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Originally published online as doi:10.1189/jlb.0704388 on October 26, 2004

Published online before print October 26, 2004
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(Journal of Leukocyte Biology. 2005;77:100-111.)
© 2005 by Society for Leukocyte Biology

Methylation of histones in myeloid leukemias as a potential marker of granulocyte abnormalities

Emilie Lukásová*, Zdenek Koristek{dagger}, Martin Falk*, Stanislav Kozubek*,1, Sergei Grigoryev{ddagger}, Michal Kozubek§, Vladan Ondrej* and Iva Kroupová

* Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno;
{dagger} Department of Internal Haematooncology and
Institute of Pathology, Masaryk University Hospital, and
§ Faculty of Informatics, Masaryk University, Brno Czech Republic; and
{ddagger} Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey

1 Correspondence: Department of Molecular Cytology and Cytometry, Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, 612 65 Brno, Czech Republic. E-mail: kozubek{at}ibp.cz


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We show that common heterochromatin antigenic protein markers [HP1{alpha}, -ß, -{gamma} and mono-, di-, and trimethylated histone H3 lysine 9 (H3K9)], although present in human blood progenitor CD34+ cells, differentiated lymphocytes, and monocytes, are absent in neutrophil granulocytes and to large extent, in eosinophils. Monomethylated and in particular, dimethylated H3K9 are present to variable degrees in the granulocytes of chronic myeloid leukemia (CML) patients, without being accompanied by HP1 proteins. In patients with an acute phase of CML and in acute myeloid leukemia patients, strong methylation of H3K9 and all isoforms of HP1 are detected. In chronic forms of CML, no strong correlations among the level of histone methylation, disease progression, and modality of treatment were observed. Histone methylation was found even in "cured" patients without Philadelphia chromosome (Ph) resulting from +(9;22)(q34;q11) BCR/ABL translocation, suggesting an incomplete process of developmentally regulated chromatin remodeling in the granulocytes of these patients. Similarly, reprogramming of leukemia HL-60 cells to terminal differentiation by retinoic acid does not eliminate H3K9 methylation and the presence of HP1 isoforms from differentiated granulocytes. Thus, our study shows for the first time that histone H3 methylation may be changed dramatically during normal cell differentiation. The residual histone H3 methylation in myeloid leukemia cells suggests an incomplete chromatin condensation that may be linked to the leukemia cell proliferation and may be important for the prognosis of disease treatment and relapse.

Key Words: human granulocytes differentiation • chromatin condensation • heterochromatin • HP1 proteins


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is generally accepted that there are two distinct structural states of chromatin in eukaryotic cell nuclei: euchromatin and heterochromatin [1 ]. In contrast to euchromatin, heterochromatin is highly condensed and transcriptionally silent. Heterochromatin proteins are associated with DNA repeats, which surround centromeres and are required for proper sister-chromatid cohesion and chromosome segregation [1 2 3 4 5 ]. Heterochromatin also stabilizes repetitive DNA elsewhere in the genome by inhibiting recombination between homologous repeats [6 , 7 ]. In addition to its role in the maintenance of genome stability, heterochromatin plays a central role in the regulation of gene expression during development and cell differentiation [8 ].

Chromatin-associated markers, distinguishing heterochromatin from euchromatin in eukaryotic chromosomes, include DNA methylation, specific histone methylation at histone H3 lysine 9 (H3K9), the absence of histone H3K9 acetylation, and the presence of heterochromatin protein 1 (HP1). Although the molecular details of the euchromatin and heterochromatin structure in proliferating cells are not fully understood, HP1, a major component of heterochromatin, is thought to establish and maintain the transcriptionally repressive heterochromatin structure [8 ]. HP1 binds to histone H3 if the lysine at position 9 (K9) is methylated [8 , 9 ]. This amino acid may be methylated to a varying degree, but the functional significance of mono-, di-, and trimethylation of lysine residues remains unclear. The results produced by Rice et al. [10 ] show that mono- and dimethylated H3K9 localize specifically to silent domains within euchromatin; in contrast to this, trimethylated H3K9 is enriched at pericentric heterochromatin. These authors further show that enzymes known to methylate H3K9 display different enzymatic properties in vivo. In mammals, G9a was found to be responsible for all detectable H3K9 dimethylation and a significant amount of monomethylation, and Suv39h1 and Suv39h2 directed trimethylation of H3K9 at pericentric heterochromatin.

The results produced by Cheutin et al. [11 ] and Festenstein et al. [12 ] show that HP1 is a highly mobile molecule. Given the rapid exchange of HP1 in heterochromatic foci, any other soluble nuclear proteins, such as transcription factors, should be able to gain access, compete with silencing factors, and potentially activate genes located within heterochromatin. These results, in contrast to previous models, indicate that interphase heterochromatin is a dynamic structure.

In blood cells, the extent of chromatin condensation increases during cell differentiation, reaching a maximum in terminally differentiated cells [13 ]. Recent results [14 , 15 ] show that condensation of chromatin in differentiated avian granulocytes and nucleated erythrocytes is not accompanied by the presence of the HP1 protein. In addition to this, HP1 is not involved in the facultative heterochromatin formation of inactivated X chromosomes in mammals [16 ]. This finding indicates that HP1 proteins are not essential for all types of heterochromatin.

As chromatin condensation and a general decrease of transcription in mature vertebrate tissues are often correlated with the appearance and accumulation of tissue-specific histone H1 subtypes, these proteins have long been considered the key factors in differentiation stage-specific chromatin condensation and gene repression [17 , 18 ]. However, gene regulation studies in cells, overexpressing or lacking certain types of histone H1, have shown that the accumulation of linker histones is not per se sufficient to cause major chromatin remodeling or a general inhibition of transcription [19 20 21 22 ].

Grigoryev et al. [14 , 23 ] found a high level of expression of the nuclear protein myeloid and erythroid nuclear termination (MENT) stage-specific protein in terminally differentiated avian blood cells, particularly in granulocytes, where it becomes the predominant nuclear nonhistone protein concentrated in peripheral heterochromatin. This developmentally regulated protein brings about condensation of the chromatin higher-order structure [23 ] in terminally differentiated avian cells. These results indicate some similarity between the HP1 and MENT proteins in their binding to methylated histone H3K9. The MENT distribution profile correlates with that of histone H3 dimethylated at lysine 9 [24 ]; HP1 also binds to methylated histone H3, although the degree of H3K9 methylation necessary for its binding is not as yet well known [25 , 26 ]. Although MENT brings about chromatin condensation and terminal differentiation in chicken granulocytes and erythrocytes [14 ], HP1 assists in maintaining constitutive and facultative heterochromatin and gene silencing in the interphase chromatin of proliferating cells [8 , 9 , 16 , 27 ]. None of these chromatin-condensation factors were found in enucleated mouse erythrocytes with abundant heterochromatin [15 ]. It is, therefore, conceivable that in these cells, chromatin condensation must be accomplished by other mechanisms without the participation of these chromatin-condensing proteins. It is interesting that the absence of HP1 proteins in avian and mouse granulocytes and erythrocytes is not accompanied by the concomitant disappearance of methylated histone H3K9 [15 , 24 ], suggesting that this type of histone modification may be the primary heterochromatin marker underlying chromatin condensation and silencing.

The differentiation of human blood cells provides a convenient system for investigating the regulation of chromatin condensation. During this process, malignant cells can arise at any stage, leading to many different types of leukemia, whose cells may retain some characteristics of the stage of differentiation at which the cells become cancerous. To determine the differences in the degree of chromatin condensation between human differentiated white blood cells possessing a certain limited proliferation capacity (lymphocytes, monocytes) and those that have completely lost this capacity (granulocytes), we studied the level and distribution of all types of histone H3 methylation at the lysine K9 (metH3K9) and the presence of HP1ß, -{gamma}, -{alpha} in white cells isolated from human peripheral blood and in CD34+ progenitor blood cells. Our studies also focused on the detection of these proteins in patients suffering from chronic myeloid leukemia (CML) and acute myeloid leukemia (AML).

Certain myeloid cell lines can be induced to differentiate to mature cells and therefore provide a useful experimental model to study histone methylation processes and "differentiation therapy" of leukemia [28 ]. The HL-60 cell line is particularly interesting, as it undergoes granulocyte differentiation following treatment with retinoic acid (RA) [29 , 30 ]. The monoblastic U-937 cells are arrested at a more advanced stage of differentiation than the HL-60 cells, and therefore, treatment of these cells with RA carries their differentiation into monocytes/macrophages [31 ]. Our goal was to find a correlation between the degree of differentiation of these two cell lines, the methylation status of histone H3K9, and the distribution of HP1 proteins and to compare the results with findings for the granulocytes of CML and AML patients.

It follows from the results presented that HP1 and metH3K9 histone are not involved in chromatin condensation during terminal differentiation of human neutrophil granulocytes and that there is a unique mechanism regulating chromatin condensation during granulocyte differentiation. This mechanism is apparently impaired during the development of myeloid leukemia and is not restored during treatment of the disease by enforced differentiation and other currently used therapies.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Separation of mononuclear cells and granulocytes from human peripheral blood
Erythrocytes were eliminated from the peripheral blood of five healthy donors by means of dextran T 500 (Amersham, Piscataway, NJ)–Telebrix N 350 (Leciva, Prague, Czech Republic) solution (density 1.095 g/ml). Separation of white cells from the remaining plasma was performed according to the methods described in Current Protocols in Immunology [32 ] by means of Ficoll-Hypaque gradient centrifugation.

Ficoll-Hypaque gradient centrifugation allows the separation of granulocytes from mononuclear cells (lymphocytes and monocytes). Monocytes, depleted from lymphocytes, by taking advantage of their capacity to adhere to plastic, were contaminated with ~5% of lymphocytes and vice versa. These two cell types can be well distinguished on the microscopic slide by the morphology of their nuclei: The nuclei of lymphocytes are spherical, and those of monocytes resemble kidneys in shape. Granulocytes have the characteristic multilobular nucleus clearly distinguishable from the nuclei of other cells. Erythrocytes, slightly contaminating granulocytes, are not observable on the microscope slide after nuclei contra-staining with TOPRO-3, as they do not contain chromatin. Owing to the characteristic morphology of the nuclei of the investigated cell types, none of the contaminating nuclei were included in the analysis of any particular cell type. Immunochemical detection of heterochromatic proteins and histone methylation was performed on sets of individual nuclei fixed on microscopic slides, enabling consideration of nuclei of a single cell type during analysis and accurate calculation of the percentage of cells manifesting a particular antigen.

A proportion of the isolated lymphocytes was stimulated to divide with phytohemagglutinin (PHA). The cells were resuspended in a complete RPMI medium containing 10% fetal calf serum (FCS; 106/ml) and incubated with 20 µl/ml PHA H15 (Murex, UK) at 37°C for 36 h.

CD34+ hematopoietic progenitor cells were isolated from concentrated mononuclear cells separated from the mobilized blood of two patients with a lymphoma. The cells were labeled with anti-CD34+ magnetic microbeads (Miltenyi Biotec, Auburn, CA) and separated in the magnetic field of the MiniMacs using a positive selection column. Granulocytes of 20 CML and three AML patients were isolated from 1.7 ml peripheral blood after erythrocyte elimination. The informed consent of all blood donors was obtained.

Cell culture and differentiation
A human HL-60 promyelocytic leukemia cell line and a U-937 monoblastic cell line were grown in RPMI-1640 medium supplemented with 10% FCS, penicillin (100 U/ml), and streptomycin (100 µg/ml) in humidified air with 5% CO2 at 37°C.

Twelve hours before the induction of differentiation, cells were harvested and resuspended in fresh medium at 2 x 105 cells/ml. Both cell types were induced to differentiate for 6–8 days with 1 µM all-trans RA (ATRA; Sigma Chemical Co., St. Louis, MO) with no change of media.

Flow cytometric detection of cell differentiation
The differentiation of HL-60 cells into granulocytes was determined after 6 days of incubation with 1 µM RA by the expression of CD11b-fluorescein isothiocyanate (FITC) and CD14-phycoerythrin (PE) surface antigen. The cells were washed two times with phosphate-buffered saline (PBS) containing 0.1% NaN3 and 1% bovine serum albumin (BSA) at 4°C. CD11b-FITC (2 µl) and 2 µl CD14-PE monoclonal antibodies (Coulter Company, Immunotech, France) were added to 50 µl cell suspension (106 of living cells/ml) in PBS containing 1% BSA and 0.1% NaN3. The cell suspension was incubated for 40 min at 4°C. The expression of CD11b and CD14 antigens was measured by a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). The same procedure was used for the detection of differentiation of U937 cells with 1 µM RA. Isotypic controls were used for result correction.

Cell fixation and immunolabeling
A dense suspension of cells in PBS (100 µl) was dropped onto positively charged microscopic slides. After attachment to the slide (~5 min), cells were fixed with 4% paraformaldehyde in PBS for 10 min, washed 4 x 5 min in PBS, permeabilized in 0.2% Triton X-100/PBS, washed 2 x 5 min, blocked with 7% normal goat serum/PBS for 30 min, and sequentially incubated with the primary and secondary antibodies. The following antibodies were used: anti-HP1ß (rabbit polyclonal), HP1{gamma} (fusion protein, mouse), HP1{alpha} (fusion protein, mouse), trimethyl H3K9 (rabbit polyclonal), dimethyl H3K9 (mouse monoclonal), monomethyl H3K9 (synthetic peptide, rabbit), acetyl H3K9 (rabbit polyclonal), histone H1 (mouse monoclonal), histone H1°/H5 (mouse monoclonal), anticentromere protein A (anti-CENP-A; rabbit, immunoaffinity-purified), all from Upstate Biotechnology (Lake Placid, NY). Antibodies were diluted 1:1000. Secondary antibodies—goat anti-rabbit-FITC and goat anti-mouse-FITC (Jackson Laboratory, Bar Harbor, ME)—were diluted 1:50. Counterstaining was performed by 1 µM TOPRO-3 (Molecular Probes, Eugene, OR) in 2 x saline sodium citrate prepared fresh from the stock solution. The percentage of cells expressing the particular antigen was calculated from 300 to 500 cells.

Fluorescence microscopy
Images were obtained by a high-resolution confocal cytometer [33 , 34 ] based on a completely automated Leica DM RXA fluorescence microscope equipped with a CSU-10a confocal unit (Yokogawa, Japan) and a CoolSnap HQ charged-coupled device camera (Photometrix, Melbourne, Australia). Forty optical sections at a 0.3-µm step were acquired for each nucleus and stored in the computer memory. The XY, XZ, YZ projections shown in Figure 1 demonstrate the three-dimensional preservation of the nuclei. The exposition time and dynamic range of the camera in the red, green, and blue channels were adjusted to the same values for all slides to obtain quantitatively comparable images. The antibody signal intensity was measured as the mean value of the green channel histogram in the dynamic range from 0 to 255 using Adobe Photoshop 5.5 software.



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  		Figure 1. Maximal XY image, XY, XZ, and YZ sections through the monocyte and neutrophil granulocyte nuclei after immunodetection of HP1ß. XY sections show one of the 40 slices through the nucleus; XZ section shows a cut along the horizontal white line through the nucleus; YZ section shows a cut along the vertical white line. Only the foci that cross the given cut are visible. Immunodetection was performed using FITC-labeled secondary antibodies. DNA was counterstained with TOPRO-3.

 
Isolation of nuclear proteins and chromatin for Western blotting
The suspensions of lymphocytes and granulocytes separated by Ficoll-Hypaque density gradient centrifugation were washed two times with PBS, centrifuged for 3 min at 1000 g, cooled on ice to 2°C, and resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 120 mM NaCl, 0.5% Nonidet P-40, and 1 mM phenylmethylsulfonyl fluoride) by vigorous vortexing for 1 min and left on ice for 30 min with intermittent homogenization with a plastic pestle, briefly (10 s) sonicated and spun down (14,000 rpm for 15 min). The supernatant was transferred into a new tube and kept on ice.

Protein electrophoresis, detection, and quantification
Proteins were separated in 15% polyacrylamide gels. Samples containing proteins and chromatin were diluted to the same value of A260 absorbance, boiled in sodium dodecyl sulfate loading buffer, and loaded on the gel. After electrophoresis, the gels were stained with Coomassie blue R-250 (Sigma Chemical Co.) or electrotransferred in Tris-glycine buffer containing 10% methanol to a nitrocellulose membrane. The membranes were blocked, treated with antidimethyl H3K9 (dilution 1:150) or with anti-HP1{gamma}, HP1{alpha} antibodies (Upstate Biotechnology; dilution 1:500), and then treated with secondary peroxidase-conjugated anti-mouse antibodies and detected with an ehanced chemiluminescence (ECL) detection system (Amersham Corp., Little Chalfont, UK). The intensity of the protein bands was compared after ECL detection using the Vilber Lourmat photodocumentation and imaging system.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Different levels of methylated histone H3K9 and HP1 immunofluorescence in the white blood cells of healthy donors
The levels and nuclear distributions of HP1ß and tri-metH3K9 in human progenitor CD34+ cells, lymphocytes, monocytes, and terminally differentiated granulocytes (neutrophils and eosinophils) are shown in Table 1 and Figure 2 . The majority of HP1ß foci are distributed through the cell nucleus with a low density of chromatin in lymphocytes and monocytes (Figs. 1 and 2) . A smaller number of foci of this protein were found in chromatin characterized by an increased intensity of TOPRO-3 fluorescence (probably heterochromatin). The foci of HP1ß were also detected in peripheral heterochromatic regions in CD34+ cells (Fig. 2) . In contrast to lymphocytes and monocytes, there was only a low signal of HP1ß in eosinophil granulocytes. HP1ß was not detected in the majority of these cells; in some eosinophils, HP1ß foci, similar to those in lymphocytes or monocytes, were present in one or both lobes (Fig. 2) . The green color of the eosinophil cytoplasm is caused by nonspecific fluorescence of the secondary antibody. The HP1ß signal was completely absent in neutrophil granulocytes (Fig. 2) . The level of tri-metH3K9 in blood cells resembled that of HP1ß (Table 1 , Fig. 2 ). Not only was the tri-metH3K9 distributed in the center of nuclei with a low density of chromatin, but some foci were also found in high-density chromatin on the nuclear periphery. In contrast, the tri-metH3K9 signal, as was the case for HP1ß, was completely lost from neutrophils and to large extent, from eosinophils. Experiments were carefully repeated, and similar results were obtained for all types of cells separated from the peripheral blood of five healthy donors.


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  		Table 1. The Mean Values of Antibody Signal Intensity in Different Cell Types from Peripheral Blood and Different Cell Lines

 


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  		Figure 2. Distribution of HP1ß and tri-metH3K9 in human peripheral blood cells (lymphocytes, monocytes, eosinophils, and neutrophils) and in human blood progenitor CD34+ cells. HP1ß and tri-metH3K9 were found in CD34+ cells, lymphocytes, and monocytes but not in neutrophils and some eosinophils. The images represent the central XY cuts through the nuclei. Immunodetection was performed using FITC-labeled secondary antibodies. DNA was counterstained with TOPRO-3. Inserted histograms represent the level of antibody signals measured as the mean value of green channel intensity in the dynamic range from 0 to 255 using Adobe Photoshop 5.5 software.

 
The absence of trimethylated H3K9 and HP1ß in granulocytes also prompted us to investigate the distribution of di- and monomethylated H3K9 as well as HP1{alpha} and -{gamma} isoforms in various blood cells. None of these proteins were detected in neutrophil granulocytes by immunodetection (Fig. 3A ); however, a low number of di-metH3K9 and HP1{gamma} foci were observed in eosinophils in which mono-metH3K9 was more frequent. The results obtained for lymphocytes, monocytes, and granulocytes were confirmed by Western blotting (Fig. 4 ). Conversely, di-, mono-metH3K9 and HP1{gamma} and -{alpha} isoforms were present in lymphocytes (nonstimulated and stimulated) and monocytes (Figs. 3B and 3C and 4 ). In contrast to the two isoforms of HP1 ({gamma}, {alpha}), preferentially located in the central part of the nucleus, the foci of di-metH3K9 and mono-metH3K9 were distributed throughout the entire nuclear volume, similarly as for trimethylated histones. This modification of histone H3K9 was not observed in the nuclei of G0 lymphocytes (detected according to Ki67 expression), although it was present in the majority of stimulated lymphocytes. Although HP1ß and -{gamma} occurred in a large number of foci (those of {gamma} were larger than those of ß), HP1{alpha} presented only several foci per nucleus of lymphocytes and monocytes. The levels of methylated histone H3K9 and HP1 proteins detected quantitatively using the intensity of green fluorescence on the cell nuclei are shown in Table 1 . The results show that in lymphocytes and monocytes, HP1ß is the most abundant, and HP1{alpha} is less abundant. Mono-metH3K9 is the most frequent and tri-metH3K9 the least frequent methylation of histone H3.



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  		Figure 3. Distribution of HP1{alpha} and -{gamma} isoforms, differently modified histone H3K9, and linker histones in human neutrophil granulocytes (A), monocytes (B), and lymphocytes (C) isolated from peripheral blood. Neither methylated H3K9 nor HP1 proteins were detected in healthy human neutrophil granulocytes. Some differences in H3K9 methylation and in the presence of HP1 were observed between neutrophils and eosinophils. H1 and CENP-A proteins are present in contrary to H1°. Similar patterns of HP1 proteins, methylated, acetylated histone H3K9, linker histone H1, its variant H1°, and CENP-A were observed in monocytes and G0-lymphocytes. H1° was present only in a part of the cell population of both cell types. Immunodetection was performed using FITC-labeled secondary antibodies. DNA was counterstained with TOPRO-3. Inserted histograms represent the level of antibody signals measured as the mean value of green channel intensity in the dynamic range from 0 to 255 using Adobe Photoshop 5.5 software.

 


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  		Figure 4. Western blot of proteins from lymphocytes (L), moncytes (M), and granulocytes (G) isolated from human peripheral blood of a healthy individual, a patient with chronic myeloid leukemia (CML), and HL-60 cells using antibodies detecting dimethyl H3K9, HP1{alpha}, and HP1{gamma}. The level of di-metH3K9 in granulocytes of CML patients determined by Western blotting is ~10 times lower than the level of this protein in lymphocytes and monocytes of healthy donors. These values are in accordance with mean relative values of the immunofluorescence measured on individual nuclei using Adobe Photoshop software in the dynamic range of green channel intensity from 0 to 255. The mean level of green channel intensity of di-metH3K9 in healthy donor lymphocytes was found ~54, that of di-metH3K9 in granulocytes of the CML patient 5.3, and the value 50 was found in HL-60 cells.

 
Approximately 100% of neutrophils from healthy donors did not show any methylation of the H3K9 histone and expression of any of the isoforms of HP1 protein, contrary to other white blood cells, where the expression of these proteins was found in 98–100% of cells. An exception to this is represented by G0 lymphocytes (not expressing the Ki67 antigen), which did not show the di-metH3K9 signal, whereas all stimulated lymphocytes did.

In lymphocytes and monocytes, histone H1 was present in a large number of foci of a similar size as that for HP1{gamma} or centromeric protein CENP-A, although it showed a much larger number of much smaller dots in granulocytes covering the entire area of chromatin (Fig. 3) . H1 was the most prominent of all the studied protein markers in granulocytes. In addition to the H1 histone, granulocytes also displayed acetylated H3K9. This protein marker was nonhomogenously distributed through the lobes of chromatin and occurred mostly in the inner part of the nucleus. A significantly higher level of acetylated H3K9 was present in lymphocytes and monocytes than in granulocytes (Table 1) . In contrast to granulocytes, some nuclei of lymphocytes and monocytes (18–20%) contained a larger amount of the histone H1° subtype (Fig. 3B and 3C) .

Changes in the methylation status of H3K9 and the levels of HP1 proteins in the granulocytes of patients with CML and AML
The methylation profiles of histone H3 at lysine K9 and the presence of HP1 proteins in the granulocytes of patients suffering from myeloid leukemia are changed relative to those in the granulocytes of healthy individuals. Strong signals from tri-, di-, and monomethylated H3K9, as well as {gamma} and ß isoforms of HP1, were detected in all the investigated patients with AML. Histone H3K9 acetylation was also prominent (Fig. 5 ), and some cells showed a higher level than others. Granulocytes of AML patients also exhibited a strong signal of histone H1, as well as several foci of its subtype H1°. HP1{alpha} gave lower intensity signals comparable with those in the lymphocytes and monocytes of healthy individuals.



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  		Figure 5. Distribution of HP1 isoforms, differently modified histone H3K9, and linker histones in granulocytes isolated from peripheral blood of AML patients. In granulocytes of AML patients, all isoforms of HP1 were detected (in different intensities) as well as tri-metH3K9, di-metH3K9, and mono-metH3K9. In contrary to linker histone H1, which is present in large amounts in small foci, its variant H1° was present only in several points (not shown). A usual pattern of centromeric protein CENP-A was observed. Immunodetection was performed using FITC-labeled secondary antibodies. DNA was counterstained with TOPRO-3. Inserted histograms represent the level of antibody signals measured as the mean value of green channel intensity in the dynamic range from 0 to 255 using Adobe Photoshop 5.5 software.

 
In the granulocytes of CML patients, the methylation status of histone H3K9 and the levels of HP1 protein staining were changed (compared with healthy granulocytes), although to a lower degree as compared with AML patients. Significant variations among individual patients were observed (Table 2 ). HP1ß was found in the granulocytes of only two of 20 investigated CML patients. These patients also showed a very high degree of H3K9 methylation. In CML patients, the only changes compared with healthy individuals were found in the levels of mono- and di-metH3K9. Mono-metH3K9 was found in rather high amounts for all patients, and di-metH3K9 varied. The patients were classified into six groups according to the amount of di-metH3K9 (Table 2) . The tri-metH3K9 was detected in samples from only several patients from all groups; all patients in blast or accelerated phase showed increased levels of this type of methylation.


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  		Table 2. Clinical Characteristics of CML Patients, the Level of Di-, Tri-metH3K9, and HP1ß in Their Granulocytes

 
Three patients had only a small number of di-metH3K9 foci in their granulocytes (the first group; the mean level of green channel intensity 0–1). The patients in this group also had a small amount of mono-metH3K9 in their granulocytes (Fig. 6 ). One of these patients had no tri-metH3K9 at all, one had tri-metH3K9 concentrated in two to five small islets in the nuclei of only some cells (~5%), and the third had several dots in most of the nuclei; this patient was in the accelerated phase.



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  		Figure 6. Distribution of methylated H3K9 in granulocytes isolated from peripheral blood of selected CML patients. Selected patients represent six groups classified according to the degree of the di-metH3K9 (Table 1) . In CML patients, tri-metH3K9 was also observed, particularly for the nuclei with a high amount of di-metH3K9. Mono-metH3K9 was observed even in patients without the other types of H3K9 methylation. Patient’s UPN number: 1, 59702; 2, 59709; 3, 59708; 4, 59715; 5, 59717; 6, 59718. Immunodetection was performed using FITC-labeled secondary antibodies. DNA was counterstained with TOPRO-3. Inserted histograms represent the level of antibody signals measured as the mean value of green channel intensity in the dynamic range from 0 to 255 using Adobe Photoshop 5.5 software.

 
In the second group (four patients; mean level of green channel intensity 1–5), the di-metH3K9 was distributed in small foci mostly in the inner part of the nuclei, where the concentration of chromatin (manifested by intense TOPRO-3 staining) is lower than in the periphery of the nucleus (Table 2 , Fig. 6 ). Two patients in this group contained a small amount of tri-metH3K9 concentrated in three to five islets per nucleus. All these patients manifested a complete cytogenetic response to the treatment with a lowering of the amount of the Ph chromosome (<1%). These patients have mono-metH3K9 in an amount similar to the patients in the first group.

The patients in the third group had an increased level (the mean level of green channel intensity 5–20) of di-metH3K9 in their granulocytes compared with the previous group (Fig. 6) . One of these patients was in the blast phase, and the others were in the chronic phase. Two of these patients showed a small amount of tri-metH3K9 in the form of islets or free foci. The foci of di-metH3K9 were small, concentrated in larger aggregates similarly as for those of tri-metH3K9.

Patients characterized by a rather high amount of di-metH3K9 (the mean level of green channel intensity 20–50) in their granulocytes were placed in the fourth group. These patients also had a large amount of mono-metH3K9, and two also had a small amount of tri-metH3K9. One patient without tri-metH3K9 is in the second chronic phase; the other is in the first chronic phase. The patient with the highest amount of tri-metH3K9 manifested complete cytogenetic response to the treatment (Ph chromosome <1%; Table 2 ).

Patients placed in the fifth group showed an extremely large amount of di-metH3K9 (the mean level of green channel intensity 50–100). Although not all of them contained tri-metH3K9, one patient had an extremely large amount of tri-metH3K9. Nonetheless, complete cytogenetic response to the treatment was observed. These patients are in the chronic phase. The data for two patients were lost from the register and are therefore incomplete in Table 2 . There was a high proportion of cells not manifesting well-developed nuclear lobulation; a lot of small, poorly differentiated forms containing a large amount of di- and mono-metH3K9 were also observed. Moreover, even the morphologically, well-differentiated granulocytes of this group contained a large amount of mono- and di-metH3K9. A small amount of HP1ß was detected in one patient, whose granulocytes were not morphologically well-differentiated and contained a large amount of tri-metH3K9.

One patient was placed in the sixth group (the mean level of green channel intensity >100 for di-metH3K9), as his granulocytes were poorly differentiated and contained an extremely large amount of mono-, di-, and tri-metH3K9 and in addition, a rather large amount of HP1ß. This patient reached the acute phase because of resistance to the treatment (Table 2 , Fig. 6 ). The methylation of histone H3K9 in the granulocytes of this patient resembled that of AML patients (Fig. 5) .

Methylation of histone H3K9 and the status of HP1 proteins in differentiated HL-60 and U-937 cells
Promyelocytic HL-60 and monoblastic U937 cells were induced to differentiate with 1 µM RA. The cell proliferation of both of these cell lines was strongly reduced on the third day of cell incubation with this agent. HL-60 cells acquired granulocyte morphology with chromatin condensed into characteristic lobes and the loss of nucleoli. The results of flow cytometric measurements of the expression of surface antigens CD11b and CD14 by HL-60 and U937 cells incubated in the presence of RA for 6 days are presented in Figure 7 . HL-60 cells show an increase in the expression of the CD11b antigen, indicating their differentiation into granulocytes. The expression of this antigen is only slightly changed in monocytes on the sixth day of incubation with RA; in contrast, there is an increase of CD14 antigen expression indicating the differentiation of these cells into macrophages. The viability of control HL-60 and U937 cells was 83% and 92%, respectively, for those treated with RA 68% and 77% on the sixth day of incubation.



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  		Figure 7. Expression of CD11b and CD14 antigens in HL-60 and U937 cells before and after incubation of cells with RA for 6 days. HL-60/K2 and U937/K2—control cells, HL-60 + ATRA, and U937 + ATRA—cells treated with RA. Modal values are shown for all cell populations.

 
The RA treatment resulted in the induction of U937 cell differentiation toward a more mature state with major characteristics of monocytes/macrophages: large cytoplasm with vacuolization, chromatin not condensed, and persistence of nucleoli. In contrast to HL-60 cells, the chromatin of U937 was not morphologically changed as compared with the original cells. Both types of differentiated cells retained this characteristic morphology during incubation with RA (8 days).

Even if the morphology of differentiated HL-60 cells displayed the characteristic lobulation, the methylation status of H3K9 of these cells was quite different from that of mature granulocytes isolated from the healthy peripheral blood (Figs. 3 and 8 ). The granulocytic forms of HL-60 differentiated by RA (distinguished on the slide by the characteristic lobulation) contained a large amount of tri-, di-, and mono-metH3K9, similar to the control HL-60 cells incubated in the absence of RA. They also contained a highly similar pattern of all three forms of the HP1 protein (high signals of ß and {gamma} and low signal of {alpha}) to the original, nondifferentiated cells (Table 1) .



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  		Figure 8. Distribution of HP1{alpha} and -ß isoforms, tri- and dimethylated H3K9 in HL-60, and U937 cells, treated and nontreated with 1 µM RA for 8 days. Immunodetection was performed using FITC-labeled secondary antibodies. DNA was counterstained with TOPRO-3. HL-60 cells show lobular morphological changes after the treatment with RA; however, the exposure of methylation and HP1 remains conserved. Inserted histograms represent the level of antibody signals measured as the mean value of the green channel intensity in the dynamic range from 0 to 255 using Adobe Photoshop 5.5 software.

 
The occurrence of mono-, di-, and trimethylated histone H3K9 and HP1 isoforms in monocytes/macrophages differentiated from U937 cells by 8-day RA treatment was also highly similar to that in control cells not induced to differentiation (Table 1) .


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results show an absence of immunofluorescence of all three isoforms of HP1 ({alpha}, ß, {gamma}) and the absence of any type of histone H3K9 methylation in human neutrophil granulocytes isolated from the peripheral blood of healthy donors. The absence of HP1 proteins and histone H3K9 methylation in healthy granulocytes was also proved by Western blotting. Only a limited amount of these proteins was found in eosinophils. In contrast to granulocytes, two other types of white blood cells, lymphocytes and monocytes, contained high levels of all these protein markers with the exception of HP1{alpha}, which is present in much lower amounts than the two other HP1 isomers. These results indicate a difference between the structure of the chromatin of human granulocytes on one hand and lymphocytes and monocytes on the other. This difference is manifested during manipulation with these cells: Neutrophil granulocytes fixed with 4% formaldehyde for 10 min are much less stable (the chromatin swells, expands to an enormous size, and loses its original shape) compared with lymphocytes, monocytes, and even eosinophils and must be subjected to immunostaining and microscopic observation immediately.

HP1 is thought to play a central role in creating a stable heterochromatic network by interacting with several other proteins, in particular, with methylated histone H3K9 [8 , 34 , 35 , 36 ]. This histone modification and the presence of HP1 are considered to be important signs of inactive chromatin regions. In addition to human granulocytes, a sharp decline in HP1 levels has also been observed in other terminally differentiated cells with highly condensed chromatin, such as chicken lymphocytes and erythrocytes from other vertebrate species [14 , 23 ]. The absence of HP1 in the heterochromatin of these terminally differentiated cells indicates that its function is not necessary for maintaining chromatin condensation, centromere function, and regulation of gene expression in these terminally differentiated blood cells in contrast to proliferating cells, even those with limited proliferating capacity (lymphocytes and monocytes). The proliferative ability of lymphocytes is well known, as is the limited proliferation of monocytes induced by a macrophage colony-stimulating factor in vitro [37 ]. In chicken granulocytes and erythrocytes, histone H3 methylation at lysine 9 (di-metH3K9) regulates chromatin condensation directly by recruitment of a serpin-like protein mature erythrocyte nuclear termination (MENT) [24 ]. In contrast to chicken granulocytes, where HP1 is replaced by MENT, and methylation of H3K9 is preserved, HP1 proteins and methylated H3K9 are not detectable in human granulocytes, as is shown in this work.

We considered the possibility of methylated histone H3 replacement by its variant CENP-A, owing to its participation in the formation of condensed centromeric heterochromatin [25 , 38 ] and its ability to replace H3 in octameric complexes with H4, H2A, and H2B and to form reconstituted nucleosomes in experiments in vitro [39 ]. However, our results show that in human granulocytes, the amount of CENP-A protein is not higher than that in lymphocytes and monocytes and that this protein is normally integrated into centromeric heterochromatin foci (Fig. 3A) . We did not detect the presence of the H1° variant of the linker histone H1 in human granulocytes, although a small amount of H1° was present in some lymphocytes and monocytes. The H1° variant was preferentially found in nondividing cells [40 ]; however, the relationship between the function of histone H1° in proliferation and differentiation has not yet been established. Variant H1°/H5 was shown by Gilbert et al. [15 ] to be increased in chicken erythrocytes during development, paralleling decreased levels in HP1s, and the authors speculate about the role of this protein in chromatin compaction in place of HP1. Nevertheless, it has been shown earlier that the accumulation of the linker histone per se is not sufficient to cause a major chromatin remodeling [17 , 18 ]. Thus, it remains an open question as to which chromosomal protein(s) controls chromatin condensation in differentiated granulocytes.

Models of heterochromatin higher-order folding in terminally differentiated cells, where only a small part of the genes remain active, have recently been revised by Grigoryev [41 ] to include an extremely compact, longitudinal folding of a 30-nm fiber or extensive lateral self-association and intercalation of nucleosome arrays [42 ] and folding of the chromatin 30-nm fiber back onto itself. Progressing chromatin folding may result in the global chromatin condensation observed in terminally differentiated cells, leaving behind only the genes protected by boundary elements [43 ] or by nuclear matrix association [44 ], separating the active chromatin from the mass of spreading heterochromatin. The histone H3 N-terminal domains are involved in nucleosome linker folding within the zig-zag nucleosome arrays [45 ]. Nucleosome linkers, along with histone H3 N-termini, should become highly inaccessible within a compact 30-nm fiber or extensively intercalated zig-zags [41 ]. Conversely, the positive detection of acetylated H3K9 (as well as CENP-A and H1) indicates that chromatin condensation in granulocytes does not prevent the penetration of antibodies. Therefore, the negative detection of the signal of mono-, di-, and tri-metH3K9 indicates rather the absence of this lysine residue methylation. This conclusion is also supported by Western blot analysis (Fig. 4) , which also failed to detect H3K9 methylation.

Our results further show that monomethylated and in particular, dimethylated H3K9 appears to a variable degree in the granulocytes of CML patients, without being accompanied by HP1 proteins. It follows from the results presented that the variable methylation of histone H3 in CML granulocytes could be an extremely important phenomenon in tumor development and progression. It is well known that CML is a hematopoietic stem cell disorder associated with the chromosomal translocation t(9;22), which progresses from a relatively benign chronic phase to a terminal blastic phase. The mechanism of the transformation of the disease from the chronic to the acute phase is not yet fully understood, although it seems that factors other than BCR/ABL are responsible for the progression of CML and evolution into an acute leukemia. In this connection, the question arises as to whether the t(9;22) translocation is the primary event or whether it occurs in an already defective stem cell. Fialkow et al. [46 ] and Raskind et al. [47 ] have studied individuals with CML, heterozygous for glucose-6-phosphate dehydrogenase (G6PD) isoenzyme expression, and their results indicate that there are patients with Ph-negative B cells expressing the same G6PD isoenzyme as their Ph-positive myeloid cells. This observation supports the idea that defective hematopoiesis could precede the t(9;22) translocation, at least in some cases.

The data published here show that strong methylation of H3K9 can be found even in patients with a very low number, if any, of BCR/ABL-positive cells. We can speculate that the impaired methylation profile is a possible manifestation of the defective hematopoiesis and therefore, can be seen even in granulocytes from CML patients without BCR/ABL translocation. On the basis of the results put forward, we can assume that the primary, structural defects are to be found in the local chromatin decondensation. This structural disorder might lead to genetic instability and to the development of further genetic rearrangement(s). The reason for the variability of the degree of methylation in CML patients is not known. The treatment of CML is focused on the elimination of the clone containing BCR/ABL translocation and does not eliminate cells with primary structural changes that may be individually variable.

The chromatin of AML granulocytes, possessing a larger proliferating capacity and accordingly higher amount of actively transcribed genes, should be much more decondensed than the chromatin of CML granulocytes. The presence of all isoforms of HP1 indicates that chromatin condensation of AML granulocytes is not as complete as in healthy granulocytes and that the mechanisms controlling chromatin condensation in AML granulocytes are rather similar to those in lymphocytes and monocytes.

The changes in the chromatin structure manifested by H3K9 methylation of granulocytes in CML and AML patients indicate that the process of differentiation in these cells has become incomplete, where incomplete differentiation was also observed in HL-60 cells induced to differentiation by RA. In spite of prolonged exposure of cells to RA (8 days), methylated forms of histone H3K9 and all isoforms of the HP1 protein remained at approximately the same level as in control cells not exposed to RA, indicating that the chromatin structure in differentiated HL-60 cells is not the same as in normal peripheral blood granulocytes. Little condensed chromatin in granulocytes differentiated by RA from HL-60 cells, as compared with normal peripheral blood granulocytes, has been revealed earlier by Olins et al. [29 , 30 ]. The authors show that granulocyte chromatin looks quite different from that of the other differentiated cell types: It has highly reduced nuclear lamina, although an increased number of lamina binding receptors (LBR). The amount of LBR increases significantly after RA treatment of HL-60 cells [30 ]. Evidence has been presented showing that LBR interacts with HP1{alpha}, which might stabilize linkage between the nuclear envelope and the peripheral heterochromatin of granulocytes [48 ]. Further evidence that HL-60 and other leukemic cell lines fail to differentiate fully toward mature granulocytes, if induced by RA, is their inability to synthesize secondary granules [49 ]. This secondary granule deficiency is an abnormality closely associated with aberrantly differentiated leukemic blasts. Aberrant differentiation of HL-60 cells to granulocytes induced by RA has also been shown in this work. This is demonstrated by the presence of mono-, di-, and tri-metH3K9 and HP1 proteins in the chromatin of induced granulocytic forms, which are absent in healthy human granulocytes isolated from the peripheral blood.

Similarly as in granulocytes differentiated by RA from HL-60 cells, the level of H3K9 methylation and the presence of HP1 were also preserved in macrophages differentiated from U937 cells. However, contrary to granulocytes, the chromatin of macrophages is not condensed, and its structure, including the presence of nucleoli, resembles that of monocytes. Similar levels of H3K9 methylation and the presence of HP1 isoforms in differentiated cells, as compared with original U937 cells, are therefore not surprising. Condensation of chromatin in these cells is apparently directed by other mechanisms than in human granulocytes.

It follows from the results presented that the absence of the methylated H3K9 marker in human granulocytes distinguishes these cells from other differentiated human blood cells. It has recently been shown [50 ] that during activation, neutrophils release chromatin and granule proteins, which assemble into extracellular fibers [neutrophil extracellular traps (NETs)], serving to bind and kill bacteria and degrade virulence factors. The specific structure of the chromatin of neutrophils may facilitate the release of DNA (a major structural component of NETs) and histones to generate these fibers.

RA is often used to stimulate some tumor cells to differentiate and undergo terminal cell division and loss of tumorigenicity. However, our results suggest that even if some leukemia cells (and presumably, some cells of other tumors that are genetically abnormal) can be reprogrammed to a nonmalignant phenotype by inducing differentiation, their chromatin structure need not be restored completely. The presence of methylated histone H3K9 in apparently mature human granulocytes derived from leukemia indicates an absence of completely condensed chromatin, implying a certain degree of instability and risk of leukemia relapse.


   ACKNOWLEDGEMENTS
 
This work was supported by the Ministry of Health of the Czech Republic (NC6987-3), the Academy of Sciences of the Czech Republic (A1065203, KSK 5052113, Z5004920, S5004010), the Grant Agency of the Czech Republic (GA202/02/0804), and the Ministry of Education (ME565). We thank L. Stixová, Laboratory of Cytokinetics, Institute of Biophysics, AS CR, Brno, for flow cytometric measurements.

Received July 8, 2004; revised September 7, 2004; accepted October 5, 2004.


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 RESULTS
 DISCUSSION
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