Myeloperoxidase Gene Expression Research Articles

Functional activity of three distinct myeloperoxidase (MPO) promoters in human myeloid cells.

The enzyme myeloperoxidase (MPO) is synthesized only in myeloid and monocytic cells, making it an important marker of the myeloid lineage. Transcription of the MPO gene is turned on early during the myeloblast stage of myeloid differentiation and is turned off when myeloid precursors are induced to differentiate along any one of a number of pathways. MPO transcripts show heterogeneity in size and sequence due, in part, to differential RNA splicing. We recently reported transfection studies which showed the presence of three distinct MPO promoters in the 5'-flanking region of the human MPO gene, suggesting that MPO transcription may also be regulated through the use of multiple promoters. We now report results of primer extension and RT-PCR experiments designed to determine if transcription of the human MPO gene is initiated at multiple promoter sites in vivo. MPO RNA obtained from myeloid cell lines was analyzed by primer extension using primers located at various sites between bp -1100 and bp +120 of the MPO gene. In addition, RT-PCR experiments were carried out using primer pairs located at intervals between bp -1000 and bp +2500 of the MPO gene. MPO transcripts were found to be initiated at three sites located about bp -920, bp -310, and bp +1 of the MPO gene, corresponding closely to our previously described P3, P2 and P1 promoters, respectively. Transcription initiated at the P1 site gave rise to large transcripts and showed the expected downregulation following induction of differentiation. On the other hand, transcripts initiated at the P3 and P2 sites did not show downregulation following induction of macrophage differentiation by TPA, and most did not appear to extend into the MPO coding region. Northern blot analysis of transcripts initiated at the P3 and P2 sites suggested that transcription at these sites was non-tissue-specific and indicated that many of these transcripts undergo premature termination. These results demonstrate that the MPO gene is transcribed in vivo primarily using the P1 promoter and that the low level of transcription occurring at the P2 and P3 sites is nonspecific and does not contribute significantly to physiologic regulation of MPO gene expression.

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Neutrophil disfunction, caused by a decreased production of effective radical oxygen species by myeloperoxidase (MPO) and NADPH oxidase within the neutrophil, may result in susceptibility to opportunistic fungal infections. In vitro, MPO produces OCl, which kills bacteria and viruses. In the case of MPO deficiency, susceptibility to Candida albicans infection was observed. Furthermore, it was demonstrated that MPO knockout mice were primarily susceptible to C. albicans infection. With regards to NADPH oxidase deficiency, such a patient was found to have severe chronic granulomatous disease (CGD) due to Aspergillus infection. This deficiency may have resulted from a gene mutation and/or abnormality of the NADH oxidase components, particularly cytochrome b558 (gp91phox and p22phox) in membrane and cytosol factors p47phox, p67phox, p40phox and others in neutrophil. Thus,irregular regulation of transcription factors for gene expression of phox molecules and MPO permits susceptibility to Aspergillus and Candida infections.

Myeloperoxidase gene expression in non-infant pro-B acute lymphoblastic leukaemia with or without ALL1/AF4 transcript

In this study, we examined myeloperoxidase (MPO) gene expression in a series of 31 non-infant pro-B acute lymphoblastic leukaemia (ALL) patients that included 16 cases with the t(4;11) translocation and/or the resultant ALL1/AF4 chimaeric gene. Sixteen out of 31 cases (51%) were MPO mRNA positive/enzyme negative. MPO mRNA was detected in nine out of 16 (56%) and seven out of 15 (47%) patients with and without the ALL1/AF4 fusion transcript respectively. The comparative study between MPO mRNA positive and negative cases showed statistically significant differences with regard to age and white blood cell (WBC) count, and was 39·5 years vs. 26·3 years (P = 0·016) and 71·4 × 109/l vs. 157·8 × 109/l (P = 0·046) in the MPO mRNA positive and negative groups respectively. The correlation analysis between MPO mRNA expression, age, WBC count and leukaemic relapse according to the presence/absence of the ALL1/AF4 fusion showed that the statistically significant differences observed in the whole group were related mostly to the ALL1/AF4-positive ALL patients. In fact, in this latter group, the mean WBC count and patients' age were 85 ± 79 × 109/l vs. 289·8 ± 102 × 109/l (P = 0·0005) and 44·8 ± 15·3 years vs. 26·7 ± 13·7 years (P = 0·01) in patients with and without MPO mRNA expression respectively. It appears, therefore, that the assessment of MPO mRNA expression enables a further dissection of leukaemia heterogeneity in apparently homogeneous genetic/immunophenotypic ALL subsets.

Myeloperoxidase gene expression in non-infant pro-B acute lymphoblastic leukaemia with or without ALL1/AF4 transcript.

In this study, we examined myeloperoxidase (MPO) gene expression in a series of 31 non-infant pro-B acute lymphoblastic leukaemia (ALL) patients that included 16 cases with the t(4;11) translocation and/or the resultant ALL1/AF4 chimaeric gene. Sixteen out of 31 cases (51%) were MPO mRNA positive/enzyme negative. MPO mRNA was detected in nine out of 16 (56%) and seven out of 15 (47%) patients with and without the ALL1/AF4 fusion transcript respectively. The comparative study between MPO mRNA positive and negative cases showed statistically significant differences with regard to age and white blood cell (WBC) count, and was 39.5 years vs. 26.3 years (P = 0.016) and 71.4 x 10(9)/l vs. 157.8 x 10(9)/l (P = 0.046) in the MPO mRNA positive and negative groups respectively. The correlation analysis between MPO mRNA expression, age, WBC count and leukaemic relapse according to the presence/absence of the ALL1/AF4 fusion showed that the statistically significant differences observed in the whole group were related mostly to the ALL1/AF4-positive ALL patients. In fact, in this latter group, the mean WBC count and patients' age were 85 +/- 79 x 10(9)/l vs. 289.8 +/- 102 x 10(9)/l (P = 0.0005) and 44.8 +/- 15.3 years vs. 26.7 +/- 13.7 years (P = 0.01) in patients with and without MPO mRNA expression respectively. It appears, therefore, that the assessment of MPO mRNA expression enables a further dissection of leukaemia heterogeneity in apparently homogeneous genetic/immunophenotypic ALL subsets.

Molecular analysis of the human myeloperoxidase promoter region.

Myeloperoxidase (MPO) is a granule protein, transiently expressed during the promyelocyte stage of myeloid differentiation. It is transcribed in a stage and lineage specific manner. Studies of MPO gene regulation can help to elucidate the mechanism of normal and abnormal myeloid differentiation. Our preliminary data indicated the lack of basal promoter activity in the region immediately 5' to the MPO cDNA. Here, we report the results of the detailed molecular studies of the human MPO promoter region. To locate potential promoter elements active in HL60 cells, we made promoter deletion constructs ranging in size from 200 bp to 4.5 kb of the 5' region of the hMPO gene, cloned into the chloramphenicol acetyl transferase (CAT) reporter vector. Following electroporation of the promoter constructs into HL60 cells, CAT enzyme production was found only in the construct containing approximately the 1 kb region upstream of the reported MPO cDNA. A separate set of constructs was made to look for putative MPO enhancer elements. Several fragments upstream of the MPO promoter showed prominent transactivation of the TK promoter, indicating a possible enhancer. Tissue specificity of MPO promoter fragments was determined in myeloid cells arrested either before induction of MPO expression (KG1), during MPO expression (HL60), or after it had ceased (U937), as well as in non-MPO expressing non-myeloid cells. The construct containing an approximate 1000 bp fragment of the 5' region of MPO was found to direct CAT expression only in HL60 cells. The 3'-truncations of this promoter region resulted in loss of tissue-specificity, while the promoter activity remained largely unchanged. A negative regulatory element was found upstream of the MPO promoter which repressed heterologous promoters in all the tested cell lines. Enhancer elements showed no tissue- or stage-specificity that were characteristic for native MPO gene. Sequence analysis of the putative MPO promoter region showed a number of potential transcription factor binding sites. Of special interest is the region containing the purine-rich site that can bind proteins from the ets-family of transcription factors and a duplicate GATA-like site. When inserted upstream of a reporter containing the minimal Herpes simplex viral thymidine kinase (HSV-TK) promoter (into pBL2CAT plasmid) this site strongly activated the TK promoter in transfected myeloid cells. Further studies showed that oligonucleotides derived from the MPO promoter region bind multiple proteins in a band-shift assay. Taken together, our experiments located the regulatory elements important for human MPO gene expression in HL60 promyelocytes.

Myeloperoxidase Gene Expression and Regulation by Myeloid Cell Growth Factors in Normal and Leukemic Cells

Myeloperoxidase (MPO) is present in azurophilic granules which appear in the promyelocyte stage of differentiation, and is the most common functional protein of myeloid cells. With progress in molecular biology, the expression and regulation of MPO have been clarified in normal myeloid and leukemic cells, not only by enzymatical activity but at the gene leveL MPO expression is affected by the differentiation of myeloid cells, and has been suggested to be regulated by myeloid cell growth factors, such as granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor and interleukin-3. In the past decade the signal transduction from their receptors has been clarified. This review describes the expression and regulation of the MPO gene in myeloid cells including myeloid disorders, such as myeloid leukemia or myelodysplastic syndromes, The effects on MPO by myeloid growth factors and signal transduction from their receptors are also presented.

Myeloperoxidase Polymorphism Is Associated with Gender Specific Risk for Alzheimer's Disease

Myeloperoxidase (MPO) is a myeloid-specific enzyme that generates hypochlorous acid and other reactive oxygen species. MPO is present at high levels in circulating neutrophils and monocytes but is not detectable in microglia, brain-specific macrophages, in normal brain tissue. However, an earlier study indicated that MPO is present in macrophage-microglia at multiple sclerosis lesions, suggesting that reactivation of MPO gene expression may play a role in neurodegenerative diseases involving macrophage-microglia. In the present study, MPO is shown to colocalize with amyloid beta (Aβ) in senile plaques in cerebral cortex sections from Alzheimer's disease (AD) brain tissue. Microglia costaining for MPO and CD68 are closely associated with plaques, suggesting that plaque components induce MPO expression in microglia. In support of this interpretation, treatment of rodent microglia with aggregated Aβ(1–42) was shown to induce MPO mRNA expression. Also, the ApoE4 allele, the major AD risk factor associated with increased Aβ deposition, was shown to correlate with increased MPO deposition in plaques (P=0.01, ANOVA). Finally, a genetic polymorphism links MPO expression to Alzheimer's risk, in that a higher expressing SpSp MPO genotype was associated with increased incidence of AD in females, and decreased incidence in males (P=0.006). These findings suggest that the MPO polymorphism is a gender-specific risk factor for Alzheimer's disease.

Prevalence of myeloperoxidase gene expression in infant acute lymphocytic leukemia.

The enzyme myeloperoxidase (MPO; donor: H2O2 oxidoreductase, EC1.11.1.7) is a well-established marker of myeloid differentiation. Most myeloid leukemias express MPO enzyme activity at the light microscopic level, whereas lymphoid leukemias characteristically lack such expression. However, the diagnostic significance of MPO RNA expression or of immunohistochemically detectable MPO protein expression in leukemic blasts is unclear. We studied the prevalence and diagnostic significance of MPO RNA and protein expression in 57 cases of MPO enzyme-negative infant B-precursor acute lymphocytic leukemia (ALL), since the blast cells in this condition have been reported to show a high incidence of coexpression of myeloid-associated antigens. MPO expression was compared with other clinical and laboratory parameters. Of the cases examined, 56% showed detectable MPO expression at the RNA or protein level or both. Most positive cases showed MPO protein in many leukemic blasts, whereas a few cases showed substantial MPO protein expression in only a few blast cells. MPO expression showed no significant correlation with other markers of myeloid differentiation. Leukemic lymphoblasts in infant ALL frequently express MPO at the RNA or protein level; this expression does not imply an overall myeloid phenotype. The leukemic blasts in infant ALL may derive from an immature hematopoietic precursor cell not fully committed to lymphoid differentiation.

Myeloperoxidase Gene Expression in Infant Leukemia: A Pediatric Oncology Group Study

A high incidence of co-expression of myeloid-associated antigens in infant B-precursor Acute Lymphocytic Leukemia (B-ALL) has been reported, but the significance of this finding is uncertain. To further assess myeloid differentiation and its prognostic significance in this disease, we investigated the frequency of myeloperoxidase (MPO) gene expression in the blast cells from 43 infants with B-ALL registered in a Pediatric Oncology Group (POG) Pilot Study for Treatment of Infant ALL, utilizing a molecular probe for detection of MPO messenger RNA (mRNA) by Northern blot hybridization and a monoclonal antibody to detect MPO-protein by immunohistochemical staining. Sufficient RNA for Northern blot was extracted from 32 bone marrow or blood samples. In two cases, MPO mRNA was determined by a reverse transcriptase-polymerase chain reaction assay and was negative in both cases. MPO-specific transcripts (MPO+) were present in 19 of 34 (56%) samples analyzed. Immunoreactive MPO protein was positive in 13 of the 20 (65%) patients studied. No correlation was found between MPO gene expression and clinical or laboratory features, karyotypic patterns or clinical outcome. The high frequency of MPO gene expression demonstrated in this study suggests that leukemogenic events in many cases of infant B-ALL appear to involve a pluripotent stem cell not yet fully committed to lymphoid differentiation.

Involvement of STAT3 in the Granulocyte Colony-stimulating Factor-induced Differentiation of Myeloid Cells

Granulocyte colony-stimulating factor (G-CSF) stimulates proliferation and differentiation of the progenitor cells of neutrophilic granulocytes. The binding of G-CSF to its receptor specifically activates JAK1 and JAK2 kinases, as well as STAT3, a signal transducer and activator of transcription (STAT). To examine the role of STAT3 in G-CSF receptor-mediated signal transduction, two different forms of the dominant negative STAT3 were introduced into a mouse myeloid cell line that exogenously expresses the mouse G-CSF receptor. In response to G-CSF, the parental myeloid cells grew for about 4 days, and then they stopped dividing and differentiated into cells with lobulated nuclei. During this period, the expression of the myeloperoxidase (MPO) gene was induced, while c-myc gene expression was down-regulated. In contrast, in the cells expressing the dominant negative STAT3, G-CSF could induce neither growth arrest nor morphological change. However, the induction of the MPO gene by G-CSF was not affected by the dominant negative STAT3. These results indicate that STAT3 activation is responsible for part of the G-CSF-induced differentiation of neutrophils but that another pathway, involving the expression of the MPO gene, that does not utilize the activated STAT3, is also required to fully differentiate the cells.

The myeloperoxidase gene proximal enhancer directs hematopoietic-specific expression in transgenic mice

The myeloperoxidase (MPO) gene is expressed specifically in immature myeloid cells. The MPO gene includes a promoter proximal enhancer which is coincident with DNaseI hypersensitive chromatin sites and is specifically active in myeloid cell lines. We developed transgenic murine lines in which 1.3 kb of murine MPO proximal 5′ flanking region DNA was linked to a TATAA homology and RNA initiation site derived from the HSV-TK promoter and to a luciferase reporter (MPOTKLUC). In each of six founder lines, high-level luciferase activity was evident in marrow, thymus and spleen. Modest- to high-level luciferase expression was also evident in brain and in the heart in several of the lines, and luciferase activity was at or near background levels in lung, liver, kidney, stomach, colon, bladder, skeletal muscle, skin and small intestine in all of the MPOTKLUC transgenic mice. Within marrow cells, luciferase activity was evident in myeloid (GR-1+), B lymphoid (B220+) and T-lymphoid (CD4+) cells. Additional regulatory regions, thus, may be required to further restrict MPO gene expression to immature myeloid cells.

Myeloperoxidase gene expression in acute lymphoblastic leukaemia.

The FAB classification of acute leukaemias is based on the light microscopic detection of myeloperoxidase (MPO) activity in blast cells. Cases with MPO activity in > or = 3% of blasts are classified as acute myeloid leukaemia. We describe two cases of acute leukaemia in which the blast cells had lymphoid morphology, ultrastructure, immunophenotype and molecular rearrangements, but expressed significant levels of the MPO gene (MPO mRNA) by reverse transcription polymerase chain reaction (RT-PCR), in the absence of translation to protein. Both cases presented a short remission duration. We discuss the incidence, features and outcome of MPO mRNA positive acute lymphoblastic leukaemia (ALL).

Multilineage gene expression precedes commitment in the hemopoietic system.

We have tested the hypothesis that multipotential hemopoietic stem and progenitor cells prime several different lineage-affiliated programs of gene activity prior to unilineage commitment and differentiation. Using single cell RT-PCR we show that erythroid (beta-globin) and myeloid (myeloperoxidase) gene expression programs can be initiated by the same cell prior to exclusive commitment to the erythroid or granulocytic lineages. Furthermore, the multipotential state is characterized by the coexpression of several lineage-affiliated cytokine receptors. These data support a model of hemopoietic lineage specification in which unilineage commitment is prefaced by a "promiscuous" phase of multilineage locus activation.

Myeloperoxidase mRNA analysis in acute lymphoblastic leukemia.

Expression of the myeloperoxidase (MPO) gene at the mRNA level is a better lineage marker than enzymatic activity in early myeloid precursors and their leukemic counterparts. Its diagnostic use depends on the specificity of expression for myeloblasts and its absence in blasts of lymphoid lineage. The present study investigates MPO mRNA expression in adult acute lymphoblastic leukemia (ALL), using reverse transcription-polymerase chain reaction. Of a total of 13 cases, six were found to have blasts positive for MPO mRNA; in all of these cases, the blasts were cytochemically negative for MPO. This unexpected finding of MPO mRNA positivity in six of 13 cases was further investigated at the molecular level. Bcr gene rearrangement analysis was positive in all six cases for the bcr breakpoint diagnostic of chronic myelogenous leukemia (CML). Only three of these six cases were cytogenetically positive for a Philadelphia (Ph) chromosome. Based on molecular analysis, these cases are considered as CML presenting in blast crisis of lymphoid lineage, as opposed to de novo ALL. The remaining seven cases were Ph negative at the cytogenetic and molecular levels; the leukemic blasts were MPO mRNA negative, confirming the lack of MPO gene expression in de novo ALL.

Control of myeloperoxidase gene expression in developing myeloid cells

Control of myeloperoxidase gene expression in developing myeloid cells

Regulation of the myeloperoxidase enhancer binding proteins Pu1, C-EBP alpha, -beta, and -delta during granulocyte-lineage specification.

We have compared the molecular architecture and function of the myeloperoxidase upstream enhancer in multipotential versus granulocyte-committed hematopoietic progenitor cells. We show that the enhancer is accessible in multipotential cell chromatin but functionally incompetent before granulocyte commitment. Multipotential cells contain both Pu1 and C-EBP alpha as enhancer-binding activities. Pu1 is unphosphorylated in both multipotential and granulocyte-committed cells but is phosphorylated in B lymphocytes, raising the possibility that differential phosphorylation may play a role in specifying its lymphoid versus myeloid functions. C-EBP alpha exists as multiple phosphorylated forms in the nucleus of both multipotential and granulocyte-committed cells. C-EBP beta is unphosphorylated and cytoplasmically localized in multipotential cells but exists as a phosphorylated nuclear enhancer-binding activity in granulocyte-committed cells. Granulocyte colony-stimulating factor-induced granulocytic differentiation of multipotential progenitor cells results in activation of C-EBP delta expression and functional recruitment of C-EBP delta and C-EBP beta to the nucleus. Our results implicate Pu1 and the C-EBP family as critical regulators of myeloperoxidase gene expression and are consistent with a model in which a temporal exchange of C-EBP isoforms at the myeloperoxidase enhancer mediates the transition from a primed state in multipotential cells to a transcriptionally active configuration in promyelocytes.

Detection of myeloperoxidase gene expression in minimally differentiated acute myelogenous leukemia (AML-M0) using in situ hybridization.

Acute leukemias containing > 3% myeloperoxidase (MPO)-positive blast cells, as detected cytochemically, are considered to be myelogenous in origin, regardless of the immunophenotypic markers expressed. Conversely, acute leukemias that express only myeloid antigens are also considered to be acute myelogenous leukemia (AML), even in the absence of MPO. These MPO-negative AMLs, designated AML-M0 in the FAB classification, currently require either immunophenotypic or electron microscopic studies for identification. To examine the association of MPO and myeloid antigen expression in AML, particularly at the early stages of myeloid cell differentiation, we have used in situ hybridization (ISH) to evaluate MPO gene expression in myeloid leukemia cell lines and a variety of well-characterized acute leukemias, including six cases of AML-M0. Strong positivity for MPO mRNA was detected in the myeloid leukemia cell line HL-60 and in 22 of 27 AMLs (three AML-M0, four AML-M1, eight AML-M2, five AML-M4, two AML-M5a). No MPO gene expression was detected in three AML-M0, one AML-M5a, one AML-M7, 5 acute lymphoblastic leukemia, the lymphoid cell lines Molt-4 and Namalwa, or in the early myeloid cell lines KG-1 and KG-1a. Ultrastructural studies for MPO activity were performed on four AML-M0; one leukemia showed both gene expression and cytochemical activity, whereas two others contained neither MPO transcripts nor enzyme. Weak MPO gene expression was evident in one AML-M0 that was negative for enzymatic activity by electron microscopy. These studies show MPO gene expression can be detected by ISH in about half of AML-M0, supporting their presumed myelocytic derivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Lineage determination of CD7+ CD5- CD2- and CD7+ CD5+ CD2- lymphoblasts: studies on phenotype, genotype, and gene expression of myeloperoxidase, CD3 epsilon, and CD3 delta.

The gene expression of myeloperoxidase (MPO), CD3 epsilon, and CD3 delta molecules, the gene rearrangement of T-cell receptor (TCR) delta, gamma, and beta and immunoglobulin heavy (IgH) chain, and the expression of cell-surface antigens were investigated in seven cases of CD7+ CD5- CD2- and four cases of CD7+ CD5+ CD2- acute lymphoblastic leukemia or lymphoblastic lymphoma (ALL/LBL) blasts, which were negative for cytochemical myeloperoxidase (cyMPO). More mature T-lineage blasts were also investigated in a comparative manner. In conclusion, the CD7+ CD5- CD2- blasts included four categories: undifferentiated blasts without lineage commitment, T-lineage blasts, T-/myeloid lineage blasts, and cyMPO-negative myeloblasts. The CD7+ CD5+ CD2- blasts included two categories; T-lineage and T-/myeloid lineage blasts. The 11 cases were of the germ-line gene (G) for TCR beta and IgH. Four cases were G for TCR delta and TCR gamma. The others were of the monoclonally rearranged gene (R) for TCR delta and G for TCR gamma or R for both TCR delta and TCR gamma. The expression or in vitro induction of CD13 and/or CD33 antigens correlated with the immaturity of these neoplastic T cells, since it was observed in all 11 CD7+ CD5- CD2- and CD7+ CD5+ CD2-, and some CD7+ CD5+ CD2+ (CD3- CD4- CD8-) cases, but not in CD3 +/- CD4+ CD8+ or CD3+ CD4+ CD8- cases. CD3 epsilon mRNA, but not CD3 delta mRNA, was detected in two CD7+ CD5- CD2- cases, while mRNA of neither of the two CD3 molecules was detected in the other tested CD7+ CD5- CD2- cases. In contrast, mRNA of both CD3 epsilon and CD3 delta were detected in all CD7+ CD5+ CD2- cases, indicating that CD7+ CD5- CD2- blasts at least belong to T-lineage. The blasts of two CD7+ CD5- CD2- cases with entire germ-line genes and without mRNA of the three molecules (MPO, CD3 epsilon, and CD3 delta) were regarded as being at an undifferentiated stage prior to their commitment to either T- or myeloid-lineage. The co-expression of the genes of MPO and CD3 epsilon in a CD7+ CD5- CD2- case MPO, CD3 epsilon, and CD3 delta in a CD7+ CD5+ CD2- case suggested the presence of some overlapping phase for T- and myeloid-lineage commitment during immature stages of differentiation. This helps understand the conversion of some T-ALL/LBL cases to acute myeloblastic leukemia (AML).(ABSTRACT TRUNCATED AT 400 WORDS)

Growth and differentiation signals mediated by different regions in the cytoplasmic domain of granulocyte colony-stimulating factor receptor

Granulocyte colony-stimulating factor (G-CSF) is a cytokine that regulates the proliferation and differentiation of neutrophils. The G-CSF receptor (G-CSFR) is a member of the hemopoietic growth factor receptor family. A G-CSFR expression plasmid was introduced into interleukin-3 (IL-3)-dependent mouse myeloid precursor FDC-P1 cells that normally do not respond to G-CSF. G-CSF stimulated proliferation of the transformants, down-regulated Thy-1 and F4 80 antigens on the cell surface, and induced expression of neutrophil-specific genes such as myeloperoxidase (MPO) and leukocyte elastase. On the other hand, neither granulocyte/macrophage colony-stimulating factor (GM-CSF) nor IL-3 induced MPO gene expression, but they inhibited G-CSFR-mediated MPO gene expression. These results suggested that the G-CSFR, but not the IL-3/GM-CSF receptors, transduced the neutrophilic differentiation signal into cells. Mutational analysis of the G-CSFR indicated that the N-terminal region of its cytoplasmic domain is sufficient to transduce the proliferation signal into cells, while the C-terminal region plays an essential role in transducing the differentiation signal.

Changes of DNA methylation and chromatin structure in the human myeloperoxidase gene during myeloid differentiation

Expression of the myeloperoxidase (MPO) gene is tightly regulated in a tissue- and development-specific manner. Accumulation of MPO messenger RNA (mRNA) occurs only at the late myeloblastic and promyelocytic stages of myeloid differentiation and is negligible at other stages of myeloid development and in other tissues. The goal of our studies was to begin to understand the events that occur to control MPO gene expression during normal granulocytopoiesis. Chromatin structure of the MPO gene was evaluated by DNase I treatment of isolated nuclei and Southern blot analysis. No detectable DNase I hypersensitive sites were found in the region of the MPO gene in non-myeloid cells. One site was present in the 5′ upstream region in myeloid cells that are developmentally too immature to transcribe MPO. Three sites of hypersensitivity in the regions of the putative MPO promoter and upstream region occurred in MPO-expressing promyelocytes. These sites were markedly reduced in terminally differentiated, non-expressing myeloid cells. Analysis of DNA methylation of the MPO gene using methylation-sensitive restriction enzymes showed that the gene was highly methylated in non-myeloid cells. Stepwise demethylation occurred in myeloid cells developmentally too immature to transcribe MPO. Maximal demethylation in the 5′ gene region occurred in MPO-expressing promyelocytes. This methylation pattern did not change in terminally differentiated, MPO non-expressing myeloid cells. A somatic hybrid cell formed by fusion of HL-60 (MPO-expressing cells) and PUT (MPO non- expressing lymphoid cells) extinguished expression of MPO and showed a chimeric pattern of MPO gene methylation, suggesting that demethylation is necessary but not sufficient for expression of the MPO gene. Our studies show that demethylation and DNase I hypersensitivity of the MPO gene were associated with a tissue-dependent potential for MPO gene expression that preceded the developmental ability to express MPO mRNA.