The Human Lysozyme Gene Undergoes Stepwise Demethylation During Phagocyte Maturation

The human lysozyme gene undergoes stepwise demethylation during phagocyte maturation

MLu¨bbert1, R Henschler1, M Kreutz2, R Andreesen2, R Mertelsmann1 and F Herrmann3

1Division of Hematology/Oncology, University of Freiburg Medical Center, Freiburg; 2Division of Hematology/Oncology, University Hospital Regensburg, Regensburg; and 3Department of Hematology/Oncology, University Hospital Ulm, Ulm, Germany

The lysozyme (LZM) gene provides a very useful model for to be differentially methylated in a development-specific man- studies of phagocyte maturation, because its protein synthesis ner.9 The vital role of DNA methylation for normal maturation is increased during myelopoiesis and thus most abundant in terminally differentiated and activated phagoyctes. LZM gene processes in vertebrates has been demonstrated in two mouse expression and DNA methylation were examined in various models: embryos with a homozygous disruption of the normal and transformed hematopoietic cells. Two shifts toward methyltransferase gene and resulting global cellular demethyl- LZM gene demethylation coincided with upregulation of ation,10 or with disruption of the gene encoding the methyl- expression: activation of expression in myeloid precursor cells CpG binding protein MeCP211 are severely disturbed in their was associated with significant demethylation at a CpG dinu- development and die in mid-gestation. A balanced presence cleotide within an Alu repeat in the 5′ flanking region; high-level expression in different types of mature phagocytic cells was of the ‘fifth base’ methylcytosine is therefore probably essen- associated with complete demethylation at two additional, tial for regulated cellular differentiation in vivo. Several mod- intragenic CpG sites contained in Alu sequences. The possi- els have been employed to delineate further the relationship bility that methylation changes occurring within the 5′ region between demethylation, gene expression and maturation.7,12 of the human lysozyme gene could be involved in the transcrip- A correlation of demethylation with transcriptional activity of tion of this gene is discussed, as well as a possible role for genes during maturation was noted in most of these studies.7 demethylation in the maintenance of distinct maturation stages during phagocyte development. We have previously shown that demethylation at five CpG Keywords: hematopoiesis; differentiation; methylation; myelo- dinucleotides within the myeloperoxidase (MPO) gene occurs poiesis; Alu repetitive elements; transcription during a distinct commitment step of granulocyte maturation.13 The MPO gene is transcribed within a narrow ‘win- dow of differentiation’, ie the late myeloblastic and promyelo- 14,15 Introduction cytic stages. No further changes of this pattern occur during terminal maturation; partial demethylation within the ′ Mechanisms underlying the coordinate regulation of gene 3 region of the gene, however, has already occurred during expression throughout the differentiation of myeloid precursor earlier commitment steps and was noted to spread toward the ′ 13,16 to peripheral blood phagocytes are not well understood.1,2 In 5 region with myeloid maturation. Similarly, a lineage- an effort to investigate the role of DNA methylation changes and expression-dependent demethylation of several CpG during distinct commitment steps of early, intermediate and dinucleotides within the second intron of the gene for macro- terminal phagocytic maturation, the lysozyme (LZM) gene phage colony-stimulating factor (M-CSF) receptor/c-fms was 17 provides an excellent system, because its gene product is one noted in monocytes and macrophages. We wished to ana- of the most abundant secretory proteins of monocytes and lyze further the role of methylation changes in this system by macrophages, accounting for 2.5% of total protein in these studying a major gene product of both types of phagocytic cells.3,4 Granulocytes store but do not secrete LZM protein, cells, ie lysozyme. and it is also synthesized in less mature myeloid cells, albeit at lower levels. The °15 kDa molecular weight human LZM protein is thus detectable at high concentrations in blood, Materials and methods tears and milk. It acts as a muramidase by hydrolyzing the linkage between N-acetylmuramic acid and N-acetyl-D-glu- cosamine in the bacterial cell wall peptidoglycan layer4 and Cells has been considered to represent a primitive humoral immune system which preceded the cellular immune system during After receiving informed consent, heparinized venous blood, evolution. The human LZM gene consists of four exons span- bone marrow or ascites samples were obtained from donors. ning a 6 kb region on chromosome 12,5 and the 3.5 kb Mononuclear cells were isolated by Ficoll–Hypaque density upstream region has been functionally characterized for pro- separation, washed several times with phosphate-buffered moter activity.6 saline (PBS) and processed further. Monocytes (Mo) were In vertebrate DNA, 60–80% of CpG dinucleotides are bio- removed from this fraction by performing adherence to plastic chemically modified at the five carbon position of cytosine.7,8 flasks for 2 h. The non-adherent, mononuclear fraction of nor- The distribution of methylated cytosines is clearly nonrandom, mal human bone marrow was composed of Ͼ85% cells of and may thus play a role as a carrier of epigenetic information the myeloid lineage (ca 10% at the promyelocytic stage); the (reviewed in Ref. 7). Human Alu sequences have been shown mononuclear cell fraction of human peripheral blood contained Ͼ90% lymphocytes, as determined by light microscopy of Wright–Giemsa-stained slide preparations. Granulocytes Correspondence: M Lu¨bbert, Division of Hematology/Oncology, Uni- (PMN) from the blood of healthy donors were purified as versity of Freiburg Medical Center, D-79106, Freiburg, Germany described previously13 and contained Ͼ95% granulocytes. In Received 8 January 1997; accepted 10 March 1997 vitro differentiation of Mo was performed as described.18 Lysozyme gene methylation during myeloid differentiation MLu¨bbert et al 991 Cell lines Dassel, Germany). Filters were baked for 2 h at 80°C and hybridized at 42°C for 16 h at conditions described pre- KG-1 early myeloblastic cells and S-LB1 T lymphoid cells viously.16 Filters were rinsed at 65°C to a final stringency of were kindly provided by Phillip Koeffler, Cedars-Sinai 0.1 × SSC and autoradiographed at −70°C using intensifying Research Center, UCLA Los Angeles; HL-60 and U-937 by screens. Inspection of ethidium–bromide-stained gels and DSM German Collection of Microorganisms and Cell Cul- rehybridization with a probe for alpha-actin served as indi- tures, Braunschweig, Germany; HOS human osteosarcoma cators for RNA integrity and equal loading. cells by ATCC, Rockville, MD, USA; HepG2 hepatocellular carcinoma cells by Dr P Hafkemeyer, Dept Gastroenterology, University of Freiburg Medical Center, Germany. All cell lines In vitro transcription assay (‘nuclear run-on’) were cultured in RPMI 1640 medium supplemented with 10% ° FCS at 37 C in a humidified atmosphere of 7% CO2 in air. Isolation and labeling of viable nuclei was performed as pre- For in vitro monocytic/macrophage differentiation of HL-60, viously described22,23 with several modifications. Cells were − U-937 and KG-1, cells were cultured in the presence of 10 7 M washed at 4°C in PBS, nuclei were isolated through lysis of 12-O-tetradecanoylphorbol 13-acetate (TPA; Sigma Bio- the cell membrane in ice-cold RSB (10 mM Tris pH 7.5, 3 mM chemical, Deisenhofen, Germany). MgCl2,5mM KCl) in the presence of 0.5% Nonidet-P40 (Sigma) for 5 min. A total of 108 nuclei were resuspended in

NSB (30% glycerol, 140 mM KCl, 5 mM mgCl2) and quick- DNA clones frozen in liquid nitrogen. For in vitro transcription, 8 ␮l each of ATP, GTP, CTP (40 mM each, Boehringer Mannheim), 2 ␮l The 700 bp PstI/RsaI restriction fragment of the pHL2 cDNA, of unlabeled UTP (40 mM) and 10 ␮l alpha-32p-UTP kindly provided by Dr S Gordon, University of Oxford, UK, (400 Ci/mmol, Amersham), 10 ␮l of ‘RNAsin’ RNAse inhibitor is complementary to 3′ exon 2 through the central, coding and 0.5 ␮l1MDTT were added. Reaction mixture was incu- portion of exon 4 of the human lysozyme gene.19 Its linearized bated at 26°C for 20 min. DNA was digested by adding 10 ␮l plasmid pGBR2.4 (pGEM3 host plasmid backbone) was used RNAse-free DNAseI (10 U/ml, Boehringer Mannheim) and ␮ ° for nuclear run-on studies. An empty linearized pBS 10 l CaCl2,20mMand further incubation at 26 C for 10 min. BlueScript vector was used as negative control in nuclear run- Elongation of nascent transcripts was stopped by adding 25 ␮l on experiments. An alpha-actin cDNA was used to check for 10 SET (5% SDS, 50 mM EDTA, 100 mM Tris pH 7.4) and 5 ␮l equal RNA balancing and integrity.20 proteinase K at 10 mg/ml (Boehringer Mannheim) in the presence of yeast tRNA. Digestion reaction was incubated at 42°C for 30 min. RNA was extracted by phenol-chloroform, precipi- DNA methylation analysis by Southern blot tated using 2.5 volume of ethanol for 1 h at −70°C. An aliquot of the labeled RNA was checked for intactness on denaturing DNA was isolated from hematopoietic cells by the method of MOPS/agarose gel. For hybridization, equal amounts of lin- Chirgwin et al,21 digested with proteinase K and further pur- earized plasmids (10 ␮g) had been immobilized by slot-blot ified by precipitations with isopropanol and 2.5 M ammonium on Nytran filter membrane after denaturation in 0.5 N NaOH. acetate as described.13 For methylation analyses by endonu- Labeled RNA normalized to equal numbers of counts was clease restriction, the following measures were used to ensure denatured at 65°C for 5 min, chilled on ice for 5 min and completeness of DNA cleavage: DNAs were initially digested hybridized to cloned DNA for 3 days at 42°C in the presence in a large reaction volume with a five-fold excess of HindIII of 50% formamide (BRL), 2 × SSC, 1% SDS, 5 × Denhardt’s for 16 h. Restrictions of HindIII-digested DNA with SmaI solution and 50 ␮g/ml yeast tRNA (Sigma). Filters were (Boehringer Mannheim, Mannheim, Germany) or XmaI (New washed in subsequent steps for 30 min each: at 55°C in the England Biolabs, Beverley, MA, USA) were performed with presence of 2 × SSC only, at 37°C with 2 × SSC and 10 ␮g/ml 10-fold and two-fold excess of enzyme, respectively (U RNAse, finally at 55°C with 0.5 × SSC. Filters were exposed enzyme/␮g DNA). After 6 h, more enzyme was added to a for 8 days at −70°C in the presence of an intensifying screen. final concentration of 20 and 4 U/␮g DNA, respectively, and Densitometry was performed as described above. digested for an additional 12 h. To sister reactions, 1 ␮gof lambda DNA was added and completeness of digestion checked on agarose gel. Restricted DNA was size-fractionated Results on Tris-acetate agarose gels, transferred to nylon-based filter membrane (GeneScreen Plus; Dupont de Nemours, Wilming- Regulation of lysozyme gene expression during ton, DE, USA) and analyzed by Southern blot technique as myelopoiesis described.13 Densitometry was performed on a Therados DCD-5 laser densitometer (Stockholm, Sweden); the areas Levels of LZM mRNA accumulation were determined in a under the curves obtained from two to three scannings were panel of cells and cell lines representing a wide spectrum of calculated and an average taken. developmental stages of phagocytes and their precursor cells. Using Northern blot analysis, the expected 1.7 kb LZM transcript was readily detectable in early promyelocyte-like HL-60 RNA isolation and Northern blot analysis and myelomonoblastic U-937 cells, and high levels in normal human bone marrow cells, peripheral blood (PB) monocytes, Total cellular RNA was extracted from leukemic cells and cell PB granulocytes and peritoneal macrophages (Figure 1, lines by the method of Chirgwin.21 RNA was dissolved in TES Table 1). In contrast, LZM transcripts were undetectable in (10 mM Tris 7.4, 1 mM EDTA, 0.1% SDS), electrophoresed on early myeloblastic KG-1 cells and in various non-myeloid denaturing formaldehyde-agarose gels and transferred to cells (lectin-stimulated blood lymphocytes, Jurkat T lymphoid nylon-based filter membrane (Nytran; Schleicher and Schuell, cell line, lung fibroblasts, HOS osteosarcoma cells; Table 1). Lysozyme gene methylation during myeloid differentiation MLu¨bbert et al 992 HL-60, U-937 and KG-1 cells were cultured for various time intervals in the presence of 12-O-tetradecanoylphorbol 13- acetate (TPA), a potent stimulator of protein kinase C24 and a strong inducer of monocyte-like differentiation of these three cells.25–27 After 12 h of TPA treatment, more than 50% of cells adhered to the plastic wall of the tissue culture flask and exhibited the decreased nuclear:cytoplasmic ratio typical of macrophage cells; after 3 days of TPA treatment, between 80 and 95% of all three cell lines exhibited these morphological features. A sharp downregulation of LZM-specific mRNA was observed in HL-60 cells after 24 h of exposure to TPA. Inhi- bition of new protein synthesis by cycloheximide did not alter LZM mRNA accumulation in either wild-type or TPA-treated HL-60 cells, suggesting that the observed downregulation is not mediated by short-lived proteins (Figure 1b). Downregulation was shown to be mediated predominantly transcriptionally, as nuclear run-on assays with HL-60 cells treated with TPA for 24 h showed a seven- to eight-fold decrease in the rate of transcription (Figure 2). A similar strong and rapid downregulation of LZM transcripts upon treatment with TPA was noted in U-937 cells; no induction of LZM transcripts by TPA was observed in early myeloblastic KG-1 cells (data not shown).

Variable methylation within the lysozyme (LZM) gene locus of LZM-expressing and non-expressing cells

DNA methylation patterns of the lysozyme gene were examined in various cell types and across an 11.3 kb region contained within a HindIII/HindIII fragment that encompasses the entire coding region of LZM as well as 5 kb of upstream flanking region containing the LZM gene promoter.6 Selective methylation analyses were performed using an isoschizomer pair of methylation-sensitive and -insensitive restriction enzymes to detect four SmaI/XmaI restriction sites (Figure 3). Each of these sites is part of one of the eight Alu repeats inserted in different orientations within and around this gene.5,19,27 The spacing of three of these sites allows for mapping of restriction fragments by Southern blot analyses using a probe located near the 3′ end of the HindIII/HindIII fragment. For all DNAs analyzed, complete digestion with XmaI was carried out in order to confirm intactness of the 6 bp restriction site and rule out sequence polymorphisms as a cause of incomplete digestion by SmaI. Methylation at site ‘S0’ located within the Alu sequence approximately 1.5 kb upstream of the transcriptional start site is not quantifiable by this assay when predominantly site ‘S1’ is cleaved by SmaI. The degree of demethylation at either SmaI/XmaI site was derived from the relative intensity of the resulting bands and quantified by densitometry.

Figure 1 Expression of lysozyme mRNA in normal and transformed myeloid cells. (a) Total RNA from peripheral blood (PB) monocytes and in vitro differentiated macrophages (cultivated through adherence to teﬂon surface for 8 days), normal peripheral blood poly- morphonuclear neutrophils (PB PMN) from healthy donors, and the T-lymphoblastoid cell line Jurkat was electrophoresed (20 ␮g/lane in all lanes except for PMN, 2 ␮g), blotted to nylon membrane and hybridized to LZM cDNA. Two different exposures were used to vis- ualize differences in expression levels. 28S rRNA as photographed from the ethidium-bromide stained gel is shown on the lower panel. (b) HL-60 promyelocytic cells were cultured in the absence or pres- − ence of TPA (10 7 M) and cycloheximide (20 ␮g/ml) for the indicated time intervals and analyzed by Northern blotting. Lysozyme gene methylation during myeloid differentiation MLu¨bbert et al 993 Table 1 Expression and methylation of the human lysozyme gene in different hematopoietic and non-hematopoietic cells and cell lines

Cells LZM S0 (%) S1 (%) S2 (%) S3 (%) mRNA

FH-109 − 80 80 Ͼ95 Ͼ95 embryonal lung fibroblasts Normal − Ͼ95 96 Ͼ95 Ͼ95 peripheral blood lymphocytes S-LB1 T- − Ͼ95 92 Ͼ95 Ͼ95 lymphoid cells HOS − Ͼ95 92 Ͼ95 Ͼ95 osteosarcoma cells HepG2 + ND 28 Ͼ95 Ͼ95 hepatoma cells KG-1 early − Ͼ95 88 Ͼ95 Ͼ95 Figure 2 Transcriptional regulation of LZM expression in HL-60 myeloblastic promyelocytes during phorbol ester-induced monocytic differen- cells tiation. 32P-labeled transcripts generated by in vitro transcription from KG-1 + TPA × 3 − Ͼ95 88 Ͼ95 Ͼ95 nuclei of HL-60 cells either untreated or treated with TPA 10−7 M for daysa 24 h were hybridized to 20 ␮gofEcoRI-linearized plasmids for U-937 ++ ND 35 Ͼ95 Ͼ95 chicken alpha-actin,19 empty Bluescript vector (pBS) and LZM19 as myelomonoblastic described in Materials and methods. Autoradiograms shown were cells generated by exposure of Kodak XR-5 film to filters in the presence U-937 + TPA × 3 − ND 35 Ͼ95 Ͼ95 of an intensifying screen at −70°C for 8 days. daysa HL-60 early + ND Ͻ15 Ͼ95 Ͼ95 promyelocytes was demethylated by 65% and greater than 85%, respectively HL-60 + TPA × 3 − ND Ͻ15 Ͼ95 Ͼ95 (Figure 4a). The density ratio of bands was unchanged during daysa monocytic differentiation and concomitant downregulation of +++ Normal human ND 10 10 50 expression with TPA treatment (Figure 4, Table 1). bone marrow MCs Phagocytic cells (high levels of LZM mRNA) exhibited a pat- PB granulocytes +++ ND Ͻ51015 tern of almost complete or complete demethylation at all three Normal PB +++ ND Ͻ5 Ͻ5 Ͻ5 SmaI restriction sites ‘S1’ to ‘S3’. This can best be appreciated monocytes by comparing the restriction patterns created by SmaI and Macrophages ++++ ND Ͻ5 Ͻ5 Ͻ5 XmaI enzymes, which were almost identical in all phagocytic cell types examined (Figure 4). In NHBM, a mixed pattern was Southern and Northern blot analyses were performed as described observed, with a major band of 5.0 kb; this most likely reflects in Materials and methods. ‘%’ indicates the degree of methylation the heterogenous cell populations within the mononuclear at a given SmaI site as determined by either densitometry or visual cell fraction of normal bone marrow. inspection. S0–S3, SmaI/XmaI sites present within the sequenced 5 region of the LZM gene locus. Relative abundance of LZM mRNA Peters et al have reported aberrant expression of LZM by as judged by visual inspection is indicated by (−)to(++++). human HepG2 hepatoma cells. As shown in Figure 5a, LZM aCells had a macrophage-like phenotype and exhibited adherence mRNA was readily detectable. Methylation analysis of the to tissue culture flask. DNA from these cells revealed significant demethylation at Ͼ MCs, mononuclear cells; ND, not determinable ( 50% cutting at restriction site ‘S1’ (Figure 5b). site ‘S1’ precludes quantitative restriction analysis 5′ of the site with the probe positioned 3′); PB, peripheral blood. Discussion As shown in Figure 4, a high degree of variability of methylation was observed in the various cell types, ranging from Cellular differentiation of hematopoietic precursor cells to complete methylation at each of the four restriction sites granulocytes and monocytic/macrophage cells is probably (presence only of the 11.3 kb HindIII fragment), to loss of all controlled on multiple levels. Several transcription factors higher molecular weight bands and presence of only the specifically involved in myeloid maturation have been 5.0 kb and 0.8 kb bands, indicating complete demethylation identified;28–30 the conformation of chromatin and its accessi- at SmaI sites 1–3. Methylation at these sites was noted in nor- bility to DNA-binding proteins may also be of importance in mal human lymphocytes (Figure 4a). In contrast, early mye- this process.13,31–34 The exact role of cytosine methylation and loblastic KG-1 cells (LZM non-expressor) exhibited approxi- its changes during myelopoiesis, however, is still unclear. mately 10% demethylation solely at the SmaI site ‘S1’ We and others have been examining the tissue- and devel- (Figure 4b, indicated by the faint presence of a single 6.9 kb opment-dependent dynamics of DNA methylation and band). This site is contained within the Alu repeat located chromatin structure within the myeloperoxidase (MPO) gene approximately 700 bp upstream of the major transcriptional locus.13,16,33–35 The human MPO gene is highly expressed in start site (‘Alu 5′a’ according to Ref. 27). This pattern was promyelocytic and late myeloblastic cells.14 It undergoes sev- highly reproducible and stable during monocytic differen- eral biochemical demodifications, which result in a spreading tiation of KG-1 cells after exposure to TPA for 3 days. Con- of demethylation from the 3′ to 5′ direction when comparing versely, in U-937 and HL-60 LZM expressor cell lines (both cells of different maturation stages. Thus, complete demethyl- expressing low to moderate amounts of LZM mRNA), this site ation of five CpG dinucleotides is present in normal and leu- Lysozyme gene methylation during myeloid differentiation MLu¨bbert et al 994

Figure 3 Map of the lysozyme gene and schematized mapping strategy for modulated methylation sites. The map plot was derived from the known genomic organization.5,27 The vertical black bars indicate exons and are numbered in order of transcription. The broken line indicates a portion of the gene where no sequence information was available. Horizontal bars indicate different restriction fragments generated by cleavage at the different restriction sites. S0-S3 indicate the SmaI/XmaI restriction sites present in the sequenced LZM gene (each is part of an Alu repeat).27

kemic promyelocytes, this state is maintained in more mature allel those noted for the MPO gene; however, complete deme- myeloid cells, in which however the MPO gene is not thylation of the LZM gene is attained at a much later stage of actively transcribed.13,16,35 differentiation, reflecting, as one could speculate, the later Felgner et al17,36 determined lineage- and expression- point of highest LZM mRNA expression. The tight association specific methylation of the macrophage colony-stimulating between demethylation of SmaI restriction site ‘S1’ and LZM factor (M-CSF) receptor gene, c-fms. Cell-specific differences expression is also present in primary leukemic cells from in DNA modification within intron 2 of this gene were noted, patients with AML.37 with several CpG residues consistently demethylated in mono- The observed fixed ratio of methylated and demethylated cytes (c-fms expressing), and to a higher degree in tissue alleles of a given cytosine nucleotide could be interpreted macrophages. In contrast, these CpG sites were methylated in according to the model of Szyf38 proposing that the degree of c-fms non-expressing peripheral blood granulocytes and lym- methylation at a given sequence is determined stochastically phocytes.17 by (1) chromatin accessibility to methyltransferase and DNA- In the present study, we examined whether DNA methyl- binding proteins; (2) the cell-specific abundance of methylation of the LZM gene (which is initially transcribed during the transferase. Chromatin analyses of the differentially methyl- early-to-intermediate stages of myeloid differentiation), would ated LZM sequences are in progress. decrease with cellular maturation, and be absent in phago- Experiments aimed at answering the question of whether cytic cells (highest mRNA expression levels of LZM). Low-to- the LZM gene methylation changes are causally involved in intermediate LZM transcript levels are detectable in HL-60 the transcriptional regulation in the different myeloid cells or early promyelocytes and U-937 monoblasts, whereas KG-1 an epiphenomenon are hampered by lack of inducible early myeloblasts do not express this gene. Compared to monocytes and granulocytes, LZM mRNA expression is highest in macrophages. Downregulation of the LZM gene was observed in HL-60 and U-937 cells treated with phorbol ester. The Figure 4 Progressive demethylation within the LZM gene flanking following patterns of progressive cytosine demethylation and coding region is associated with its expression and with terminal appeared: a significant degree of demethylation solely at a myeloid differentiation. (a) DNA was isolated from normal peripheral CpG dinucleotide within an Alu repeat contained in the 5′ blood lymphocytes (nl PBL); U-937 myelomonoblastic cells, HL-60 −7 flanking region of the LZM gene (site ‘S1’ in Figure 3) was promyelocytic cells (both also cultured with TPA (10 M) for 3 days); associated with LZM expression in late myeloblastic, myelo- normal peripheral blood granulocytes (nl PMN); peripheral blood (PB) monocytes; macrophages derived by in vitro culture from peripheral monoblastic and promyelocytic cells. Almost complete or blood mo. (b) DNA was isolated from human diploid embryonal lung complete demethylation of all three CpGs sites amenable to fibroblasts; KG-1 early myeloblastic cells (also cultured with TPA − analysis in the gene locus was strictly associated with granulo- (10 7 M) for 3 days), normal human bone marrow (NHBM); peritoneal cytic and monocytic/macrophage phenotype. The observed macrophages. DNA was purified as described in Materials and ratios of demethylation were highly reproducible for a given methods, first cleaved with HindIII alone (shown for U-937 in a and cell type. Downregulation of LZM gene expression in leu- peritoneal macrophages in b, similar results, not shown, were obtained with the other cells) and then with either SmaIorXmaI (the kemic cells lines after phorbol-ester treatment was not asso- latter only shown for macrophage DNA; similar results, not shown, ciated with any detectable methylation changes. The stepwise, were obtained from the other cell types). DNA was analyzed for LZM- regional changes of the LZM gene towards demethylation par- specific band patterns by Southern blot technique. Lysozyme gene methylation during myeloid differentiation MLu¨bbert et al 995 Lysozyme gene methylation during myeloid differentiation MLu¨bbert et al 996 protein–DNA interactions mediating progressive LZM demethylation. The SmaI site and surrounding repetitive element inserted in the 5′ to 3′ orientation approximately 700 bp upstream of the lysozyme transcriptional start site appears of particular interest in this regard. Thus far, there are no stable genotypic indicators of myeloid maturational stages in normal precursors and myeloid leukemias that extend the information yielded by morphologic, cytochemical, and immunological diagnostic tools. The methylation status of LZM, MPO, c-fms and possibly other myeloid-specific genes, as well as the M-bcr gene may thus provide lineage-specific molecular markers of hematopoietic differentiation.48–50 In addition, it has recently become clear that cancer-associated hypermethylation resulting in silencing of expression is detectable in the promoter region of tumor suppressor genes.51 In summary, methylation analyses might prove useful in treatment monitoring of myeloid malignancies; this appears of particular relevance when drugs with known methyltransferase-inhibiting activity such as 2′-desoxy-5-azacytidine and 5-azacytidine are administered.

Figure 5 Aberrant expression of LZM in hepatoma cells is associa- Acknowledgements ted with partial demethylation. (a) RNA was extracted from HepG2 human hepatoma cells and probed (20 ␮g) for LZM mRNA expression by Northern blot as described in the legend to Figure 1. (b) DNA from This study was supported in part by Deutsche Krebshilfe these cells was assayed for LZM gene methylation status as described (grants W48/92/Lu¨1, W16/94/Br1) and Deutsche Forschungs- in the legend to Figure 4. gemeinschaft (SFB 364). We wish to thank Dr Constanze Bon- ifer, Dept of Biology, University of Freiburg for critical reading of the manuscript, and acknowledge the technical efforts of J methylation changes in the cell lines studied. However, func- Rommelt and R Ruszynski. tional evidence for an important regulatory role of methylation ¨ changes within the murine LZM gene during myeloid differentiation stems from work by Klages et al.39 They demonstrated the presence of a single HpaII restriction site in the 3′ flanking References region, overlapping an enhancer element, at which demethylation is induced by in vitro differentiation of murine FDCP- 1 Blau HM. Differentiation requires continuous active control. Annu A4 cells and is accompanied by a strong induction of LZM Rev Biochem 1992; 61: 1213–1230. mRNA. Furthermore, in vitro methylation of this portion of the 2 Metcalf D. The molecular control of cell division, differentiation, commitment, and maturation in hematopoietic cells. Nature 1989; gene resulted in dramatically reduced enhancer function as 339: 27–29. well as interference with binding of the ets-related GA binding 3 Gordon S, Todd T, Cohn ZA. In vitro synthesis and secretion of protein (GABP) transcription factor.40 lysozyme by mononuclear phagocytes. J Exp Med 1974; 139: For the human LZM gene, in a model of normal peripheral 1228–1248. blood progenitor cells induced to differentiate ex vivo, our 4 Jolle´s P, Jolle´s J. What’s new in lysozyme research? Mol Cell results suggest that the first shift toward demethylation appears Biochem 1984; 63: 165–189. 41 5 Peters CWB, Kruse U, Pollwein R, Grzeschik KH, Sippel AE. The at least simultaneously with increased LZM expression, thus human lysozyme gene: sequence, organization and chromosomal suggesting that it might be a necessary step for the initiation localization. Eur J Biochem 1989; 182: 507–516. of development-specific LZM transcription but not for tran- 6 Clarke S, Greaves DR, Chung LP, Tree P, Gordon S. The human scriptional shut-off. lysozyme promoter directs reporter gene expression to activated All SmaI/XmaI restriction sites studied are located within myelomonocytic cells in transgenic mice. Proc Natl Acad Sci USA four of the eight Alu sequences which are clustered in both 1996; 93: 1434–1438. ′ 7 Bird A. The essentials of DNA methylation. Cell 1992; 70: 5–8. orientations within and 5 of the LZM gene. The functional 8 Clark SJ, Harrison J, Frommer M. CpNpG methylation in mam- role of this family of repetitive sequences for the control of malian cells. Nat Genet 1995; 10: 20–27. chromatin formation and transcription is under active investi- 9 Hellmann-Blumberg U, Hintz MF, Gatewood JM, Schmidt CW. gation.42 Methylation of Alu repeats from several tissue- Developmental differences in methylation of human Alu repeats. specific genes can transcriptionally silence gene Mol Cell Biol 1993; 13: 4523–4530. expression9,42–45 and an Alu binding protein preventing CpG 10 Li E, Bestor TH, Jaenisch R. Targeted mutation of the DNA methyl- 46 transferase gene results in embryonic lethality. Cell 1992; 69: methylation has recently been identified. One of several 915–926. moderately conserved repeat elements located within the 11 Tate P, Skarnes W, Bird A. The methyl-CpG binding protein chicken lysozyme has been implicated in the ‘silencing’ of MeCP2 is essential for embryonic development in the mouse. Nat LZM gene expression.45 It is tempting to speculate that pro- Genet 1996; 12: 205–208. gressive spreading of demethylation within LZM and other 12 Chu C, Shen CKJ. DNA methylation: its possible functional roles myeloid-specific genes might reflect a ‘cogwheel’ function in developmental regulation of human globin gene families. In: Jost JP, Saluz HP (eds). DNA Methylation: Molecular Biology and during cellular maturation. Several proteins have been ident- Biological Significance. Birkha¨ user Verlag: Basel, 1993, pp 385– ified which bind DNA depending on cytosine methyl- 403. ation.11,47 Further studies are necessary to unravel the role of 13 Lu¨bbert M, Miller CW, Koeffler HP. Changes of DNA methylation Lysozyme gene methylation during myeloid differentiation MLu¨bbert et al 997 and chromatin structure in the human myeloperoxidase gene dur- 33 Chang K-S, Zhao S, Wang Y, Lu J, Trujillo JM, Stass SA, Freireich ing myeloid differentiation. Blood 1991; 78: 345–356. EJ. Downregulation of myeloperoxidase gene associated with 14 Tobler A, Miller CW, Johnson KR, Selsted ME, Rovera G, Koeffler specific nuclease hypersensitive sites during TPA-induced differ- HP. Regulation of gene expression of myeloperoxidase during entiation of HL-60. Leukemia 1991; 5: 205–209. myeloid differentiation. J Cell Phys 1988; 136: 215–225. 34 Jorgenson KF, Antoun GR, Zipf TF. Chromatin structural analysis 15 Fouret P, Du Bois R, Bernaudin J, Takahashi H, Ferrans V, Crystal of the 5′ end and contiguous flanking region of the myeloperoxi- RG. Expression of the neutrophil elastase gene during human bone dase gene. Blood 1991; 77: 159–164. marrow cell differentiation. J Exp Med 1989; 169: 833–845. 35 Hashinaka K, Yamada M. Undermethylation and DNase I hyper- 16 Lu¨bbert M, Oster W, Ludwig W-D, Ganser A, Mertelsmann R, sensitivity of myeloperoxidase gene in HL-60 cells before and after Herrmann F. A switch toward demethylation is associated with differentiation. Arch Biochem Biophys 1992; 293: 40–45. the expression of myeloperoxidase in acute myeloblastic and pro- 36 Felgner J, Kreipe H, Heidorn K, Jaquet K, Zschunke F, Radzun myelocytic leukemias. Blood 1992; 80: 2066–2073. H-J, Parwaresch MR. Increased methylation of the c-fms proto- 17 Felgner J, Kreipe H, Heidorn K, Jaquet K, Heuγ R, Zschunke F, oncogene in acute myelomonocytic leukemias. Pathobiol 1991; Radzun H-J, Parwaresch MR. Lineage-specific methylation of the 59: 293–298. c-fms gene in blood cells and macrophages. Leukemia 1992; 6:37Lu¨bbert M, Mertelsmann R, Herrmann F. Cytosine methylation 420–425. changes during normal hematopoiesis and in acute myeloid leuke- 18 Kreutz M, Krause SW, Hennemann B, Rehm A, Andreesen R. mia. Leukemia 1997; 11 (Suppl. 1): 12–18. Macrophage heterogeneity and differentiation: defined serum-free 38 Szyf M. DNA methylation patterns: an additional level of infor- culture conditions induce different types of macrophages in vitro. mation? Biochem Cell Biol 1991; 69: 764–767. Res Immunol 1992; 143: 107–115. 39 Klages S, Mo¨llers B, Renkawitz R. The involvement of demethyl- 19 Chung LP, Keshav S, Gordon S. Cloning the human lysozyme ation in the myeloid-specific function of the mouse M lysozyme cDNA: inverted Alu repeat in the mRNA and in situ hybridization gene downstream enhancer. Nucleic Acids Res 1992; 8: 1925– for macrophages and Paneth cells. Proc Natl Acad Sci USA 1988; 1932. 85: 6227–6231. 40 Nickel J, Short ML, Schmitz A, Eggert M, Renkawitz R. Methylation 20 Schwarz RJ, Haran JH, Rothblum KN, Dugaiczik A. Regulation of the mouse M lysozyme gene downstream enhancer inhibits het- of muscle differentiation: cloning of sequencing from alpha-actin erotetrameric GABP binding. Nucleic Acids Res 1995; 23: messenger ribonucleic acid. Biochemistry 1980; 19: 5883–5889. 4785–4792. 21 Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ. Isolation 41 Lu¨bbert M, Brugger W, Mertelsmann R, Kanz L. Developmental of biologically active ribonucleic acid from sources enriched in regulation of myeloid gene expression and demethylation during ribonuclease. Biochemistry 1979; 18: 5294–5299. ex vivo culture of peripheral blood progenitor cells. Blood 1996; 22 Henschler R, Brennscheidt U, Mertelsmann R, Herrmann F. Induc- 87: 447–455. tion of c-jun expression in the myeloid leukemia cell line KG-1 42 Englander EW, Wolffe AP, Howard BH. Nucleosome interactions by 1-beta-D-arabinofuranosylcytosine. Mol Pharmacol 1991; 39: with a human Alu element. Transcriptional repression and effects 171–176. of template methylation. J Biol Chem 1993; 268: 19565–19573. 23 Koeffler HP, Gasson J, Tobler A. Transcriptional and post-tran- 43 Kochanek S, Renz D, Doerfler W. DNA methylation in the Alu scriptional modulation of myeloid colony-stimulating factors by sequences of diploid and haploid primary human cells. EMBO J tumor necrosis factor and other agents. Mol Cell Biol 1988; 8: 1993; 12: 1141–1151. 3432–3438. 44 Kochanek S, Renz D, Doerfler W. Transcriptional silencing of 24 Nishizuka Y. The role of protein kinase C in cell surface signal human Alu sequences and inhibition of protein binding in the box transduction and tumor promotion. Nature 1984; 308: 693–697. B regulatory elements by 5′-CG-3′ methylation. FEBS Lett 1995; 25 Rovera G, O-Breine TA, Diamond L. Induction of differentiation in 360: 115–120. human promyelocytic leukemia cells by tumor promoting agents. 45 Baniahmad A, Muller M, Steiner C, Renkawitz R. Activity of two Science 1979; 204: 868–870. different silencer elements of the chicken lysozyme gene can be 26 Koeffler HP, Bar-Eli M, Territo M. Phorbol ester effect on differen- compensated by enhancer elements. EMBO J 1987; 6: 2297– tiation of human myeloid leukemia cell lines blocked at different 2303. stages of maturation. Cancer Res 1981; 41: 919–926. 46 Chesnokov IN, Schmid CW. Specific Alu binding protein from 27 Riccio ML, Rossolini GM. Unusual clustering of Alu repeats within human sperm chromatin prevents DNA methylation. J Biol Chem the 5′-flanking region of the human lysozyme gene. DNA 1995; 270: 18539–18542. Sequence 1993; 4: 129–134. 47 Tate PH, Bird AP. Effects of DNA methylation on DNA-binding 28 Hromas R, Davis B, Rauscher FJ, Klemsz M, Tenen D, Hoffman proteins and gene expression. Curr Opin Gen Devel 1993; 3: S, Xu D, Morris JF. Hematopoietic transcriptional regulation by 226–231. the myeloid zinc finger gene, MZF-1. Curr Top Microbiol Immu- 48 Litz CE, Goldfarb AN, Strickler JG, Brunning RD. Presence of cell nol 1996; 211: 159–164. lineage-specific hypomethylated sites in the major breakpoint 29 Zhang D-E, Hohaus S, Voso MT, Chen H-M, Smith LT, Hethering- cluster region. Cancer Res 1990; 50: 4984–4990. ton CJ, Tenen DG. Function of PU.1 (Spi-1), C/EBP, and AML1 in 49 Ohyashiki JH, Ohyashiki K, Kawakubo K, Tauchi T, Shimamoto early myelopoiesis: regulation of multiple myeloid CSF receptor T, Toyama K. The methylation status of the major breakpoint clus- promoters. Curr Top Microbiol Immunol 1996; 211: 137–148. ter region in human leukemia cells, including Philadelphia chro- 30 Okuda T, van Deursen J, Hiebert SW, Grosveld G, Downing JR. mosome-positive cells, is linked to the lineage of hematopoietic AML1, the target of multiple chromosomal translocations in cells. Leukemia 1993; 7: 801–807. human leukemia, is essential for normal fetal liver hematopoiesis. 50 Mills KI, Sproul AM, Burnett AK. Methylation of the major break- Cell 1996; 84: 321–330. point cluster region (M-bcr) in Philadelphia-positive CML. Leuke- 31 Doerfler W. DNA methylation and gene activity. Annu Rev mia 1993; 7: 707–711. Biochem 1983; 52: 93–124. 51 Herman JG, Merlo A, Mao L, Lapidus RG, Issa JP, Davidson NE, 32 Bonifer C, Hecht A, Saueressig H, Winter DM, Sippell AE. Sidransky D, Baylin SB. Inactivation of the CDKN2/p16/MTS1 Dynamic chromatin: the regulatory domain organization of euka- gene is frequently associated with aberrant DNA methylation in ryotic gene loci. J Cell Biochem 1991; 47: 99–108. all common human cancers. Cancer Res 1995; 55: 4525–4530.