (4°C) water, pooled, and then dialyzed against HEG buffer containing 100 mM KCl. When the conductivity of the protein solution decreased to that of HEG buffer containing 150 mM KCl, Nonidet P-40 (l/1000 volume of a lo%, v/v, solution) was added to a final concentration of 0.01% (v/v), and the mixture was diluted with HEG buffer (without KCl) until the conductivity of the solution was equivalent to that of HEG containing 100 mM KCl. In- soluble material was removed by centrifugation in a Sorvall SS-34 rotor at 10,000 rpm for 10 min, and typically, one-half of sample (protein derived from 40 g embryos) was subjected to chromatography on a S-Sepha- rose Fast Flow resin (the other half of the sample was frozen in liquid nitrogen, stored at -lOO”C, and purified when needed): column volume = 0.4 ml; column dimen- sions (diameter X height) = 0.5 X 2 cm; flow rate = 0.1 ml/min; fraction size = 0.25 ml; full scale absorbance range at 280 nm = 0.2 absorbance unit. The sample was applied to the resin equilibrated with HEG buffer containing 0.1 M KCl, and the column was washed with 15 column volumes (6 ml) of the same buffer. Protein was eluted with a linear gradient of 0.1 to 1.0 M KC1 over 7 column volumes (2.8 ml), and the column was washed with an additional 4 column volumes (1.6 ml) of HEG buffer containing 1.0 M KCl. (After the 1 M KC1 wash, the S-Sepharose resin can be reused several times.) Nonidet P-40 (l/100 volume of a l%, v/v, solution) was added to a final concentration of 0.01% (v/v) to each of the column fractions. The fractions containing Hl, as estimated by the A 280nm profile, were dialyzed against HEMG buffer containing 0.1 M KC1 and 0.01% (v/v) Nonidet P-40, frozen in liquid nitrogen, and stored at -100°C. Hl elutes from the S-Sepharose resin from 0.4 to 0.5 M KCl. The purity of the Hl was determined by SDS-polyacrylamide gel electrophoresis. A typical preparation of Hl from 80 g of embryos yields 1.6 mg of protein of greater than 95% homogeneity. Note: when handling or diluting purified Hl, it is important to in- clude 0.01% (v/v) Nonidet P-40 in the buffer solutions. Otherwise, the Hl will irreversibly adsorb to the plastic- ware and glassware. However, Nonidet P-40 should not be added to solutions with moderate concentrations (>0.5 M) of ammonium sulfate because such conditions result in the formation of a precipitate.

Purification of HI from HeLa Cells

Hl was purified from a standard in vitro transcription extract derived from HeLa cells. A HeLa nuclear extract was prepared from 2 X 10” exponentially growing cells (36 liter culture) according to the procedure of Dig- nam et al. (18), except that 0.42 M KC1 was used instead


of 0.42 M NaCl in the nuclei extraction buffer. At 0.42 M monovalent salt concentration, both transcription factors (18) and histone Hl (20) are extracted from nuclei. The crude nuclear extract was subjected to precipitation with 2.1 M ammonium sulfate, and the transcription factors were present in the pellet whereas the Hl was predominantly in the supernatant. Glycerol was added to the supernatant fraction to a final concentration of 10% (v/v), and the material was frozen in liquid nitrogen and stored at -100°C. (In one instance, the addition of glycerol to the supernatant fraction caused the formation of an insoluble material. The formation of this precipitate did not, however, decrease the yield of Hl.) Hl was purified from this fraction by phenyl-sepharose and S-Sepharose chromatography as described above for the Drosophila Hl. The chromatographic properties of the HeLa Hl were indistinguishable from those of the Drosophila protein. Furthermore, the specific activity for repression of RNA polymerase II transcription of HeLa Hl was similar to that of Drosophila Hl. In a typical preparation of Hl from 2 X lOlo HeLa cells, roughly 0.8 mg of protein of greater than 95% homogeneity was obtained.

Purification of Hl from Calf Thymus

Fresh calf thymus was obtained from the Avila Meat Packing Co. (Newman, CA), and the tissue was frozen in liquid nitrogen and stored at -1OO’C. Hl was purified in conjunction with the core histones. Nuclei were prepared by homogenization in a Waring Blender as described by Kim and Dahmus (26) and then digested with micrococcal nuclease to give chromatin fragments. The chromatin fragments were separated on a 5 to 30% su- crose gradient in the presence of 0.5 M NaCl, as described by Butler and Thomas (9), and the fractions containing histone Hl, as determined by SDS-polyacrylamide gel electrophoresis, were pooled. Hl was present in the upper portion of the gradient along with other low-molecular-weight species. (The fractions containing oligonucleosomes were also used to prepare core histones by the procedure of Simon and Felsenfeld (27)). The mixture containing Hl was subjected to precipitation with 2.26 M ammonium sulfate. To the supernatant containing Hl, glycerol was added to a final concentration of 10% (v/v), and the solution was frozen in liquid nitrogen and stored at -100°C. Hl was then purified by phenyl-Sepharose and S-Sepharose chromatography as described above for the Drosophila protein. The chromatographic properties of calf thymus Hl were identical to those of the Drosophila and HeLa proteins. From 50 g of calf thymus, roughly 0.4 mg of Hl can be obtained with this procedure.

RESULTS AND DISCUSSION

It has been generally known that crude nuclear extracts from HeLa cells and Drosophila embryos contain

the general RNA polymerase II transcriptional machin- ery (for reviews, see Refs. (28,29)), numerous promoter- and enhancer-binding factors (for reviews, see Refs. (30,31)), and a nonspecific DNA-binding inhibitor of transcription (for example, see Refs. (18,25)). We have recently purified the DNA-binding transcriptional inhibitor and identified the protein as histone Hl (17). Thus, Hl was purified on the basis of its properties as a DNA-binding inhibitor of RNA polymerase II transcription without prior knowledge of its identity. Conse- quently, the procedure that we developed to purify Hl differed from the well-established methods for Hl purification, such as perchloric acid extraction (for example, see Refs. (32,33)) and ion-exchange chromatography (for example, see Refs. (27,33-35)). There are potential disadvantages to the use of perchloric acid in the preparation of Hl histones since perchloric acid is both a strong oxidizing agent as well as a strong acid. Differ- ences in the properties of perchloric acid-extracted Hl and salt-extracted Hl have been noted (for instance, see Ref. (35)). The simple and reliable method described here yields Hl that is active for repression of RNA polymerase II transcription and may also be useful for pro- viding native Hl for studies of the reconstitution of chromatin.

Purification of Hl from Drosophila Embryos

Crude extracts of salt-treated nuclei from Drosophila embryos (19,22) contain both RNA polymerase II transcription factors and Hl, and these proteins can be isolated as co-products from a single preparation. The key step in the separation of the transcription factors from the Hl is a 2.26 M ammonium sulfate precipitation. The transcription factors are precipitated with 2.26 M ammonium sulfate (19,22), whereas the Hl is present in the supernatant of the ammonium sulfate precipitation. To purify Hl, we directly subjected the ammonium sulfate supernatant of a standard Drosophila nuclear extract (22) to chromatography on a phenyl-Sepharose CL-4B resin (Fig. 1). Since Hl is a highly charged protein, it eluted relatively early from the phenyl-Sepharose column with a linear salt gradient (Fig. 1A). Phenyl-Se- pharose chromatography yielded nearly loo-fold purification of Hl, as monitored by the increase in the specific activity of Hl-mediated repression of transcription (Fig. lA, Table 1). As shown in Fig. lB, the Drosophila Hl was nearly pure after the phenyl-Sepharose column. (In fact, at this stage, the protein is of sufficient purity for many applications, such as repression of RNA polymerase II transcription). The identity of the two pre- dominant polypeptides of M, 45 to 50 kDa in the ammonium sulfate supernatant (Fig. lB, lane 2) is not known. Although the calculated molecular mass of Drosophila Hl (including the initiator Met1 residue) is 26,343 Da (36-38), the Drosophila embryo protein migrates with

166 CROSTON, LIRA, AND KADONAGA

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FIG. 1. Phenyl-Sepharose CL-4B chromatography of Drosophila histone Hl. (A) Elution profile of material during chromatography. The solid line indicates the absorbance at 280 nm; the dashed line represents the ammonium sulfate concentration, as determined by measurement of the conductivity of the eluate; and the dotted line denotes transcriptional repression activity, as defined under Experimental Procedures. (B) Analysis of protein eluting from the phenyl-Sepharose column. Protein fractions were precipitated with 20% (w/v) trichloroacetic acid, and the samples were then subjected to electrophoresis on a 12% polyacrylamide-SDS gel. The protein was visualized by staining with Coomassie blue. Lane 2 contains 3 pg of the ammonium sulfate supernatant fraction. Lanes 3 to 11 contain 10 11 each of the indicated fractions from the representative Hl purification that is described under Experimental Procedures. The position of Hl migration is denoted by a bracket. Lanes 1 and 12 contain molecular mass markers. The sizes of the markers are indicated in kilodaltons.

an apparent molecular mass of 39 kDa on a polyacryl- The peak fractions from the phenyl-Sepharose col- amide-SDS gel (Fig. 1B). In addition, transcriptional umn (Fig. lB, lanes 6 to 8) were then subjected to chro- repression activity (Fig. 1A) co-eluted with Hl protein matography on a S-Sepharose Fast Flow resin (Fig. 2). (Fig. lB), and the majority of the transcriptional re- The S-Sepharose chromatography concentrates the Hl pression activity in the nuclear extracts was due to Hl preparation about 25fold, and as a matter of conve- (data not shown). nience (due to the restrictions imposed by the dimen-


TABLE 1

Purification of Histone Hl from Drosophila Embryos (Based on 80 g Embryos)

Fraction

Total Total volume protein

(ml) (md

Total activity (units)

Specific activity

(units/mg) Yield

60) Purification

(fold)

Ammonium sulfate supernatant 102 340 17340 51 100 1 Phenyl-Sepharose 24 3.2 15840 9778 91 95 S-Sepharose 1.0 1.6 11430 14288 66 140

sions and flow rates of our chromatography columns), we typically perform two successive cycles of S-Sepha- rose chromatography, with each cycle using one-half of the phenyl-Sepharose peak fractions. The protein fractions were adjusted to a conductivity equivalent to that of buffer containing 0.1 M KC1 by a combination of dialy- sis and dilution, and Nonidet P-40 was added to 0.01% (v/v) concentration. The protein was applied to the S- Sepharose resin at 0.1 M KC1 and then eluted with a linear gradient from 0.1 to 1.0 M KC1 (Fig. 2). There were two major protein peaks, as monitored by the absorbance at 280 nm, and the second, higher salt peak represents Hl. (The extinction coefficient of histone Hl at 280 nm is relatively small due to the paucity of amino acid residues containing aromatic side chains.) Nonidet P-40 (to 0.01% (v/v) concentration) was added directly to each of the S-Sepharose fractions, and the fractions corresponding to the second A,, nm peak were dialyzed to 0.1 M KCl, frozen in liquid nitrogen, and stored at -100°C. At this stage, the Hl is of greater than 95%

homogeneity (Fig. 3, lane 1). This procedure is also highly ‘reproducible. We have successfully carried out seven independent preparations of Hl from Drosophila embryos (80 to 250 g scale). In each of these experiments, the chromatographic properties of Hl were identical. Furthermore, the different Hl preparations possessed the same specific activity for repression of RNA polymerase II transcription. The purification of Drosophila Hl is summarized in Table 1.

Although there are an estimated 110 copies of the Hl gene in Drosophila melanogaster (39,40), it appears, at present, that there is only a single version of Hl in the embryo. First, we have not been able to separate different Drosophikz Hl species with either SDS-polyacrylamide gels or other polyacrylamide gel electrophoresis systems that are designed for separation of Hl subtypes (data not shown). Second, we have determined the amino acid sequence of 231 amino acid residues out of the total predicted (including Metl) 257 amino acid residues of Drosophila Hl by automated Edman degrada-

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FIG. 2. FIG. 2. Elution profile of material during S-Sepharose chromatography of Drosophila histone Hl. The solid line indicates the absorbance at Elution profile of material during S-Sepharose chromatography of Drosophila histone Hl. The solid line indicates the absorbance at 280 nm; the dashed line represents the KC1 concentration, as measured by the conductivity of the eluate; and the dotted line denotes the 280 nm; the dashed line represents the KC1 concentration, as measured by the conductivity of the eluate; and the dotted line denotes the transcriptional repression activity, as defined under Experimental Procedures. transcriptional repression activity, as defined under Experimental Procedures.

168 CROSTON. LIRA, AND KADONAGA

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FIG. 3. Hl histones purified from three different organisms. HI histones (0.4 ag each) were subjected to electrophoresis on 12% polyacrylamide-SDS gels, and protein was visualized by staining with Coomassie blue. Lane 1 contains Hl from Drosophila embryos; lane 2 contains Hl from HeLa cells; and lane 3 contains Hl from calf thymus. Lane 4 contains molecular mass markers. The sizes of the markers are indicated in kilodaltons.

tion and plasma desorption mass spectrometry of pep- tides generated by digestion with either trypsin or Staphylococcus aureus V8 protease (17). In the course of sequencing Drosophila Hl, we did not detect a single peptide with a primary sequence which varied from that predicted from two independent Drosophila clones (36- 38). Curiously, the nucleotide sequence of the two Dro- sophila Hl clones is identical in the coding region (36- 38). It is not known, however, how many of the estimated 110 Hl genes possess this particular sequence. Notwithstanding, our current evidence suggests that there is only a single version of Hl polypeptide in embryos of Drosophila melanogaster.

Purification of HI Histones from HeLa Cells and Calf Thymus

By using the same strategy that was employed for the purification of Drosophila Hl, we have also purified Hl histones from HeLa cells and calf thymus (Fig. 3). The chromatographic properties of the HeLa and calf thymus Hl histones were indistinguishable from those of Drosophila Hl. The purified HeLa and calf thymus Hl histones also repressed RNA polymerase II transcription in a manner similar to that of the Drosophila protein (data not shown). Unlike Drosophila Hl, which ap-

peared to consist of a single polypeptide, the HeLa and the calf thymus Hl preparations contained at least five different subtypes, as determined by electrophoresis with various polyacrylamide gel systems (Fig. 3; data not shown). Hence, it is apparent that the new method for purification of Hl histones is generally applicable for the purification of Hl from different organisms.

In summary, we have described a new procedure for the purification of Hl histones. This method was origi- nally devised as a means of purifying a repressor of RNA polymerase II transcription, which was later identified as Hl (17). By monitoring the transcriptional repression activity of Hl, we were able to purify Hl to greater than 95% homogeneity with a 66% overall recovery of activity (Table 1). This high recovery of activity, along with the use of mild conditions to isolate the protein, suggests that the Hl is in its native form. In addition, this procedure can be used to purify Hl histones, including various subtypes, from Drosophila embryos, HeLa cells, and calf thymus. By use of the strategy out- lined in this paper, Hl can be purified as a co-product of RNA polymerase II transcription factors from either Drosophila embryos or HeLa cells. Thus, this method is ideal for studies of the role of Hl upon transcription by RNA polymerase II. Moreover, since the Hl is in a bio- chemically active form, it may also be useful for experiments involving reconstitution of chromatin.

ACKNOWLEDGMENTS

We thank Rohinton Kamakaka and Paul Laybourn for advice on the purification and properties of Hl, and Kathy Jones for the gener- ous gift of HeLa extracts. G.E.C. is a National Science Foundation Predoctoral Fellow. This work was supported in part by National Institutes of Health Grant GM 41249. J.T.K. is a Lucille P. Markey Scholar, and this work was supported in part by the Lucille P. Markey Charitable Trust.

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A general method for purification of H1 histones that are active for repression of basal RNA polymerase II transcription