INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

Linker histone is an abundant basic protein present in almost all eukaryotes. The protein is involved in the formation of higher order structure in the chromatin (1) and the maintenance of the overall chromatin compaction (2). In general, linker histone has a tripartite structure: a central globular domain flanked by N- and C-terminal tail domains (3). The globular domain binds the linker DNA and interacts with the nucleosome where DNA enters and exits the nucleosome (4, 5). Unlike core histones, linker histones diverge significantly in sequence and structure (6, 7).

Numerous developmentally regulated variants of linker histone have been defined. These variants can be subdivided in three major groups in vertebrates as a function of their expression during development and cell differentiation (8). First, an embryonic form of linker histone is present during the oogenesis and the early development in amphibians. Replication-dependent types are present in all tissues during the life of the organism, and finally the differentiation-specific group accumulates in differentiating cells. Some members of this later group are tissue- and species-specific, like histones H5 and H1t. Others, like histone H1⁰, are widely expressed in many tissues and in almost all vertebrates (9).

Previously, we have shown that there is a tight correlation between the type of linker histone expressed and the proliferative capacities of cells during early Xenopus development. Histone H1⁰ appears relatively late and concomitant with a dramatic decrease in the cell proliferation during the tail bud-tadpole transition period (10). Therefore, crucial periods in development can be characterized by a transition in the linker-histone variants within chromatin. Nothing is known concerning the role of these variants in specific organization of the chromatin structure. Immunolocalization of these proteins using specific polyclonal and monoclonal antibodies provided interesting information concerning the distribution of a given linker histone variant in the nucleus (11-13). However, chromatin organization is extremely dynamic and is subject to permanent remodeling. One of the most striking examples of this phenomenon is early embryonic development. Indeed, transition periods have been defined during development that are characterized by the modification of both chromatin constituents and the proliferative capacities of cells (8). Moreover, later during development and in adult tissues, chromatin remodeling continues as adult type linker histones accumulate in cells (9). It is therefore of great importance to understand the nature of these remodeling processes and to evaluate their role in the expression of specific genetic programs.

The aim of this work was the identification of monoclonal antibodies raised against histone H1⁰, showing specific abilities in recognizing this protein in chromatin. Individual anti-H1⁰ monoclonal antibodies were screened in an immunolocalization assay to isolate clones able to recognize H1⁰ in a differentiation-dependent manner. Antibodies characterized in this work appeared to be probes that are useful for monitoring chromatin structure modifications occurring concomitantly with regulatory events such as the onset of a differentiation program and the arrest of cell proliferation.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Cell Culture-- Murine erythroleukemia (MEL)¹ cells from clone G9, a subclone of F4NW0, were maintained in culture in minimum essential medium (Life Technologies, Inc.) containing 10% fetal calf serum (14). To induce differentiation, MEL cells in exponential growth were treated with hexamethylene-bis-acetamide (Sigma) as described previously (14). Clone 6 cells, a rat embryonic fibroblast cell line transformed by ras, were maintained in RPMI 1640 (Boehringer) supplemented with 5% fetal calf serum and glutamin 4 mM and grown in a humidified atmosphere of 95% air, 5% CO₂ normally at 37 °C or shifted to 32 °C to induce the cell growth arrest (15).

Purification of Nuclei and Oligonucleosomes-- Nuclei were extracted from untreated or hexamethylene-bis-acetamide-treated MEL cells. Cells were collected and washed with phosphate-buffered saline. After centrifugation at 200 × g for 5 min, cells were lyzed in a lysis buffer containing 15 mM Tris-HCl, pH 7.4, 60 mM KCl, 15 mM NaCl, 0.65 mM spermidine, 2 mM EDTA, 0.5 mM EGTA, 0.34 M sucrose, 0.05% (v/v) Triton X-100, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride. After centrifugation at 1500 × g for 5 min, nuclei were rinsed with the same buffer lacking EDTA, EGTA, and Triton (buffer D).

Preparation of Digested and Recombinant Histone H1⁰-- Mouse full-length histone H1⁰ cDNA (16) or cDNAs corresponding to mutated H1⁰ were cloned in pET expression vector (Novagen). All mutations and deletions were performed by polymerase chain reaction. Briefly, for N-terminal deletion mutants, oligonucleotides (33 bases) containing the desired sequence (removing an increasing number of amino acids from the N-terminal part of the protein, see Fig. 1) and an additional NdeI restriction site were used to amplify cDNAs by polymerase chain reaction. The 3 primer had the sequence of the stop codon region and an additional XhoI site. The proline-valine mutant as well as the 24-30 deletion mutant were produced according to the method described in Refs. 17 and 18. Polymerase chain reaction products were digested with NdeI and XhoI and cloned, and their sequences were verified.

Antibodies-- Anti-H1⁰ antibodies were monoclonal antibodies produced in our laboratory as described previously (19). Antibodies used in this work were from clones 27E 8E10 (clone 27 antibody) and 34B10H4 (clone 34 antibody). For immunostaining, hybridoma supernatant was used, and for gel shift assays, Igs were purified as follows. Ascites were induced in BALB/c mice after the intraperitoneal injection of hybridoma cells (5 × 10⁶ cells/mouse). Ascitic fluid was collected and centrifuged at 3000 × g for 10 min, and the supernatant was collected. Igs were then purified as described (20). Briefly, albumin and other non-Ig proteins were first precipitated with caprilyc acid. Then Igs were precipitated by ammonium sulfate.

Protein Electrophoresis and Immunodetection-- H1 histones were analyzed by SDS-15% polyacrylamide gel electrophoresis (21). Proteins were transferred to a Hybond C extra membrane (Amersham Corp.) at 24 V (0.2 A) for 1 h with a semi-dry electrotransfer apparatus. The membrane was blocked in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl (Tris-buffered saline) containing 3% bovine serum albumin. The membrane was incubated with specific antibodies and then with an horseradish peroxidase-linked sheep anti-IgG. Detection was performed by enhanced chemiluminescence kit (ECL, Amersham Corp.). Oligonucleosomes fractionated on sucrose gradient were dotted on Hybond C extra membrane (10 µg of chromatin for each fraction) and subjected to immunodetection as above.

Gel Shift Assays-- The four-way junction DNA was obtained by annealing four oligonucleotides synthesized according to the sequence published by Teo et al. (22). The four oligonucleotides were annealed in 10 mM Tris-HCl, 1 mM EDTA, and 50 mM NaCl, gel purified, and labeled by polynucleotide kinase and [-³²P]ATP.

Total RNA Extraction and Northern Blot Analysis-- RNAs were extracted from cells and analyzed by Northern blot by methods described by Khochbin et al. (14).

Immunostaining-- Cells were collected and fixed with 3% paraformaldehyde in phosphate-buffered saline for 20 min at room temperature, permeabilized with 0.25% (v/v) Triton X-100 (Sigma), then incubated with monoclonal antibody against H1⁰. The second antibody was a goat fluorescein isothiocyanate-conjugated F(ab)₂ fragment against mouse IgG Fc fragment (Jackson Immunoresearch Lab). After immunolabeling, DNA was stained with DNA-specific fluorochrome Hoechst 33258 (Sigma). When immunostaining was carried out on nuclei, purified nuclei were obtained as described above, resuspended in 20 mM Tris, pH 7.4, 6 mM MgCl₂ containing either 100 mM, 200 mM or 300 mM NaCl, at room temperature for 30 min then fixed with 3% paraformaldehyde in the same buffer for 1 h at +4 °C. Nuclei were then doubly stained according to the procedure described above.

Analysis by Flow Cytofluorimetry-- The doubly stained cells were analyzed in a FACStar Plus (Becton Dickinson) using a dual laser configuration: the Hoechst fluorescence was excited at 340-360 nm by the first argon laser, and the fluorescein isothiocyanate fluorescence at 488 nm by the second argon laser. The fluorescence intensities were collected in a list mode. To determine the mean specific H1⁰ fluorescence per cell we used ProCyt®, a computer program developed in our laboratory (available on request) (24).

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Screening of Monoclonal Antibodies for H1⁰ Recognition in Chromatin-- Previously, we have reported the preparation of monoclonal antibodies raised against histone H1⁰ (19). In this work we undertook a screening of these antibodies for their ability to recognize the protein in chromatin. The first screening was performed using immunodetection of the protein in undifferentiated MEL cells. This work allowed us to define two clones: one (34B10H4) able to recognize H1⁰ in chromatin and the second one (27E8E10) not able to bind the protein in this environment (both antibodies recognizing H1⁰ in cellular or nuclear extracts with high specificity; data not shown). For simplicity, in the text we will refer to 34B10H4 and 27E8E10, as clone 34 and clone 27 antibodies, respectively. Preliminary mapping based on the recognition of peptides obtained by partial cleavage of H1⁰ by cyanogen bromide (cleaving the protein at methionine 31) showed that the N-terminal part of the protein is essential for recognition; neither antibody recognized the protein fragment containing the amino acids 31-193 (Fig. 1A). We therefore focused our attention on the N-terminal part of histone H1⁰. A series of experiments were planned to map precisely the epitopes recognized by these two antibodies and to elucidate the basis for the differential recognition of the protein in chromatin.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Region of H10 involved in the recognition of the protein by monoclonal antibodies (clone 27E8E10 and 34B10H4 antibodies). A, partial cyanogen bromide cleavage of H10. Peptides were resolved on 15% SDS-polyacrylamide gel (Stain) and transferred to a membrane, and an immunodetection was carried out using the two antibodies (right panel). B, schematic representation of the bacterially expressed H10 bearing deletions and mutations (50 N-terminal AA are considered). The lines represent the N-terminal tail domains, and the boxes represent the globular domains. The two arrowheads in the Pro-Val mutant show the region where the mutation is placed. The gap in the 24-30 mutant represents the region deleted. C, the various mutants were analyzed on 15% SDS-polyacrylamide gel (Stain) and transferred to a membrane, and an immunodetection was carried out using the two antibodies and revealed by ECL system (27E8E10 and 34B10H4). WT means wild type mouse H10, and 10, 15, 20, 38, 41, and 44 stand for proteins having 10, 15, 20, 38, 41, and 44 N-terminal AA deleted respectively. Pro-Val (B) and P-V (C) mean a mutation converting proline 26 into valine, and 24-30 indicates a protein having a deletion covering the 24-30 AA region.

Precise Epitope Mapping-- Mouse H1⁰ cDNAs encoding the wild type protein or proteins bearing mutations affecting the N-terminal tail and the globular domain were cloned into prokaryotic expression vectors to obtain purified recombinant proteins (Fig. 1B). The wild type recombinant protein was efficiently recognized by both antibodies, and moreover, the complete deletion of the N-terminal tail domain did not affect protein recognition (Fig. 1C, 20). However, the removal of 18 AA from the N-terminal part of the globular domain completely abolished the binding of these antibodies (Fig. 1C, 38). A portion of the protein covering the AA 20-38 region therefore plays an essential role in the recognition of the protein by both antibodies.

View larger version (43K): [in this window] [in a new window]   Fig. 2.   Involvement of the AA 20-30 region in the recognition of the protein by the two antibodies. A, the sequences of the AA 21-39 region of H10 from human (34), mouse (16), rat (35), and Xenopus (36) were compared with the sequence of the corresponding region of the sea urchin H1 (25). The identical AA are shaded. B, total H1 extracted from Xenopus and sea urchin tissues as well as from MEL cells were resolved on 15% SDS-polyacrylamide (Stain) and transferred into a membrane, and an immunodetection was carried out with the two antibodies (34B10H4 and 27E8E10, respectively). C, the scheme summarizes data presented in Fig. 1 and in A and B of this figure. The AA 20-30 and AA 24-30 regions appear to be essential for the recognition of the protein by the clone 34 antibody and the clone 27 antibody, respectively. N stands for N-terminal tail domain, and G stands for the globular domain.

24-30

Interaction of H1⁰ with Four-way Junction DNA and the Recognition of the Target Epitope by Clone 34 and 27 Antibodies-- The high affinity binding of H1 to four-way junction DNA (23, 28) allows the study of the specific aspects of H1-DNA interactions (29). We took advantage of this model to determine how the defined target epitopes are recognized by our antibodies when H1⁰ interacts with DNA. Conditions for a complete shift of the labeled four-way junction DNA upon the addition of recombinant H1⁰ (wild type or mutated) were determined, excesses of the clone 27 and 34 antibodies were added to the mixture, and the shift was examined. Upon the addition of the clone 34 antibody, three different cases were observed: 1) a fraction of DNA-H1⁰ complex interacts with the antibody and is super-shifted (Fig. 3A, WT panels, lanes +H1⁰+Ab); 2) a fraction of DNA-H1⁰ complex is not recognized by the antibody; and 3) a fraction is dissociated as indicated by the release of free DNA.

View larger version (58K): [in this window] [in a new window]   Fig. 3.   Binding of H10 to four-way junction DNA and the recognition of the complex by the clone 27 and 34 antibodies. A, 32P-labeled four-way junction DNA was incubated with wild type H10 (WT) or the indicated mutants (+H10 lane), and the indicated antibody is added (+H10+Ab lane). Complexes were analyzed in 4% polyacrylamide gel. Antibody used for the analysis is indicated above each panel. WT, 10, and 20 represent the wild type H10 and the 10 and 20 mutants. B, the same experiment as above was carried out except that the Pro-Val and the 24-30 mutant were used, and the ability of the antibodies to supershift and dissociate the complex was analyzed (+H10+Ab34 and +H10+Ab27, respectively).

panels 10

20

panels 10

20

Differential Recognition of H1⁰ within Chromatin during the Induced Differentiation of MEL Cells-- MEL cells are virus-transformed erythroid precursors able to undergo a differentiation program under the action of a large variety of chemical inducers (30). We used this differentiation model to monitor H1⁰ recognition by our antibodies during cell differentiation. Uninduced MEL cells or cells treated with the inducer (4 mM hexamethylene-bis-acetamide) for 6, 8, 16, 24, 32, and 48 h were fixed, and the immunofluorescence was monitored by flow cytofluorimetry after immunostaining with clone 27 and 34 antibodies. In uninduced MEL cells, whereas H1⁰ is efficiently recognized by the clone 34 antibody, the protein is not recognized by clone 27 antibody (Fig. 4A, 0 h). In these cells, clone 27 antibody-related immunofluorescence corresponds to the background fluorescence, which is observed when anti-H1⁰ antibody is omitted (not shown).

View larger version (47K): [in this window] [in a new window]   Fig. 4.   Flow cytofluorimetric analysis of the recognition of H10 by the two antibodies during the induced differentiation process in MEL cells. A, histograms show the clone 27 and 34 antibody-related immunofluorescence (upper and lower panels, respectively) recorded from uninduced cells (0 h) or cells induced for indicated times with 4 mM hexamethylene-bis-acetamide. Each histogram corresponds to the analysis of 10,000 cells (y axis). B, RNA samples prepared from cells taken at the indicated time after the induced differentiation were analyzed according to the Northern blot procedure. The blot was hybridized with -globin and GAPDH probe, respectively. C, effect of the salt-induced modification of the chromatin structure on the recognition of H10 by the two antibodies. Nuclei were isolated from the uninduced cells and incubated for 30 min in a buffer containing the indicated concentration of NaCl. They were then subjected to immunostaining and analyzed as above.

View larger version (56K): [in this window] [in a new window]   Fig. 5.   Differential recognition of H10 by the two antibodies in fractionated chromatin from undifferentiated MEL cells or cells induced to differentiate. A, chromatin obtained after the micrococcal nuclease digestion of the nuclei purified from MEL cells and cells induced for 48 h was fractionated on a sucrose gradient. Fractions were collected and DNA from each fraction was purified and analyzed on 1% agarose gels. B, equivalent amounts of oligonucleosomes (10 µg of chromatin) corresponding to the above fractions were dotted in duplicate, and an immunodetection using the two anti-H10 antibodies was carried out (anti-H10 panel). The same blots were then washed and another immunodetection was carried out using an anti-H1 antiserum (anti-H1 panel). As a control 5, 10, and 20 ng of purified H10 were loaded on the same membrane (purified H10 panel).

Modification of the H1⁰ Recognition during the P53-mediated Arrest of Cell Proliferation-- Clone 6 cells are rat embryonic fibroblasts transformed by ras and a thermosensitive P53 mutant. At 37 °C, P53 is in a mutated conformation that is responsible for the appearance of a transformed phenotype. At 32 °C, P53 exhibits the property of the wild type protein and triggers an arrest of cell proliferation (15). We used this system to monitor H1⁰ accessibility during this process. A flow cytofluorimetric analysis of H1⁰ immunolabeling using clone 34 antibody was performed (Fig. 6A). H1⁰ was detected by indirect immunofluorescence (y axis) and DNA by Hoechst fluorescence (x axis). DNA fluorescence reflects the position of cells in the cell cycle (G₁ cells are around channel 60, G₂ cells are found around channel 120, and S phase cells are in between). A general increase of H1⁰ immunofluorescence is observed during the cell cycle indicating the normal doubling of cell constituents when cells accomplish DNA replication and enter the G₂/M phases of the cell cycle (Fig. 6A, panel 37 °C). However, 8 h after the transfer of cells at 32 °C, a clear increase in the H1⁰ immunofluorescence intensity was observed, specifically visible in the G₂/M cell populations (32 °C panel, dot plot representation, compare 0 and 8 h). It is precisely in this phase of the cell cycle that the first accumulation of cells is observed (Fig. 6A, note the accumulation of cells in the G₂/M phase of the cell cycle, 32 °C panel, 8 h histogram). After 16 h at 32 °C, these cells enter the G₁ phase and stop proliferating. An increase in the H1⁰ immunofluorescence is visible in these arrested cells (panel 32 °C, lane 16 h). After 24 h at 32 °C, almost all cells are in the G₀/G₁ phase of the cell cycle, and a clear increase of the H1⁰ related immunofluorescence intensity is observed in these cells compared with the control cells kept at 37 °C (panel 32 °C, lane 24 h). Clone 27 antibody did not recognize H1⁰ either in proliferating cells or in arrested cells (data not shown).

View larger version (68K): [in this window] [in a new window]   Fig. 6.   Cell cycle-dependent modulation of the chromatin structure during the induced arrest of cell proliferation. Clone 6 cells expressing thermosensitive P53 were shifted to 32 °C for the indicated times (0, 8, 16, and 24 h), and the control cells were maintained at 37 °C during this period. A, flow cytofluorimetric analysis of the immunodetection of H10 was carried out as described in the legend of Fig. 4, except in this case cells are doubly stained for H10 by indirect immunofluorescence and for DNA with the DNA-specific dye Hoechst 33258. This method (dual analysis of cells by cytofluorimetry; dot plot representations shown in the left panel for each temperature) allows monitoring of H10 immunoreactivity as a function of the position of cells in the cell cycle. For each temperature, the modification of the cell cycle parameters as a function of time is visualized in histograms showed on the right side. These histograms represent the number of cells present at different positions in the cell cycle (DNA fluorescence). B and C, the induced arrest of cell proliferation is not associated with an increase in the amount of H10 encoding mRNA and protein. RNA prepared from cells maintained at 37 °C or shifted to 32 °C was analyzed according to the Northern blot procedure. The blot was probed with a H10, H4, or 28 S rRNA probe (B). Total H1 was extracted from proliferating cells (0 h) or after the shift of temperature (8, 16, and 24 h). Proteins were analyzed on SDS-15% polyacrylamide gels stained with Coomassie Blue and by Western blotting.

Differential Pattern of H1⁰ Immunolocalization by Clone 27 and Clone 34 Antibodies-- To know if the differential recognition of H1⁰ by these antibodies correlates also with a specific pattern of immunodetection, we performed a microscopic analysis of immunolabeled nuclei. Xenopus embryos were first used in this experiment to examine the situation in an in vivo context. H1⁰ accumulates relatively late during the Xenopus development, and the first detectable accumulation of the protein is tissue-specific, observed in the nervous tissue, somites, and the cement gland. Later during development, at tadpole stage, the accumulation of the protein was observed in many different tissues (10). The analysis of the immunolabeled cells observed on a section of the cement gland (for example, Fig. 7A, arrowhead) shows that nuclei are immunolabeled with both antibodies and that, moreover, foci could be observed in clone 27 antibody-immunolabeled nuclei (Fig. 7B, bottom left panel) in contrast to a relatively homogenous labeling for nuclei labeled by the clone 34 antibody. Nuclei of cells forming the neighboring tissue are negative for H1⁰ detection (Fig. 7B, compare the anti-H1⁰ column with the Hoechst column). The same pattern of immunofluorescence was observed when we examined the pattern of clone 27 and 34 antibody immunolabeling in nuclei of differentiated MEL (data not shown). These observations suggest that the recognition of H1⁰ by clone 27 antibody occurs only on restricted regions that could be sites of specific chromatin remodeling.

View larger version (61K): [in this window] [in a new window]   Fig. 7.   Immunostaining of H10 by the clone 27 and 34 antibodies in Xenopus embryos. A, Xenopus embryos were taken at the stage 36 of development and fixed, and cryosections were obtained. Two successive sections were subjected to immunostaining with the two anti-H10 antibodies and counterstained with Hoechst, which allows the detection of all nuclei (nuclei are shown from one section at low magnification). The white arrowhead shows the cement gland that is also shown at high magnification in B. B, consecutive 10-µm sections obtained from stage 36 embryos were used for the immunodetection of H10 by the clone 34 and 27 antibodies (left panels) and counterstained with the DNA-specific fluorochrome Hoechst 33258 (right panels).

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

In this work we have precisely mapped a region of histone H1⁰ located at the entry of the globular domain and involved in the recognition of the protein by two monoclonal antibodies. One of them, clone 34 antibody, recognizes the protein dependent on the AA 20-30 region. Recognition by the second antibody (clone 27 antibody) has been shown to be dependent on only 7 AA (AA 24-30) within this region. Moreover, an essential role of the proline 26 has been illustrated by site-directed mutagenesis. Indeed, the replacement of this proline by a valine completely abolished the recognition of the protein. These data suggest that the proline-mediated structure is important for the recognition of the protein by this antibody. Interestingly, this antibody does not recognize H1⁰ within the chromatin of undifferentiated MEL cells. However, after the commitment in the differentiation program, H1⁰ becomes recognizable. The AA 20-30-dependent recognition of the protein by clone 34 antibody is efficient in uninduced as well as in differentiated MEL cells.

Two explanations can be proposed for the differential recognition of H1⁰ by the clone 27 antibody during cell differentiation. First, modification of the chromatin structure in differentiated cells can render H1⁰ accessible to this antibody. However, clone 34 antibody, for which the recognition of H1⁰ is also highly dependent on the AA 24-30 region, binds the protein in uninduced cells as well as in differentiated cells. Therefore, a simple modification of the accessibility of the 24-30 region cannot satisfactorily account for the differentiation-dependent reactivity of H1⁰ toward the clone 27 antibody. The proline-valine replacement experiment suggests that recognition by clone 27 antibody could be dependent on a structure in the AA 24-30 region. Therefore, a modification of the structure of the N-terminal domain of H1⁰ occurring during the induced differentiation of MEL cells could be a reason for the observed differential recognition by the clone 27 antibody.

The importance of the N-terminal tail of H1⁰ in the recognition of the protein by our antibodies is also suggested by analyzing their ability to recognize H1⁰-four-way junction DNA complex in vitro. At least two different kinds of H1⁰-DNA complexes have been found. One is able to interact with the antibody and is supershifted in a gel retardation assay. In the rest of the population of H1⁰-DNA complexes, the addition of the antibody creates a competition between the antibody and DNA for interaction with H1⁰. This competition is accompanied with a release of DNA from the complex. The observed displacement of the DNA is enhanced when the N-terminal tail is shortened. The removal of 10 AA or the whole N-terminal tail domain (20) facilitated greatly the displacement of DNA by the clone 34 antibody. This facilitated displacement could be due to a decrease in the strength of DNA-protein interaction or to a better recognition of the protein by the antibody. The first possibility is unlikely because the removal of 10 AA, although it dramatically increases the dissociation of the H1⁰-DNA complex, does not eliminate any of the 58 lysine/arginine residues present in the protein or any of the conserved residues showed to be involved in the interaction of H1 with DNA (29). Moreover, the shortening of the N-terminal tail domain does not affect the clone 27 antibody-dependent dissociation of the complex. However, it influences the formation of the ternary complex.

These data suggest that the N-terminal tail domain of histone H1⁰, which is nonessential for the recognition of the free protein by our antibodies, can influence the protein recognition when it interacts with DNA and strengthens the possibility of a differential recognition of the protein in chromatin due to a modification of the N-terminal tail conformation during critical stages of cell life.

A different pattern of the immunolabeling is also observed when we compared clone 34 and clone 27 antibody immunostained nuclei. Clone 27 antibody is able to reveal foci of immunoreactivity within the nuclei. Under the same conditions clone 34 antibody shows a more homogenous nuclear labeling. The appearance of foci after the immunolabeling by clone 27 antibody is observed in different Xenopus tissues, as well as in cells in culture (not shown). Because in MEL cells, labeling by clone 27 antibody is differentiation-dependent, one can assume that these foci of H1⁰ immunolabeling correspond to sites of specific chromatin remodeling, rendering the N-terminal part of H1⁰ recognizable by the clone 27 antibody.

The salt treatment experiment (Fig. 4C) showed that the recognition of H1⁰ by clone 34 antibody is more sensitive to modification of chromatin structure than that of clone 27 antibody. Clone 34 antibody shows an enhanced H1⁰ recognition after the P53-mediated arrest of cell proliferation, whereas in these cells (cycling or arrested), H1⁰ is not recognized by clone 27 antibody. These observations suggest that chromatin remodeling events of a different nature are associated with cell arrest and differentiation.

Several reports described the use of monoclonal and polyclonal antibodies raised against different parts of histone H1, H5, and H1⁰, as well as the use of immobilized proteases to investigate the accessibility of the linker histones in chromatin. The majority of these works showed a lower accessibility of the globular domain compared with the N- and C-terminal tail domains of linker histone in the chromatin (32, 33). Therefore, recognition of H1s by antibodies raised against the globular domain of the protein are expected to be much more sensitive to different chromatin remodeling events than that of antibodies raised against tail domains. The work presented here shows that modifications of the chromatin structure occur at precise periods during different important cellular events, such as the commitment of cells in a particular differentiation program or the arrest of cell proliferation. The clone 27 antibody can immunolabel H1⁰ in MEL cells precisely at the onset of -globin gene expression during the induced differentiation. This observation shows that a modification of the chromatin structure occurs concomitant with the reinitiation of erythropoiesis. The clone 34 antibody allowed us to show a modification of the chromatin structure occurring during a restricted period of the cell cycle before the arrest of cell proliferation. The described antibodies therefore seem to be valuable tools for monitoring precise timing of chromatin remodeling associated with different regulatory events controlling the cell fate and enable investigation of these modifications in more detail.