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• W2000606095 abstract "Terminal cell differentiation is correlated with the extensive sequestering of previously active genes into compact transcriptionally inert heterochromatin. In vertebrate blood cells, these changes can be traced to the accumulation of a developmentally regulated heterochromatin protein, MENT. Cryoelectron microscopy of chicken granulocyte chromatin, which is highly enriched with MENT, reveals exceptionally compact polynucleosomes, which maintain a level of higher order folding above that imposed by linker histones. The amino acid sequence of MENT reveals a close structural relationship with serpins, a large family of proteins known for their ability to undergo dramatic conformational transitions. Conservation of the “hinge region” consensus in MENT indicates that this ability is retained by the protein. MENT is distinguished from the other serpins by being a basic protein, containing several positively charged surface clusters, which are likely to be involved in ionic interactions with DNA. One of the positively charged domains bears a significant similarity to the chromatin binding region of nuclear lamina proteins and with the A·T-rich DNA-binding motif, which may account for the targeting of MENT to peripheral heterochromatin. MENT ectopically expressed in a mammalian cell line is transported into nuclei and is associated with intranuclear foci of condensed chromatin. Terminal cell differentiation is correlated with the extensive sequestering of previously active genes into compact transcriptionally inert heterochromatin. In vertebrate blood cells, these changes can be traced to the accumulation of a developmentally regulated heterochromatin protein, MENT. Cryoelectron microscopy of chicken granulocyte chromatin, which is highly enriched with MENT, reveals exceptionally compact polynucleosomes, which maintain a level of higher order folding above that imposed by linker histones. The amino acid sequence of MENT reveals a close structural relationship with serpins, a large family of proteins known for their ability to undergo dramatic conformational transitions. Conservation of the “hinge region” consensus in MENT indicates that this ability is retained by the protein. MENT is distinguished from the other serpins by being a basic protein, containing several positively charged surface clusters, which are likely to be involved in ionic interactions with DNA. One of the positively charged domains bears a significant similarity to the chromatin binding region of nuclear lamina proteins and with the A·T-rich DNA-binding motif, which may account for the targeting of MENT to peripheral heterochromatin. MENT ectopically expressed in a mammalian cell line is transported into nuclei and is associated with intranuclear foci of condensed chromatin. base pair(s) phosphate-buffered saline micrococcal nuclease polymerase chain reaction open reading frame nuclear localization signal In eukaryotic cells, DNA in association with histones and other nuclear proteins forms a DNA-protein complex or chromatin that exhibits varying levels of compaction (1van Holde K.E. Chromatin. Springer-Verlag New York Inc., New York1988Google Scholar, 2Wolffe A.P. Chromatin Structure and Function. Academic Press, Inc., New York, NY1995Google Scholar). Chromatin is folded hierarchically, with the basic level represented by a repeated structural unit, the nucleosome, comprising 200 ± 40-bp1 DNA of which 146 bp make about 1.7 superhelical turns around the histone octamer (3Luger K. Mader A.W. Richmond R.K. Sargent D.F. Richmond T.J. Nature. 1997; 389: 251-260Crossref PubMed Scopus (6883) Google Scholar), and the remainder (linker DNA) is not constrained by core histones. Linker DNA is usually associated with the ninth (linker) histone, which brings the core-proximal segments of linker DNA together (4Allan J. Hartman P.G. Crane-Robinson C. Aviles F.X. Nature. 1980; 288: 675-679Crossref PubMed Scopus (524) Google Scholar, 5Bednar J. Horowitz R.A. Dubochet J. Woodcock C.L. J. Cell Biol. 1995; 131: 1365-1376Crossref PubMed Scopus (139) Google Scholar, 6Hamiche A. Schultz P. Ramakrishnan V. Oudet P. Prunell A. J. Mol. Biol. 1996; 257: 30-42Crossref PubMed Scopus (157) Google Scholar). In vitro, nucleosome arrays fold into ∼30-nm-wide chromatin fibers having a characteristic zigzag organization at low ionic strength with separate nucleosomes connected by extended linkers (7Thoma F. Koller T. Klug A. J. Cell Biol. 1979; 83: 403-427Crossref PubMed Scopus (1179) Google Scholar, 8Worcel A. Strogatz S. Riley D. Proc. Natl. 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Imbalzano A. Genes Dev. 1996; 10: 905-920Crossref PubMed Scopus (404) Google Scholar), replication (20Holmquist G.P. Am. J. Hum. Genet. 1987; 40: 151-173PubMed Google Scholar), transposition (21Zou S. Voytas D.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7412-7416Crossref PubMed Scopus (82) Google Scholar), and perhaps other template-dependent functions. It has been known for many years that, in interphase nuclei, the chromatin is not uniformly decondensed but contains a considerable amount of more compact material, called heterochromatin (22Heitz E. Jahrb. Wissench. Bot. 1928; 69: 762-818Google Scholar), which generally increases during the terminal stages of eukaryotic cell differentiation (23Frenster J.H. Busch H. The Cell Nucleus. 1. Academic Press, Inc., New York1974: 565-581Google Scholar). Much cytological and genetic evidence suggests that chromatin compaction (spreading of heterochromatin) is associated with position-specific genetic inactivation (24John B. Verma R.S. Heterochromatin: Molecular and Structural Aspects. Cambridge University Press, Cambridge1988: 1-147Google Scholar, 25Lohe A. Hilliker A.J. Curr. Opin. Genet. Dev. 1995; 5: 746-755Crossref PubMed Scopus (74) Google Scholar, 26Elgin S.C.R. Curr. Opin. Genet. Devel. 1996; 6: 193-202Crossref PubMed Scopus (198) Google Scholar, 27Henikoff S. Curr. Opin. Cell Biol. 1997; 9: 388-395Crossref PubMed Scopus (85) Google Scholar, 28Singh P.B. Huskisson N.S. Dev. Genet. 1998; 22: 85-99Crossref PubMed Scopus (26) Google Scholar). A surprisingly high diversity of structurally distinct heterochromatin-associated proteins has been reported. For example, SIR3, SIR4, and RAP1 proteins are involved in telomeric silencing in Saccharomyces cerevisiae (29Hecht A. Laroche T. Strahl-Bolsinger S. Gasser S. Grunstein M. Cell. 1995; 80: 583-592Abstract Full Text PDF PubMed Scopus (694) Google Scholar, 30Cockell M. Palladino F. Laroche T. Kyrion G. Liu C. Lustig A.J. Gasser S.M. J. Cell Biol. 1995; 129: 909-924Crossref PubMed Scopus (155) Google Scholar), Pdd1p is involved in chromatin condensation and elimination in Tetrahymena thermophila (31Madireddi M.T. Coyne R.S. Smothers J.F. Mickey K.M. Yao M.-C. Allis C.D. Cell. 1996; 87: 75-84Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), and the numerous Su(var) and Polycomb Group proteins of Drosophila melanogaster (26Elgin S.C.R. Curr. Opin. Genet. Devel. 1996; 6: 193-202Crossref PubMed Scopus (198) Google Scholar, 32Reuter G. Spierer P. BioEssays. 1992; 14: 605-612Crossref PubMed Scopus (264) Google Scholar, 33Pirrotta V. Cell. 1998; 93: 333-336Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar) are implicated in chromosomal locus-specific genetic repression mediated by chromatin structure, as are the related “chromo” domain-containing vertebrate proteins (34Wreggett K.A. Hill F. James P.S. Hutchings A. Butcher G.W. Singh P.B. Cytogenet. Cell Genet. 1994; 66: 99-103Crossref PubMed Scopus (179) Google Scholar, 35Alkema M.J. Bronk M. Verhoeven E. Otte A. van't Veer L.J. Berns A. Lohuizen M. Genes Dev. 1997; 11: 226-240Crossref PubMed Scopus (228) Google Scholar). Although several studies have indicated that stable alterations of chromatin higher order folding distinguish the organization of euchromatin and heterochromatin (13Weintraub H. Cell. 1984; 38: 17-27Abstract Full Text PDF PubMed Scopus (177) Google Scholar, 36Weith A. Chromosoma. 1985; 91: 287-296Crossref PubMed Scopus (24) Google Scholar, 37Wallrath L.L. Elgin S.C.R. Genes Dev. 1995; 9: 1263-1277Crossref PubMed Scopus (408) Google Scholar), the structural determinants and the molecular mechanism of heterochromatin formation and spreading are still obscure. Terminal cell differentiation provides a convenient system for biochemical studies of the coordinated formation of heterochromatin and associated genetic inactivation. In particular, blood cell differentiation or hemopoiesis has been extensively studied both for its applicability to gene regulation and for its intrinsic significance for oncology (38Metcalf D. Nature. 1989; 339: 27-30Crossref PubMed Scopus (865) Google Scholar, 39Sachs L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4742-4749Crossref PubMed Scopus (120) Google Scholar). In the normal developmental sequence, the number of expressed genes becomes drastically reduced (40Tobin A.J. Selvig S.E. Lasky L. Dev. Biol. 1978; 67: 11-22Crossref PubMed Scopus (13) Google Scholar, 41Beaulieu A. Paquin R. Rathanaswami P. McColl S.R. J. Biol. Chem. 1992; 267: 426-432Abstract Full Text PDF PubMed Google Scholar), the nuclear chromatin undergoes a considerable increase in condensation (see reviews in Ref. 42), and nuclear matrix components, which are associated with transcriptional activity, are progressively lost (43LaFond R.E. Woodcock C.L.F. Exp. Cell Res. 1983; 147: 31-39Crossref PubMed Scopus (42) Google Scholar). Based on the hypothesis that the extensive heterochromatin spreading in blood cells is caused by one or more of the relatively few proteins that are still expressed during terminal differentiation, we have sought highly abundant nuclear proteins that specifically interact with repressed chromatin and are confined to the late stages of cell differentiation. Our study has yielded an abundant developmentally regulated 42-kDa nuclear protein, MENT (myeloid anderythroid nuclear termination stage-specific protein). This protein is expressed in terminally differentiated blood cells, is associated with repressed chromatin, and is able to induce large scale condensation of nuclear chromatin and the dissociation of inactivated chromatin from the nuclear matrix in vitro (44Grigoryev S.A. Solovieva V.O. Spirin K.S. Krasheninnikov I.A. Exp. Cell Res. 1992; 198: 268-275Crossref PubMed Scopus (30) Google Scholar, 45Grigoryev S.A. Woodcock C.L. Exp. Cell Res. 1993; 206: 335-343Crossref PubMed Scopus (22) Google Scholar). MENT is present in all three main avian blood cell types (erythrocytes, lymphocytes, and granulocytes) and is especially abundant in granulocytes, where it becomes the predominant nuclear nonhistone protein (∼2 molecules/nucleosome) and is concentrated in the compact peripheral heterochromatin (46Grigoryev S.A. Woodcock C.L. J. Biol. Chem. 1998; 273: 3082-3089Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). MENT-like polypeptides have also been found in chromatin of mammalian leukocytes. 2S. Grigoryev, unpublished observations. 2S. Grigoryev, unpublished observations. We have isolated the compact granulocyte chromatin in a soluble form and applied cryoelectron microscopy, a powerful imaging technique permitting the visualizing of the native organization of unfixed biological material (47Dubochet J. Adrian M. Chang J.J. Homo J.C. Lepault J. McDowall A.W. Schultz P. Q. Rev. Biophys. 1988; 21: 129-228Crossref PubMed Scopus (1764) Google Scholar) to the study of heterochromatin conformation. For the first time, we have documented a direct link between heterochromatin and higher order folding of nucleosome arrays that is attributed to the accumulation of a single nonhistone protein, MENT, in chromatin fibers. We have cloned the MENT cDNA, deduced the protein primary structure, and identified the protein structural motifs that may account for the molecular interactions of MENT. The combined results suggest a molecular basis for the formation of MENT-directed heterochromatin. Polymorphonuclear granulocytes were isolated from peripheral blood of adult white leghorn chicken as described (46Grigoryev S.A. Woodcock C.L. J. Biol. Chem. 1998; 273: 3082-3089Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). COS-7 cells (CV-1 simian cells transformed with origin-defective SV-40 virus expressing wild-type T antigen, ATCC number CRL-1651) were grown to 80% confluency in Dulbecco's modified Eagle's medium (D-5796; Sigma) containing 10% fetal calf serum (F-2442; Sigma) and 1 mm pyruvate. Cell cultures were washed 2 times in PBS containing 0.14 m NaCl, 2.7 mm KCl, 10 mm Na2HPO4, 1.7 mm KH2PO4, pH 7.5, and the cells were resuspended in PBS using a cell scraper. To isolate the nuclei, cell suspensions of granulocytes or COS-7 cells in PBS were centrifuged for 3 min at 1000 × g and resuspended in reticulocyte standard buffer containing 10 mm NaCl, 3 mm MgCl2, 10 mm Tris-HCl, pH 7.5, plus 0.5% Nonidet P-40 (Nonidet P-40; Life Technologies, Inc.) and 1 mm phenylmethylsulfonyl fluoride. With granulocyte nuclear preparations, the NaCl concentration varied between 0 and 0.3m. 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 reticulocyte standard buffer plus 1 mm phenylmethylsulfonyl fluoride. Isolated nuclei could be stored for a week at +2 °C without a detectable DNA or protein degradation. Micrococcal nuclease (MNase) digestion of all types of nuclei was conducted as described (46Grigoryev S.A. Woodcock C.L. J. Biol. Chem. 1998; 273: 3082-3089Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) and terminated by adding EDTA to 10 mm. To obtain soluble chromatin, the nuclei were digested with MNase to obtain an average DNA fragment size between 400 and 6000 bp and centrifuged for 5 min at 10,000 × g. The supernatant S1 was removed, and the pellet was resuspended in 1 ml of TEN (Tris-EDTA-NaCl buffer containing 10 mm Tris-HCl, pH 7.5, 1 mm EDTA, 10 mm NaCl). Supernatant S2 containing more than 50% of the input nuclear chromatin was then obtained after centrifugation for 5 min at 10,000 × g. The nuclear pellets were washed once more with 1 ml of TEN buffer with centrifugation to provide the S3 supernatant, and the nuclear remnant fraction was obtained by dissolving the final pellet in 1 ml of 0.5% SDS. DNA electrophoresis in agarose, polyacrylamide gel electrophoresis of proteins, detection of proteins and nucleic acids, Western blotting, probing with anti-MENT antibodies, and quantitative densitometry of electrophoregrams were conducted as before (46Grigoryev S.A. Woodcock C.L. J. Biol. Chem. 1998; 273: 3082-3089Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Protein to DNA ratios were estimated from parallel measurements of DNA concentration by UV spectrophotometry (A 260 = 1 for 50 μg/ml DNA). For size fractionation of soluble chromatin, 0.5 ml of S-150 sample containing 0.2 mg/ml DNA was loaded on a 5–25% sucrose gradient (12 ml) containing TAEW (25 mm sodium acetate, 2 mm Na2-EDTA, 20 mm Tris acetate, pH 7.4) or HEN (HEPES-EDTA-NaCl buffer containing 40 mm NaCl, 1 mm EDTA, 10 mm HEPES, pH 7.5). Ultracentrifugation was carried out in an SW-41 rotor on a Beckman L8–50 ultracentrifuge for 3 h at 4 °C and 35,000 rpm. 1-ml fractions were collected and used for protein and DNA electrophoreses. The chromatin pellet (fraction 1) was resuspended from the bottom of the tube in 1 ml of 0.5% SDS. Cryoelectron microscopy using soluble chromatin samples (50–100 μg/ml in TEN buffer) was conducted as described (5Bednar J. Horowitz R.A. Dubochet J. Woodcock C.L. J. Cell Biol. 1995; 131: 1365-1376Crossref PubMed Scopus (139) Google Scholar, 48Woodcock C.L. Horowitz R.A. Methods Companion Methods Enzymol. 1997; 12: 84-95Crossref Scopus (27) Google Scholar). Specifically, 3-μl chromatin samples were applied to holey carbon films, blotted with Whatman 52 filter paper, and plunged into liquid ethane held just above its freezing point in liquid nitrogen. Grids were transferred under liquid nitrogen to a cryoholder (model 626; Gatan Inc., Pleasanton, CA), and observed at −170 °C in an electron microscope (CM10; Philips Electronic Instruments Co., Mahwah, NJ) at a nominal magnification of × 45,000. Tilt pairs of micrographs (angular separation 30°) at 1.1–1.5-μm defocus, were recorded in low dose mode, on film S0–163 (Eastman Kodak Co., Rochester, NY) and developed in full-strength D-19 (Kodak) for 12 min. MENT protein was isolated from the nuclei of unfractionated chicken blood cells as described (44Grigoryev S.A. Solovieva V.O. Spirin K.S. Krasheninnikov I.A. Exp. Cell Res. 1992; 198: 268-275Crossref PubMed Scopus (30) Google Scholar). Five peptides derived from isolated MENT were sequenced. For PCR-mediated cloning of MENT cDNA, we designed redundant oligonucleotide primers as described (49Wilkie T.M. Aragay A.M. Watson A.J. Simon M.I. Methods Enzymol. 1994; 237: 327-344Crossref PubMed Scopus (9) Google Scholar). The 20–22-nucleotide-long primers were deduced from MENT regions with minimally degenerate codons. Total RNA and poly(A)+mRNA was isolated from 1 g of chicken bone marrow using Trizol reagent (Life Technologies, Inc.) and the Message Maker RNA isolation kit (Life Technologies), and the first strand cDNA synthesis was conducted using the Life Technologies Superscript preamplification system essentially as described in the vendor's manual. PCR amplifications with degenerate primers were carried out using the AmpliTherm polymerase and MasterAmp PCR optimization kit (Epicentre Technologies, Madison, WI). Amplification cycles were carried out in 30-μl samples using a high melting wax for “hot start” conditions. The first denaturation for 5 min at 94 °C was followed with 35 cycles, each containing three 1-min steps at 94, 42, and 72 °C, and finally for 10 min at 72 °C. PCR products were cloned into pCRII vector (Invitrogen) and sequenced using the Sequenase II system (Amersham Pharmacia Biotech). One of the sequenced PCR products amplified between the primers specific for peptides 1 (ATIGGIAA(C/T)TT(C/T)ACIGTIGA) and 3 (TGIAT(A/G)TT(C/T)TCIGC(C/T)TG(C/T)TC) also included the sequence of peptide 2, indicating that this PCR product was derived from the target MENT cDNA. To restore the 5′-end of the cDNA, we employed the 5′-rapid amplification of cDNA ends PCR technique (50Frohman M.A. Dush M.K. Martin G.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8998-9002Crossref PubMed Scopus (4332) Google Scholar) essentially as described (51Schuster D.M. Buchman G.W. Rashtchian A. Focus. 1992; 14: 46-52Google Scholar). The 3′-end of the cDNA was restored using the 3′-rapid amplification of cDNA ends PCR method (50Frohman M.A. Dush M.K. Martin G.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8998-9002Crossref PubMed Scopus (4332) Google Scholar). The PCR-amplified DNA fragments were inserted into pCRII vector (Invitrogen) and sequenced on both strands, revealing a 1230-bp open reading frame (ORF) encoding all five independently sequenced MENT peptides. To verify the ORF sequence, PCR amplification was repeated with two nonredundant primers flanking the MENT ORF, and the product was inserted in the pCRII vector and sequenced on both strands with the Sequenase II system. MENT ORF was amplified by reverse transcription-PCR with oligonucleotide primers containing appropriate restrictase sites and inserted in pRc/CMV vector (Invitrogen) to provide a MENT-expressing plasmid, pSG109. COS-7 cells were transfected with pSG109 and SuperFect transfection media (Qiagen) as described in the vendor's manual. Transfected cells were grown for 48 h. Fixing of cells attached to the cover glasses with methanol/acetone and staining with anti-MENT antibodies and DNA dyes (10 μg/ml Hoechst 33258) was performed essentially as described (45Grigoryev S.A. Woodcock C.L. Exp. Cell Res. 1993; 206: 335-343Crossref PubMed Scopus (22) Google Scholar). For double-staining with anti-MENT and anti-fibrillarin (52Pollard K.M. Lee D.K. Casiano C.A. Bluthner M. Johnston M.M. Tan E.M. J. Immunol. 1997; 158: 3521-3528PubMed Google Scholar) antibodies, cells were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. Fluorescence and confocal microscopy were conducted as described (45Grigoryev S.A. Woodcock C.L. Exp. Cell Res. 1993; 206: 335-343Crossref PubMed Scopus (22) Google Scholar). A search for DNA and protein homologies in the available data banks was performed using the BLAST program (53Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (70277) Google Scholar). Sequence alignments were conducted using the Wisconsin package version 9.1 (Genetics Computer Group, Madison, WI). The protein pI was calculated as described (54Bjellqvist B. Basse B. Olsen E. Celis J.E. Electrophoresis. 1994; 15: 529-539Crossref PubMed Scopus (403) Google Scholar). To build the three-dimensional protein model, we employed the ProModII software (55Peitsch M.C. Biochem. Soc. Trans. 1996; 24: 274-279Crossref PubMed Scopus (899) Google Scholar, 56Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9554) Google Scholar). The model-building resources are provided by the automated protein modeling server, Swiss Model (Glaxo Wellcome Experimental Research, Geneva, Switzerland), which is accessible through the Internet. The protein models were visualized using the Swiss-PDB viewer program compatible with Windows 95TM. Modeling of electrostatic and surface properties of the molecules was performed using the GRASP program (Graphical Representation and Analysis of Surface Properties (57Nicholls A. Sharp K.A. Honig B. Proteins Struct. Funct. Genet. 1991; 11: 281-296Crossref PubMed Scopus (5315) Google Scholar)) running on a Silicon Graphics Indigo workstation with operating system IRIX 5.3. Highly condensed chromatin from chicken granulocyte nuclei has a nucleosome repeat (192 ± 2 bp) and a core and linker histone content (46Grigoryev S.A. Woodcock C.L. J. Biol. Chem. 1998; 273: 3082-3089Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) similar to that of many cells with much more active genomes (1van Holde K.E. Chromatin. Springer-Verlag New York Inc., New York1988Google Scholar). Unlike most other types of chromatin, which are readily solubilized after MNase digestion and removal of divalent cations, granulocyte nuclei digested to DNA fragment sizes between 200 and 20,000 bp did not release any soluble chromatin after repeated washing in low salt/high pH media. Our previous work suggested that MENT, an extremely abundant granulocyte chromatin protein (2.1 molecules/nucleosome) was the most likely factor inhibiting chromatin solubility (46Grigoryev S.A. Woodcock C.L. J. Biol. Chem. 1998; 273: 3082-3089Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). To address the impact of auxiliary proteins on chromatin conformation, it was essential to isolate granulocyte chromatin from the nuclei while maintaining its association with MENT. We therefore determined the optimal salt concentration allowing chromatin solubilization with a minimal loss of associated chromatin proteins by preparing a series of granulocyte chromatin fractions eluted from nuclease-digested nuclei with varying concentrations of NaCl. An abrupt increase in chromatin solubility occurred between 100 and 150 mm NaCl (Fig.1, a and b). The amount of MENT retained in chromatin also changed very considerably between these two NaCl concentrations, as shown by Western blotting of nuclear proteins probed with anti-MENT antibodies (Fig.1 c). Granulocyte nuclei washed with 150 and 300 mm NaCl (samples CG-150 and CG-300) retained, respectively, 25 and 1.4% of the original MENT and were used for isolating soluble chicken granulocyte chromatin after limited MNase digestion. Further characterization was carried out on the soluble chromatin fraction S2 (see “Materials and Methods”), which contained about 50–90% of total nuclear DNA. The MENT/DNA ratio was the same in the TEN-washed nuclei as in the soluble chromatin fractions. Depending on the extent of micrococcal nuclease digestion, the average size of nucleosome chains (N av) in S2 preparations varied between 3 and 50 without affecting the protein composition of the solubilized material (data not shown). Since the isolation of granulocyte chromatin required a considerable depletion of MENT, it was important to determine if the residual protein was bound firmly enough to be considered as an integrated architectural element of soluble polynucleosomes. We subjected soluble granulocyte polynucleosomes (CG-150, N av = 20) to ultracentrifugation in 5–25% sucrose gradients containing 50 mm monovalent ions and analyzed the gradient fractions by DNA electrophoresis and Western blotting (Fig.2, a and b). All input MENT cosediments with chromatin (Fig. 2 b), none being found at the top of the gradient. Thus, the behavior of MENT is in sharp contrast to the ubiquitous architectural nonhistone chromatin protein, HMG-1, most of which does not co-sediment with chromatin under these conditions (58Falciola L. Spada F. Galogero S. Langst G. Voit R. Grummt I. Bianchi M. J. Cell Biol. 1997; 137: 19-26Crossref PubMed Scopus (111) Google Scholar). We observed a significant enrichment in MENT (0.84 molecules of MENT/nucleosome) in the fastest sedimenting fraction containing long (>20-mer) polynucleosomes. Small oligonucleosomes contained less MENT, with practically none recovered from the mononucleosome fraction. As discussed below, the distribution of MENT is consistent with the greater compaction of long polynucleosomes (Fig.3, a–f). The low level of MENT in short oligonucleosomes appears to reflect their origin in less condensed “euchromatin” rather than their inability to bind the protein; when we ran a sucrose gradient of a granulocyte chromatin sample from the same nuclei (CG-150) but obtained after extensive cleavage by MNase (N av = 3), MENT was abundant in both the oligonucleosome and mononucleosome fractions, and again no free protein was detected (data not shown). Therefore, the most likely explanation for the preferential association of MENT with long polynucleosomes is that this fraction originated from heterochromatic nuclear domains relatively protected from nuclease digestion (MENT inhibits MNase digestion and is unevenly distributed in granulocyte nuclei, being preferentially associated with compact heterochromatic areas (46Grigoryev S.A. Woodcock C.L. J. Biol. Chem. 1998; 273: 3082-3089Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar)). As the heterochromatin becomes more extensively digested, MENT then appears in the oligonucleosome and mononucleosome fractions. To determine whether any other proteins besides MENT are associated with compact granulocyte chromatin, we analyzed the protein composition of nuclear and soluble chromatin released after the 150 mmNaCl treatment (Fig. 2 c, lanes 1 and2) and after sedimentation of oligonucleosomes (N av = 3) through a sucrose gradient (lane 3). Densitometry of the Coomassie-stained gel shows that soluble CG-150 chromatin (lane 2) retains 26% of the MENT present in untreated granulocyte nuclei (lane 1), which, from the previous estimation of 2.1 MENT molecules/nucleosome in granulocytes (46Grigoryev S.A. Woodcock C.L. J. Biol. Chem. 1998; 273: 3082-3089Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), indicates that about 0.5 molecules of MENT/nucleosome are associated with total soluble chromatin. After centrifugation, MENT remains the only prominent band among the nonhistone proteins (Fig. 2 c,lane 3). All other abundant nonhistone proteins such as actin and Mim-1 present in the crude nuclear preparation (46Grigoryev S.A. Woodcock C.L. J. Biol. Chem. 1998; 273: 3082-3089Abstract Full Text Full Text PDF PubMed Scopu" @default.
• W2000606095 created "2016-06-24" @default.
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• W2000606095 creator A5018186705 @default.
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• W2000606095 date "1999-02-01" @default.
• W2000606095 modified "2023-09-30" @default.
• W2000606095 title "MENT, a Heterochromatin Protein That Mediates Higher Order Chromatin Folding, Is a New Serpin Family Member" @default.
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