Chromatin Structure in Granulocytes
A LINK BETWEEN TIGHT COMPACTION AND ACCUMULATION OF A HETEROCHROMATIN-ASSOCIATED PROTEIN (MENT)*

Sergei A. GrigoryevDagger and Christopher L. Woodcock

From the Biology Department, University of Massachusetts, Amherst, Massachusetts 01003

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

To study the mechanism of heterochromatin formation in vertebrate cells, we isolated nuclei from chicken polymorphonuclear granulocytes and examined the chromatin organization. We found granulocyte chromatin to remain insoluble after nuclease digestion and to be resistant to swelling in low salt/high pH media. Both insolubility and resistance to swelling were lost after washing with 0.3 M NaCl, a procedure that released two abundant tissue-specific proteins from granulocyte nuclei. One of them (42 kDa) is identified as MENT, a protein previously shown to be associated with repressed chromatin from mature chicken erythrocytes. We show that MENT is immunolocalized in granulocyte heterochromatin, where it is one of the most abundant chromatin proteins (~2 molecules/200 base pairs of DNA). MENT is the first nuclear protein structurally related to the serine protease inhibitor family. The other abundant protein is similar to or identical with mim-1, a myeloid-specific protein that is known to be stored in cell granules and to associate with isolated nuclei. MENT (but not mim-1) binds chromatin and free DNA, and, at its physiological protein/DNA ratio, enhances compaction and the reversible Mg2+-dependent self-association of nucleosome arrays. MENT appears to promote the formation of heterochromatin by acting as a "glue" within and between chains of nucleosomes.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

In eukaryotic chromosomes, the DNA double helix is folded by proteins in a hierarchical manner. At the basic folding level, DNA periodically makes superhelical turns around an octamer of histones, generating repeating nucleoprotein particles called nucleosomes. The x-ray structure of a synthetic nucleosome core particle at 2.8-Å resolution has recently been reported (1). All nucleosomes contain four pairs of evolutionarily conserved core histones, H2A, H2B, H3, and H4. In addition to the core histone octamer, nucleosomes generally contain a ninth basic protein, histone H1, also called linker histone. Linker histones are required for nucleosome chains to form the next level of chromatin folding, the 30-nm chromatin fiber (2-4).

Genetic inactivation of many chromosomal loci in eukaryotic cells is correlated with a tighter compaction of chromatin, forming what is often referred to as heterochromatin (5). Recent genetic studies have revealed a complexity of protein factors that are potentially involved in heterochromatin formation in multicellular eukaryotes (6, 7). However, neither the mechanism of heterochromatin formation nor the proteins directly involved in this level of chromatin condensation are presently known.

The extent of chromatin condensation increases during cell differentiation, reaching its maximum in terminally differentiated quiescent cells where it often involves the bulk of interphase chromatin. This is typified by vertebrate blood cells, which provide a convenient experimental system to investigate the mechanism(s) of chromatin condensation by both biochemical and cytological approaches.

Since chromatin condensation and general decrease in transcription in mature vertebrate tissues are often correlated with the appearance and accumulation of tissue-specific histone H1 subtypes (for reviews, see Refs. 8-10), these proteins have long been considered one of the key factors in differentiation stage-specific chromatin condensation and gene repression. However, gene regulation studies in cells either overexpressing or lacking certain types of histone H1 have greatly undermined the concept that linker histones act as general chromatin repressors (11-15). It may now be concluded that the accumulation of linker histones per se is not sufficient to cause a major chromatin remodeling or a general inhibition of transcription in vivo and that additional factors must be involved in priming chromatin condensation. It also remains to be determined whether linker histone concentration is increased in all cases where extensive heterochromatin formation occurs.

Previously, we observed that, in addition to the erythrocyte-specific linker histone H5, the condensed and repressed chromatin from terminally differentiated chicken erythrocytes contained a 42-kDa polypeptide that was absent from the active chromatin fraction (16). This protein, which was also abundant in polymorphonuclear granulocytes, was designated as MENT (myeloid and erythroid nuclear termination stage-specific protein). In both types of blood cell, MENT was located exclusively inside the nuclei, forming a number of foci in erythrocytes and a dense layer at the periphery of granulocyte nuclei (17).

Granulocyte chromatin is highly condensed and forms a dense heterochromatic layer at the nuclear periphery (18-20). Since chicken granulocytes give a very strong immunofluorescent reaction with anti-MENT antibodies, it seemed likely that the extensive heterochromatization in these cells might be associated with a high level of MENT accumulation.

Here we report the purification of chicken granulocytes, isolation of their nuclei, and an analysis of granulocyte chromatin organization. Despite extensive heterochromatization, granulocyte nuclei contain no more linker histone than is normally found within chromatin of actively proliferating cells, including undifferentiated promyelocytic precursors. However, granulocyte heterochromatin accumulates a large amount of MENT, sufficient to induce substantial compaction of both nuclear chromatin and soluble polynucleosomes in vitro. In addition, granulocyte nuclei are greatly enriched with another myeloid-specific protein, mim-1, which is normally found within heterophil granules (21, 22). Based on our studies of the interaction of purified MENT and mim-1 proteins with DNA and chromatin in vitro, we argue that it is the hyperaccumulation of MENT in chicken granulocyte nuclei that induces and maintains the high level of chromatin condensation in vivo. Chromatin remodeling during terminal differentiation in granulocytes thus may require only one major additional chromosomal protein (MENT).

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Fractionation and Isolation of Chicken Blood Cells-- Fresh chicken blood was purchased from Pel-Freez (Rogers, AR) and used within 24 h of exsanguination. Erythrocytes were obtained from blood of adult chicken or from 19-day-old chicken embryos by centrifugation at 800 × g for 5 min. in the presence of 2% sodium citrate and washing three times with PBS1 medium (0.14 M NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.7 mM KH2PO4, pH 7.5) plus 1% sodium citrate. The upper layers (buffy coats) were removed after each centrifugation and either discarded or used for isolation of white blood cells. The bottom layer containing pure erythrocytes was used for nuclear isolation.

To fractionate chicken blood cells, 200 ml of whole blood containing 2% sodium citrate was centrifuged for 5 min at 800 × g, and the buffy coats enriched with leukocytes were removed, resuspended in PBS with 1% sodium citrate, centrifuged as above, and again separated from the bottom erythrocyte layer. The leukocyte-enriched material was applied to a 40-ml gradient of 60% Percoll containing PBS. The Percoll gradient was prepared 2 h before the experiment by the mixing of 24 ml of stock Percoll suspension (density 1.128 g/ml, Pharmacia Biotech Inc.) with 16 ml of 2.5 × PBS in a 50-ml centrifuge tube and by centrifugation of the resulting mixture at 20 °C for 30 min at 23,400 × g in a SS34 rotor (Sorvall, Boston, MA). The leukocyte-enriched material was loaded on the preformed gradient and centrifuged at 1000 × g for 20 min at room temperature. At the end of the centrifugation, five distinct zones were obtained (Fig. 1, left). The cell fractions corresponding to each zone were taken from the gradient, resuspended in 40 ml of PBS, centrifuged at 1000 × g for 5 min, and finally resuspended in 2 ml of PBS. Smears of the cell fractions were analyzed under a light microscope. Zone I contained thrombocytes and many cell aggregates; zone II contained predominantly lymphocytes; zone III was a mixture of different mononuclear white blood cells; zone IV contained a highly enriched granulocyte population; and zone V contained the residual erythrocytes (see Fig. 1).

Isolation of Cell Nuclei and Chromatin-- Cell nuclei were isolated from chicken erythrocytes as described (16) and stored in RSB (10 mM NaCl, 3 mM MgCl2, 10 mM Tris-HCl, pH 7.5) containing 50% glycerol at -20 °C.

To isolate the leukocyte nuclei, the suspensions of lymphocytes (gradient zone II) and of granulocytes (gradient zone IV) in PBS were centrifuged for 3 min at 1000 × g and resuspended in 0.5% Nonidet P-40 in RSB with 1 mM PMSF at 2 °C. The cell suspensions were homogenized by 20-30 strokes of pestle A in a Dounce homogenizer over 30 min on ice. Nuclei were centrifuged for 10 min at 7600 × g, and the nuclear pellets were resuspended in RSB plus 1 mM PMSF. Nuclei could be stored for 1 week at 2 °C without a detectable DNA or protein degradation.

For micrococcal nuclease digestion, an aliquot of the nuclear preparation (A260 = 20) was resuspended in 5 ml of RSB containing 0.5 mM PMSF. CaCl2 was added to give a final concentration of 1 mM, micrococcal nuclease (Boehringer Mannheim) was added at 3-30 units/ml depending on the nature of the nuclei and the desired extent of digestion, and the reaction was carried out at 37 °C .

To obtain soluble chicken erythrocyte chromatin (mean number of nucleosomes in a chain, n = 6) the reaction used 5 units of enzyme/ml and was terminated after 10 min by adding 0.5 ml of 0.1 M Na-EDTA and 5 ml of ice-cold TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.6). The digested nuclei were pelleted for 10 min at 12,000 × g. The supernatant (A260 = 50) was loaded onto a 5-25% sucrose gradient containing 10 mM HEPES, pH 7.5, and centrifuged for 22 h at 60,000 × g in a SW-27 rotor (Beckman). The gradient fraction containing chains of 6 ± 1 nucleosomes was collected and dialyzed against 10 mM HEPES, pH 7.5.

Protein Electrophoresis, Detection, and Quantitation-- Protein electrophoresis in 16% polyacrylamide gels was carried out as described (23). Soluble chromatin and nuclear samples were boiled in SDS-containing sample buffer and applied without separation of DNA and proteins. After electrophoresis, the gels were either stained with Coomassie Blue R-250 (Sigma) or electrotransferred in Tris-glycine buffer containing 10% methanol to polyvinylidene difluoride membranes (Immobilon P, Millipore Corp., Bedford, MA) as described by Harlow and Lane (24). The filters were blocked, treated with anti-MENT antibodies (dilution 1:5000) or with anti-H1 antibodies (dilution 1:100), and then treated with secondary peroxidase-conjugated anti-rabbit antibodies and detected with the ECL detection system (Amersham Corp.) as described in the vendor's manual. Rabbit anti-MENT antibodies were obtained and purified on a Protein A-Sepharose column as described (17). Rabbit antibodies raised against calf thymus histone H1 (25) were kindly provided by Dr. C. Mura and were incubated with an excess of membrane-attached mim-1 protein to reduce the cross-reaction with mim-1. Rabbit antibodies against mim-1 were kindly provided by Dr. S. Ness. For quantitation of the proteins in the nuclear samples, the serial dilutions of purified proteins with known concentrations were electrophoresed on the same gels. The Coomassie-stained gels or the autoradiographs after the ECL detection were digitized, and the intensity of protein bands was quantitated using the NIH Image software package. Calibration curves were made by plotting the intensities of control protein bands against samples of known concentration and used to estimate the concentration of unknown samples. Protein/DNA ratios were estimated from parallel measurements of DNA concentration by UV spectrophotometry (A260 = 1 for 50 µg/ml DNA).

Histone Isolation-- All preparative procedures in this section were conducted at 2 °C, and all solutions contained 0.5 mM PMSF as a protease inhibitor. Core histones (an equimolar mixture of H2A, H2B, H3, and H4) were isolated from adult chicken erythrocyte nuclei by 2 M NaCl treatment essentially as described by Von Holt et al. (26) and stored in 2 M NaCl at -70 °C. To isolate histone H5, chicken erythrocyte nuclei were first washed in 0.35 M NaCl and pelleted at 1460 × g for 5 min to remove the loosely bound nonhistone proteins (the 0.35 M NaCl extract). To remove histone H1, the nuclear pellet was resuspended in 0.55 M NaCl and centrifuged in an SS-34 rotor at 27,000 × g for 30 min. The pellet was washed in 0.55 M NaCl, centrifuged again for 10 min at 27,000 × g, and finally solubilized in 0.65 M NaCl. DNA and core histones were removed by centrifugation at 200,000 × g for 16 h (Ti-70 rotor, Beckman, Palo Alto, CA). The 0.65 M NaCl supernatant containing more than 95% pure histone H5 was stored at -70 °C. Histone concentrations were measured spectrophotometrically (A230 = 2.3 for 1 mg/ml histone H5).

DNA Isolation, Reconstitution, and Electrophoresis-- A plasmid vector pAT153 containing the ~1.2-kilobase pair hexamer tandem repeat of a 198-bp fragment of Lytechinus variegatus 5 S rDNA was obtained from Dr. R. Simpson (27). Plasmids were isolated from 2-liter cultures of Escherichia coli by an alkaline lysis method (28). The resulting plasmids were additionally purified by treatment with phenol/chloroform and ethanol precipitation. The isolated plasmids were treated with the restriction endonuclease, HhaI, which excises the tandemly repeated 5 S rDNA insert from the plasmid vector. The 1.2-kilobase pair-long insert was purified by gel-filtration on a Sephacryl S-500 (Pharmacia) column as described (29). To reconstitute oligonucleosomes, the 1.2-kilobase pair-long fragment (10 µg) was mixed with 1.05 mol of histone octamer/198 bp of DNA in 2 M NaCl and dialyzed for 16 h, during which the concentration of NaCl was gradually decreased to 0.5 M (30). Nonbound histone was removed by repeatedly concentrating the reconstitutes using a 100-kDa filtration membrane (Microcon-100, Amicon, Beverly, MA). Finally, the reconstitutes were dialyzed overnight against 10 mM HEPES, pH 7.5, 5 mM NaCl. For H5-containing reconstitutes, a 50-µl portion of the washed sample in 0.5 M NaCl was mixed with 1 mol of histone H5/1 mol of octamer in 0.65 M NaCl, and the mixture was dialyzed overnight against 10 mM HEPES, pH 7.5, 5 mM NaCl. Nuclease digestion mapping, DNA labeling, and analysis in denaturing polyacrylamide gel electrophoresis were conducted as described by Meersseman et al. (30). DNA electrophoresis in agarose gels (Sigma, type I) was conducted in Tris acetate buffer (31). Electrophoreses of DNP (DNA-protein complexes) were run in 1% agarose (Sigma, type IV) in 20 mM HEPES, pH 8.0, 0.1 mM EDTA.

Isolation and Sequence Analysis of MENT and mim-1 Proteins-- MENT and mim-1 proteins were isolated from nuclei obtained from unfractionated "buffy coat" cells collected from the top of chicken blood pellets as described above. Nuclei were suspended in RSB plus 0.5 mM PMSF at A260 = 200, and an equal volume of RSB containing 0.7 M NaCl was added to make the final NaCl concentration 0.35 M. After mixing for 30 min on ice, the nuclear suspension was centrifuged for 10 min at 23,400 × g (Sorvall SS-34). The supernatant was taken, dialyzed against 0.2 M NaCl, 20 mM HEPES, pH 7.6, and applied to an S-Sepharose FF ion exchange column (Pharmacia) equilibrated with 0.2 M NaCl, 20 mM HEPES, pH 7.6. Proteins were eluted with 20 mM HEPES, pH 7.6, in a 0.2-1.0 M NaCl gradient. Peaks containing MENT and mim-1 polypeptides were collected, dialyzed against 20 mM HEPES, pH 7.6, containing either 0.05 M NaCl (for MENT) or 0.2 M NaCl (for mim-1), and repurified on the S-Sepharose FF column as above. The protein concentration was estimated by Bradford's method (32), calibrated by a quantitative amino acid analysis. For protein peptide microsequencing, 200 pmol of MENT was electrophoresed and electrotransferred as described above to a Problot polyvinylidene difluoride membrane (Applied Biosystems) and subjected to amino acid analysis, peptide separation, mass spectrometry, and amino acid microsequencing (Harvard Microchemistry Facility, Cambridge MA). For mim-1 microsequencing, 40 µl of 1 mg/ml column-purified protein was treated for 30 min at 37 °C with 0.2 mg/ml trypsin (Sigma; 10,000 benzoyl-L-arginine ethyl ester units/mg), electrophoresed, and transferred on polyvinylidene difluoride membrane. The major polypeptide bands were excised and microsequenced (Columbia University Protein Chemistry Facility, New York, NY).

Reassociation of Isolated Nonhistone Proteins to Nuclei and Soluble Chromatin-- For nuclear reconstitution experiments, 0.1 ml of nuclear suspension (0.25 mg/ml DNA) was mixed with 32 µl of 0.25 mg/ml MENT (1 mol of MENT/nucleosome) or with 28 µl of 1 mg/ml mim-1 (4.5 mol of protein/nucleosome) in RSB containing 0.2 M NaCl and 1 mM PMSF and stirred at 4 °C for 2 h, during which the samples were gradually diluted five times with RSB. The nuclei were then centrifuged for 3 min at 1000 × g and resuspended in 0.1 ml of RSB.

For binding studies with naked sea urchin 5 S DNA (1.2 kb) and reconstituted nucleosome hexamers, 30 µl of 20 mM HEPES, pH 7.5, containing various concentrations of MENT or mim-1 were mixed with 0.1 ml of native oligonucleosome preparation (0.1 mg/ml DNA) in 10 mM HEPES, pH 7.5, 50 mM NaCl, 2 mM MgCl2 and dialyzed against 5 mM HEPES, pH 7.5. Reactions with soluble native oligonucleosomes were conducted similarly, but MgCl2 was omitted from the reaction buffer. No dialysis was performed before loading on agarose gels.

Light, Fluorescence, and Electron Microscopy-- For light microscopy, smears of whole blood or of Percoll gradient-fractionated cells were fixed and stained with the Leukostat blood staining kit (Fisher). Unfixed nuclear suspensions in appropriate buffers were stained with 10 µg/ml DAPI (Sigma) and examined with a Nikon Optiphot microscope equipped with epifluorescence and differential interference contrast optics.

For immunogold labeling, a preparation of chicken granulocytes was fixed with 1% glutaraldehyde and 4% paraformaldehyde for 20 min at 20 °C and 1 h on ice. Fixed cells were embedded in Unicryl resin (33) polymerized by UV irradiation. Ultrathin (80-nm) sections of the resin-embedded cells were obtained, mounted on electron microscopic grids, blocked, exposed to anti-MENT antibodies (Ref. 17; optimal dilution, 1:200), and then exposed to gold-conjugated goat anti-rabbit antibodies (10-nm diameter gold particles, Sigma) as described by Erickson et al. (34) with OsO4 poststaining for 1.5 min, uranyl acetate for 12 min, and lead citrate for 5 min.

For electron microscopy of soluble chromatin, samples were fixed by adding 0.1% glutaraldehyde for at least 20 h at 2 °C, applied to glow-discharged thin carbon films, and stained with aqueous uranyl acetate or ethanolic phosphotungstic acid (35). Samples were examined in a Philips CM10 electron microscope, and images were either recorded on a CCD camera (Gatan Inc.) or on film.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Isolation of Chicken Granulocytes and Immunolocalization of MENT-- To obtain a pure population of chicken granulocytes, we fractionated a leukocyte-enriched portion of the cells pelleted from chicken blood ("buffy coat") on a gradient of 60% Percoll. Microscopic examination of stained cell smears from five distinct Percoll gradient cell zones showed that zone IV contained a 97% pure population of polymorphonuclear granulocytes, as judged by their characteristic nuclear morphology (Fig. 1).


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Fig. 1.   Fractionation of chicken blood and isolation of granulocytes. Left, scheme of chicken blood fractionation on 60% Percoll. Right, zone IV contains polymorphonuclear granulocytes. Scale bar, 5 µm.

For immunolocalization of MENT, ultrathin sections of isolated granulocytes were treated with Protein A-Sepharose-purified anti-MENT antibodies (17) followed by gold-conjugated anti-rabbit IgG. The immunogold label was abundant in the nuclei of all granulocyte cells examined and absent from other cell compartments (Fig. 2, top). No specific labeling was obtained when preimmune serum was used (Fig. 2, middle). At higher magnification, a strong preferential localization over the more electron-dense heterochromatin was apparent (Fig. 2, bottom).


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Fig. 2.   Immunolocalization of MENT in granulocyte heterochromatin. Immunogold labeling of ultrathin section of granulocytes by anti-MENT antibodies (top and bottom panels) and with preimmune serum (central panel). Gold particles are seen as black dots, 10 nm in diameter. Nuclei (N), cytoplasm (C), and granules (G) are marked. Scale bars, 0.5 µm.

Isolation of Granulocyte Nuclei and Unusual Insolubility of the Chromatin in Low Ionic Strength Media-- Nuclei were isolated from the preparations of erythrocytes and granulocytes. The morphology of the nuclear samples remained intact throughout the isolation, and the granulocyte nuclei frequently maintained their multilobed shape (Fig. 3, central top panel). Anti-MENT antibodies produced a strong immunofluorescence reaction with all nuclei having the granulocyte morphology, suggesting that MENT was present at a comparable level in all granulocytes. The control preimmune serum did not react with granulocyte nuclei (not shown).


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Fig. 3.   Chromatin in granulocyte nuclei is resistant to swelling and insoluble after nuclease digestion. Top panels, DAPI stain. Isolated nuclei of chicken erythrocytes are highly swollen in LS (left panel). Isolated granulocyte nuclei do not swell in LS (central panel) unless washed in RSB containing 0.3 M NaCl and resuspended in LS (right panel). Scale bar, 5 µm. Bottom panels, percentage of total nuclear DNA (A260) recovered in solution after digestion of chicken erythrocyte nuclei (left), granulocyte nuclei (center), and 0.3 M NaCl-washed chicken granulocyte nuclei (right) with micrococcal nuclease (7.5, 30, and 5 units/ml, respectively) for 20 min. Columns in each panel (from left to right) represent the percentage of soluble DNA released in the course of digestion (RSB) and after two sequential washings in TE buffer followed with two washings in LS. NR represents the percentage of DNA remaining insoluble after the washings.

Typically, isolated nuclei swell and chromatin disperses when divalent cations are removed and the pH is raised above neutrality. For mature chicken erythrocytes, swelling starts in "physiological" phosphate buffer above pH 7.4 (36), and the nuclear diameter increases dramatically (Fig. 3, top left panel) in low salt media such as LS (1 mM Tris, 0.1 mM EDTA, pH 8.5), a buffer used to spread nuclear chromatin for electron microscopy (37). However, no morphological changes were observed when granulocyte nuclei were exposed to LS (Fig. 3, top central panel). Treatment of the granulocyte nuclei by 0.3 M NaCl, which elutes MENT from chromatin (17), did not alter their compactness, but upon transfer of 0.3 M NaCl-washed nuclei to LS, considerable swelling was observed (Fig. 3, top right panel), suggesting that factors removed by 0.3 M NaCl were responsible for the unusual resistance of the granulocyte nuclei to swelling.

In an attempt to isolate soluble granulocyte chromatin, the purified nuclei were treated with micrococcal nuclease to produce DNA fragments averaging from 200 to 2000 bp. Granulocyte nuclei were much more protected from digestion than erythrocyte nuclei, about 5 times more enzyme being needed to achieve a comparable level of digestion in the former than in the latter. The nucleosome repeat produced by nuclease cutting of granulocyte nuclei (192 ± 2 bp) was about 20 bp shorter than in erythrocytes (compare lanes 13 and 14 on the DNA gel in Fig. 4).


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Fig. 4.   Micrococcal nuclease cutting analysis of granulocyte chromatin. Shown is agarose electrophoresis of DNA isolated from chicken granulocyte nuclei treated with 60 units/µl micrococcal nuclease for 1 (lane 2), 2 (lanes 3 and 13), 5 (lane 3), 10 (lane 4), 20 (lane 5), and 50 (lane 6) min and DNA isolated from chicken erythrocyte nuclei treated with 30 units/µl micrococcal nuclease for 1 (lane 7), 2 (lanes 8 and 14), 5 (lane 9), 10 (lane 10), and 20 (lane 11) min. Lanes 1, 12, and 15, DNA molecular size markers (Life Technologies, Inc.). G and E, equivalent digests of granulocyte (3 min) and erythrocyte (1 min) chromatin for comparison of nucleosome repeat lengths.

After micrococcal nuclease digestion, nuclear chromatin is typically solubilized when divalent cations are removed. This occurs even with highly condensed chicken erythrocyte nuclei (Fig. 3, bottom left panel). In contrast, granulocyte nuclei digested to contain mono- and oligonucleosomes (Fig. 4, lane 5, on DNA electrophoresis) did not release soluble chromatin even after repeated washing in TE buffer, followed by washing with LS. However, when the digested nuclei were exposed to 0.3 M NaCl after the low salt washes, most of the chromatin was released into solution. Pretreatment of intact granulocyte nuclei with 0.3 M NaCl resulted in an ~4-fold increase in the subsequent micrococcal nuclease digestion rate without affecting the nucleosome repeat length and also led to chromatin solubilization of the nuclease-digested chromatin in TE buffer (Fig. 3, bottom right panel). The unprecedented insolubility of nuclease-digested granulocyte chromatin can thus be attributed to factor(s) released from nuclei by 0.3 M NaCl.

A Tissue-specific Nonhistone Chromatin Protein, MENT, Is Extremely Abundant in Isolated Chicken Granulocyte Nuclei-- When nuclear proteins from chicken erythrocytes, lymphocytes, and granulocytes were run on SDS-polyacrylamide gel electrophoresis and stained with Coomassie dye, it was evident that granulocyte nuclei are strongly enriched in three polypeptides with apparent molecular masses of 42, 41, and 30 kDa. The 42- and 30-kDa proteins are almost completely removed from the nuclei by treatment with 0.3 M salt (Fig. 5). Western blotting demonstrated that the 42-kDa protein was identical to MENT, which was also present in nuclei of all major blood cell types. Densitometry of the Western blots showed that granulocytes have a MENT/DNA ratio about 10-fold higher than lymphocytes and 80-fold higher than erythrocytes. The concentration of MENT in granulocyte nuclei was estimated by densitometry of Coomassie-stained gels in comparison with a serial dilution of isolated MENT standard in which the protein concentration was measured by amino acid analysis. Granulocyte nuclei were found to contain 2.1 molecules of MENT/nucleosome (average of four different isolations). This appears to be one of the most extreme cases of hyperexpression of a single nuclear protein in somatic eukaryotic cells.


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Fig. 5.   Electrophoresis of proteins from blood cell nuclei. Top panels, SDS-polyacrylamide gel electrophoresis of proteins from total erythrocyte (E), lymphocyte (L), and granulocyte (G) nuclei and from granulocyte nuclei washed with 0.35 M NaCl. P, pellet; S, supernatant. Coomassie R-250 stain was used. Bottom panels, Western blots of proteins from the pellet (P) and supernatant (S) of granulocyte nuclei washed with 0.35 M NaCl were probed with anti-MENT, anti-mim-1, anti-H1, and anti-actin IgG as indicated.

The 30-kDa protein was the other major tissue-specific polypeptide whose removal by 0.3 M NaCl was correlated with chromatin decondensation. To identify this protein, we have isolated it from a 0.35 M extract of granulocyte nuclei by ion exchange chromatography to an apparent 95% homogeneity. The protein was further subjected to a limited trypsin digestion and microsequencing. The resulting peptide sequence, GEKHKGVDVIXTDGS, matched a single protein in the data base, mim-1 (21). Western blotting probed with anti-mim-1 antiserum (kindly provided by Dr. S. Ness) confirmed the identity of the 30-kDa band (Fig. 5). The protein band with electrophoretic mobility close to histone H2A that is seen among the proteins removed by 0.3 M NaCl from granulocyte nuclei, also reacts with anti-mim-1 antibodies and is found in purified mim-1 samples (data not shown). Therefore, we consider this polypeptide to be a proteolytic fragment of mim-1. This protein has been previously shown to be abundant in chicken heterophil granules and also to associate with isolated nuclei in vitro (22, 38). It thus appears likely that mim-1 contaminated the granulocyte nuclear preparation during isolation and is not associated with chromatin. Further in vitro experiments demonstrating no interactions between mim-1 and DNA and chromatin (Fig. 6) have confirmed this conclusion.


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Fig. 6.   MENT but not mim-1 interacts with DNA and reconstituted hexanucleosomes. Top panels, agarose electrophoresis of DNA and reconstituted hexanucleosomes. Lanes 1-5, 1.2-kb 5 S rDNA mixed with MENT at the following ratios per 200 bp: 0 (lane 1), 1 (lane 2), 3 (lane 3), 9 (lane 4), 27 (lane 5). Lanes 6-10, supernatant (top panel) and pellet redissolved in TE after centrifugation at 10,000 × g for 5 min (bottom panel) of reconstituted hexanucleosomes mixed with MENT at the following ratios per 200 bp of DNA: 0 (lane 6), 0.1 (lane 7), 0.3 (lane 8), 1 (lane 9), and 3 (lane 10). Lanes 11-15, 1.2 kb of 5 S rDNA mixed with mim-1 at the following ratios per 200 bp: 0 (lane 11), 0.3 (lane 12), 1 (lane 13), 3 (lane 14), 10 (lane 15). Lanes 16-20, reconstituted nucleosome hexamers rDNA mixed with mim-1 at the following ratios per 200 bp DNA: 0 (lane 16), 0.3 (lane 17), 1 (lane 18), 3 (lane 19), and 10 (lane 20). Bottom panel, electron microscopy of nucleosome hexamers. Samples were fixed in 50 mM NaCl, 20 mM HEPES, pH 7.5, and stained with aqueous uranyl acetate. Upper row, without MENT. Bottom row, core histones plus 1 molecule of MENT/200 bp of DNA. Scale bar, 30 nm.

The abundant polypeptide seen at ~41 kDa in both lymphocyte and granulocyte nuclear samples strongly reacts with anti-actin antibodies. Also consistent with its being cytoplasmic actin is its appearance in cytoplasm extracts of the cells and its depletion from nuclei by repeated low salt washing.

By densitometry of stained gels with nuclear protein (e.g. Fig. 5, top panels), combined with parallel measurements of A260 of the nuclear samples, we estimate that the granulocyte nuclei have a core histone/DNA ratio similar to that in erythrocytes and lymphocytes. The ratio of total linker histones to core histones estimated by densitometry of Western blots probed with the anti-H1 antibodies appeared to be notably smaller in granulocytes (0.8) than the 1.4 value reported for adult chicken erythrocytes (39), showing that the extensive heterochromatization of granulocyte nuclei should not be attributed to an increased concentration of linker histones.

MENT Interacts with Naked DNA and Oligonucleosomes-- To determine whether the abundant granulocyte nuclear proteins interact with DNA in a completely defined in vitro system, we reconstituted specifically positioned hexanucleosomes on a 1.2-kb-long DNA fragment containing hexamer tandem repeats of a strong nucleosome positioning sequence from the 5 S rDNA of L. variegatus (27). By electrophoretic and spectral criteria, the reconstitutes (after separation from unbound core histones) contained one histone octamer per nucleosomal repeat (198 bp of DNA). Nucleosome positions were mapped by micrococcal nuclease and restriction nuclease cutting (data not shown) and found to be in agreement with the results of Meersseman et al. (30).

MENT or mim-1 was then allowed to interact with either the naked 1.2-kb DNA fragment or with the reconstituted hexanucleosomes. Mixing of up to 10 molecules of mim-1 per 200 bp with either DNA or oligonucleosomes did not induce any change in electrophoretic mobility. In contrast, MENT binding to the 1.2-kb naked DNA resulted in a considerable retardation on agarose gels (Fig. 6). Retardation was also observed with other sequences and lengths of DNA and did not show detectable sequence specificity. When increasing amounts of MENT were added to reconstituted hexanucleosomes at ratios of 0.3-1 MENT/nucleosome, the resulting complexes were retarded in agarose gels. Adding MENT at input ratios of 2 or more caused the particles to precipitate in the presence of 2 mM MgCl2, but the recipitation was reversed when Mg2+ was chelated by EDTA (Fig. 6, lane 10; compare top and bottom panels). MENT-containing oligonucleosomes closely resemble those reassociated with a similar amount of histone H5 both in terms of the extent of retardation in agarose and precipitation in the presence of divalent cations. Since previously we observed that during electrophoresis of native chromatin, MENT was associated with oligonucleosomes of various length but did not bind to nucleosome core particles containing 146 bp of DNA (16), we conclude that the histone-free linker DNA rather than nucleosome cores mediated the interactions of MENT with nucleosomes.

For direct visualization of the conformational changes caused by MENT, we compared the ultrastructure of hexanucleosomes containing only core histones with hexanucleosomes reconstituted with 1 molecule of MENT/nucleosome. Reconstitutes were fixed with glutaraldehyde, applied to thin carbon films, and positively stained with uranyl acetate or ethanolic phosphotungstic acid (35). With core histone-containing reconstitutes, hexanucleosomes appeared as more or less linear arrays of closely juxtaposed nucleosomes after fixation in solutions containing 50 mM NaCl (Fig. 6, bottom panel, upper row). The addition of 1 molecule of MENT/nucleosome to hexanucleosomes containing core histones resulted in the condensation of the nucleosome chains into tightly packed spherical clusters (Fig. 6, bottom panel, lower row).

MENT Promotes Tight Packing of Nuclear and Soluble Chromatin at Low Ionic Strength without Divalent Cations-- From previous experiments, we knew that at the protein/DNA ratio of 1 molecule/10 kb of DNA found in chicken erythrocytes, MENT facilitated chromatin condensation in the nuclei of immature erythrocytes in the presence of divalent cations (16) but did not prevent nuclear swelling in low salt/high pH media. To explore the potential effect of the much higher concentration found in granulocytes, chicken embryo erythrocyte nuclei containing less than 1 molecule of MENT/100 nucleosomes were allowed to interact with 1 molecule of MENT/200 bp of DNA in RSB, after which the nuclei were transferred into LS. In LS, the volume of the untreated erythrocyte nuclei increased ~80-fold, while the MENT-associated chicken erythrocyte nuclei experienced only a ~6-fold increase (Fig. 7, top row).


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Fig. 7.   MENT promotes chromatin compaction in low salt media. Top three panels, DAPI stain of isolated chicken embryo erythrocyte nuclei (CE) in RSB (left) and in LS (center). Right panel, chicken embryo erythrocyte nuclei reassociated with 1 MENT/200 bp of DNA and transferred into LS. Bottom three panels, electron microscopy of isolated oligonucleosomes fixed in 5 mM HEPES-native (left panel) and after mixing with 0.5 (central panel) and 1 molecule of MENT (right panel) per 200 bp of DNA.

We then explored the effect of MENT on chromatin conformation by adding the protein to native oligonucleosomes isolated from chicken erythrocyte nuclei. Under conditions of low ionic strength (5-20 mM NaCl), the native oligonucleosomes have the expected zig-zag conformation with clearly distinguished nucleosome cores and linker DNA (Fig. 7, lower left panel). Upon the addition of 0.5 molecule of MENT/nucleosome, a reduction of internucleosome distance and formation of more tightly packed nucleosome clusters was observed (Fig. 7, lower central panel). Particles of larger than hexanucleosome size presumably resulting from self-association of oligonucleosomes were frequent. Extended linker DNA was still seen occasionally, suggesting an uneven distribution of MENT within groups of nucleosomes. When 1 molecule of MENT/nucleosome was added, extended linkers were no longer observed, and the chromatin was seen as electron-dense particles of heterogeneous shape and size (Fig. 7, lower right panel). We conclude that at the concentration found in granulocyte nuclei, MENT clearly has a very significant condensing effect on chromatin.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The demonstration that a developmentally regulated protein, MENT, accumulates in mature chicken granulocyte nuclei at a level sufficient to cause a total compaction of nuclear chromatin is one of the most significant results of this work. MENT is the first heterochromatin-associated protein shown to be capable of inducing chromatin structural transitions in vitro at its physiological protein/DNA ratio. As previously observed with erythroid cells (16), MENT expression appears to be strictly confined to the terminal stage of cell differentiation; only a trace of MENT was observed in either proliferating or differentiating chicken promyelocytic cell lines: HD-13, HD-50, and 1A1 (40). It is noteworthy that promyelocyte nuclei have little if any heterochromatin as judged from light microscopy of stained cells. On the basis of this evidence, we propose that MENT is responsible for the extensive chromatin condensation observed during the terminal differentiation of myeloid cells.

In metazoan eukaryotes, heterochromatin formation is imposed on polynucleosomes already containing some histone H1 or similar linker histones (e.g. Refs. 4, 8, 9). A level of chromatin compaction above that usually associated with the presence of linker histone would therefore be expected. The extensive compaction and self-association of nucleosome arrays is thought to occur via a reduction of electrostatic repulsion between DNA negative charges brought about by a combination of low molecular weight counterions and the positively charged domains of core and linker histones, which altogether provide the cationic "glue" for contacts between neighboring nucleosomes (41-44). Even in the absence of linker histones, the compaction and self-association of reconstituted oligonucleosomes may be achieved if negative charges are neutralized by divalent cations (45). In the nuclei of maturing chicken erythrocytes, the main increase of net positive charge is driven by histone H5, which raises the linker histone to nucleosome ratio by about 40% (39) with most of the extra H5 deposited in repressed chromatin domains but not in transcriptionally active chromatin (46, 47). In the case of granulocyte heterochromatin, the level of linker histone is not increased, but instead, there is an extensive accumulation of another basic protein, MENT (pI 9.2). As deduced from the amino acid composition, MENT has more positive charges than histone H5, suggesting that the extent of DNA neutralization in granulocytes may exceed that found in erythrocytes. This is consistent with the high level of compaction and resistance of granulocyte nuclei to dispersion (see above). It thus seems likely that chromatin condensation can proceed via a common electrostatic mechanism but be driven by different proteins in different cells, suggesting that heterochromatin formation evolved independently even in cells sharing a common differentiation pathway, such as myeloid and erythroid cells.

A remarkable feature of chromatin condensation by MENT is that it proceeds through the highly selective association of the protein with a distinct chromatin fraction. MENT is strictly confined to the repressed chromatin in chicken erythrocytes, in contrast to H5, which is only partially enriched in this fraction (16). In vitro, MENT also recognizes the repressed chromatin isolated from chicken erythrocytes and binds to it in a highly selective manner. In granulocytes, where the concentration of MENT is high, its selective association with compact heterochromatin is clearly observed with electron microscopy (Fig. 2) Recently, we isolated a fraction of soluble granulocyte chromatin, which was selectively associated with MENT and had a highly compact higher order folding in low ionic strength media, while other granulocyte polynucleosomes that had lost MENT during isolation adopted a typical zig-zag conformation in vitro,2 thus confirming the uneven interaction of MENT with chromatin.

Although the exact mechanism of selective recognition of chromatin by MENT remains unclear, we suggest that, in chromatin, MENT recognizes the conformation of already inactivated chromosomal domains rather than specific DNA sequences. Thus, when reassociated with isolated nuclei, MENT selectively binds to repressed chromatin and interferes with the chromatin organization of the chicken c-myc gene in immature erythrocytes, where the gene is silent, but not in erythroblasts, where c-myc is transcriptionally active (16). When reassociated with immature erythrocyte nuclei, MENT did not inhibit transcriptional elongation ("run-on" assays) of either total erythrocyte nascent RNA or of beta -globin or histone H5 RNA.3 From these data, as well as from the analogy with linker histones that do not act as general transcriptional repressors (11-15), we propose that MENT may not necessarily play a direct role in transcriptional silencing. Rather, the main function of chromatin-condensing factors (MENT as well as linker histones) could be to "lock down" the already inactivated areas of genome, ensuring stringent repression of protooncogenes and other potentially harmful genetic loci involved in proliferation but down-regulated by the time of terminal differentiation. In this respect, it is worth noting that in myeloid cells, proteins involved in chromatin decondensation (and thus potential MENT antagonists) are linked to neoplastic transformations (48-51). Myeloid differentiation thus emerges as a very interesting system to study the physiological importance and interactions of chromatin-remodeling proteins.

In addition to linker histones and MENT, several other proteins have been implicated in the regulation of chromatin condensation and genetic repression associated with heterochromatin (reviewed in Refs. 3 and 5-7). However, the molecular mechanism(s) driving the formation of heterochromatin by these proteins is unknown. The only well studied (both genetically and biochemically) case of extensive structural alterations of chromatin is that associated with the yeast silencing complex (52). A common feature of the interaction of yeast silencers and MENT with chromatin is the formation of nearly stoichiometric complexes between nucleosomes and the chromatin-modifying proteins. This implies that the formation of compact heterochromatin in general might require binding of specific "architectural" proteins that act by altering the local conformation of nucleosome chains.

To look for structural similarities with other chromatin-binding proteins, we obtained amino acid sequences for several peptides derived from purified MENT. A computer search (53) of the available protein and DNA sequence data bases showed that MENT was a unique protein. No sequence homology was observed with the proteins of the yeast silencing complex (52) or other heterochromatin-associated proteins such as Polycomb (54), which resemble MENT in their association with repressed chromatin and in their focal localization within nuclei.

Among the homologies to other known sequences, the strongest ones were with proteins related to serine protease inhibitors (serpins) (55, 56). Three of the five peptides had strong homologies with the elastase inhibitor, while another two were closer to other serpin family members. The smallest sum probability, p(N), of stochastic matching of MENT peptides with horse elastase inhibitor in the PIR data base (accession number S34062), was 1.6e-09, making the chance of coincidental matching between MENT and the serpins highly unlikely.

We have tested the inhibitory activity of MENT toward the most common serine proteases with different cleavage specificity including trypsin, chymotrypsin, elastase, and thrombin in assays with synthetic protease substrates (57-59). No inhibition of any of the proteolytic reactions tested was observed. In collaboration with Dr. A. Greenberg (Winnipeg, Manitoba, Canada), MENT was also shown to have no inhibitory activity against Granzyme B and Granzyme 3, the serine proteases associated with natural killer activity whose protein targets are localized in cell nuclei (60). We thus conclude that, despite its strong sequence similarity with the serpin family, MENT does not share their protease inhibitory properties. Although no serpin-like protein has been previously reported to be located in the nucleus, it is not uncommon for serpins to lack a protease inhibitory activity and be involved in other cellular and extracellular functions (reviewed in Ref. 56).

A complete sequencing of MENT should finally clarify its relationship with other proteins and aid in identification of similar or homologous sequences in other organisms. We have already observed an abundant chromosomal protein with a strong antigenic relationship to MENT that may fulfill a similar function in condensed chromatin of mammalian leukocytes. Understanding the genetic and sequence organization of MENT and the molecular mechanism(s) underlying chromatin condensation in terminally differentiated mammalian cells are among the most important goals of our future work.

    ACKNOWLEDGEMENTS

We are grateful to Lucy Yin for planning and conducting the immunogold labeling experiments, Dr. A. Greenberg for tests of proteinase inhibition, Dr. R. T. Simpson for providing L. variegatus 5 S rDNA, Dr. C. Mura and Dr. S. Ness for providing antibodies, and T. Nikitina for technical assistance.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants GM-51352 (to S. G.) and GM-43786 (to C. W.). The Microscopy and Imaging Facility, University of Massachusetts, Amherst, is supported in part by National Science Foundation Grant BBS 87-14235.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger On leave from the Department of Molecular Biology, M. V. Lomonosov Moscow State University, Moscow, Russia. To whom correspondence should be addressed: Biology Dept., University of Massachusetts, Amherst, MA 01003. Tel.: 413-545-2878; Fax: 413-545-1696; E-mail: sergei{at}bio.umass.edu.

1 The abbreviations used are: PBS, phosphate-buffered saline; RSB, reticulocyte standard buffer; TE buffer, Tris-EDTA buffer; DAPI, 4',6-diamidino-2-phenylindole; PMSF, phenylmethylsulfonyl fluoride; LS, low salt medium; bp, base pair(s); kb, kilobase pair(s).

2 S. Grigoryev, C. Woodcock, and J. Bednar, manuscript in preparation.

3 S. Grigoryev and C. Woodcock, unpublished observations.

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Discussion
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