- Email: [email protected]
EBP and C-Myb sites are important for the functional activity of the human myeloperoxidase upstream enhancer
Biochemical and Biophysical Research Communications 371 (2008) 309–314
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
C/EBP and C-Myb sites are important for the functional activity of the human myeloperoxidase upstream enhancer Congjun Yao a,b, Zhenyu Qin c,1, Kimberly N. Works a, Garth E. Austin a,b,2, Andrew N. Young a,b,* a
Pathology and Laboratory Medicine Service, Veterans Affairs Medical Center, Decatur, GA 30033, USA Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA c Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA b
a r t i c l e
i n f o
Article history: Received 8 April 2008 Available online 22 April 2008
Keywords: Myeloperoxidase Enhancer Promoter C/EBP c-Myb
a b s t r a c t Myeloperoxidase (MPO), an enzyme active against bacterial and fungal infections, is expressed specifically in myeloblasts and promyelocytes and minimal in other cell types. We recently identified and partially characterized an upstream enhancer located between 4100 and 3844 bp of the MPO gene. We showed that an AML1 site contributes to enhancer activity and specificity. We now demonstrate three additional footprints within the MPO enhancer and provide evidence that C/EBP and c-Myb sites contribute to its functional, tissue-specific activity. This distal enhancer appears to play an important role in the control of MPO transcription during differentiation of myeloid cells. Ó 2008 Elsevier Inc. All rights reserved.
Tissue-specific and maturation stage-specific gene expression in myeloid cells are dependent in large part upon the specific interactions of transcription factors with particular cis-elements within or near the promoter regions of the genes whose expression they control. We have been examining the mechanisms responsible for tissue-specific gene expression in immature myeloid cells by determining the regulation of myeloperoxidase (MPO) gene expression during differentiation of granulocytes and monocytes. MPO (donor: H2O2 oxidoreductase, EC1.11.1.7) is an enzyme which plays an important role in the defense against bacteria and fungi [1]. Since the MPO gene is transcribed only during the myeloblast and promyelocyte stages of myeloid maturation and MPO is expressed in myeloid cells but generally minimal in lymphoid cells, MPO enzyme activity has been widely used in diagnosis and classification of acute leukemias [2]. We previously identified and characterized a basal promoter in the 50 -flanking region of the human MPO gene, lying between 128 and +11 bp. This promoter appears to be responsible for initiation of most full length MPO transcripts [3]. We also described a series of cis-elements in the proximal 1 kb of 50 -flank* Corresponding author. Address: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA. Fax: +1 404 6169913. E-mail address: [email protected] (A.N. Young). 1 Present address:Department of Medicine, College of Medicine, University of Cincinnati, Cincinnati, OH 45267, USA. 2 Deceased. 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.04.065
ing MPO gene which appear to modulate the function of this basal promoter [4,5]. More recently, we described two additional human MPO promoters (P2 and P3) lying within the proximal 1 kb of DNA directly upstream from the major (P1) MPO promoter [6], although the P2 and P3 promoters may play only a minor role in the physiologic, tissue-specific transcription of the human MPO gene [7]. The possible existence of an upstream enhancer in the MPO gene was first suggested by early studies of the chromatin structure of the MPO gene in HL-60 cells by Lubbert et al. [8] and Jorgenson et al. [9]. These studies demonstrated the existence of a DNase I hypersensitive site in the human MPO gene approximately 4 kb upstream from the transcription start site. The DNase I hypersensitivity of this site was reduced following induction of terminal differentiation of these cells by DMSO or 1,25-dihydroxyvitamin D3, suggesting that this region might contain regulatory elements involved in the control of MPO gene expression. In response to these studies, we investigated an MPO enhancer, and reported its identification and partial characterization at this location in the human MPO gene, approximately 4000 bp upstream from the transcription initiation site [10]. We showed that this enhancer increases the activity of the minimal human MPO P1 promoter in leukemic cells and is involved in the decrease in MPO promoter activity in HL-60 cells after treated with chemical inducers of myeloid maturation. We also demonstrated that an AML1 binding site contributes substantially to the activity of the upstream enhancer. However, it is unknown whether other binding sites in the human
310
C. Yao et al. / Biochemical and Biophysical Research Communications 371 (2008) 309–314
upstream MPO enhancer could contribute to its activity and specificity. We now report results of further analysis of the human MPO upstream enhancer. We demonstrate the existence of three additional footprints within the upstream enhancer, and we show that C/EBP and c-Myb sites within one of these footprints and a second AML1 site within another footprint contribute to the tissue-specific and maturation stage-specific activity of the human MPO upstream enhancer. Materials and methods Cells. The human cell lines HL-60 (myeloblastic), K562 (erythroleukemic), U937 (histiocytic), and HeLa (squamous carcinoma) were cultured as previously described [7]. Construction of luciferase reporter plasmids containing normal or mutated segments of the 50 -flanking sequence of the human MPO gene. To prepare luciferase reporter plasmids containing portions of MPO promoter DNA, selected segments of the MPO gene were amplified using human genomic DNA as template. Promoter segments were inserted into the multiple cloning site of pGL3-Basic or phRG-Basic vectors (Promega). Enhancer segments were amplified by PCR and inserted into the TA cloning vector pCR2.1 (Invitrogen). The segments were digested and inserted into upstream from the MPO ‘‘minimal” promoter in the Basic vectors. Site directed mutants of the upstream enhancer were prepared using the Altered SitesÒ II in vitro mutagenesis system (Promega) as described previously [10]. The selection of sites to mutate was guided by DNase I footprinting and gel shift experiments and by computerized analysis of DNA motifs and of sites of sequence homology between human and murine upstream enhancers. The enhancer DNA fragments containing mutated sites were restricted by EcoRI and ligated to the pGL3-128 plasmid vector at its EcoRI site. Dideoxy terminator sequencing was performed to check the correct sequence of all mutations. Transient transfection by electroporation or using Lipofectamine reagentÒ. Leukemic cell lines were transfected by electroporation as previously described [3,4], using a Gene Pulser System (BioRad, Richmond, CA), at 250 V, 960 lF, and Tau of 39 ms. HeLa cells were transfected using Lipofectamine Reagent (Gibco BRL). To normalize for efficiency of transfection, cells were co-transfected with plasmid pSV-b-galactosidase control vector (Promega) [11]. After overnight incubation, luciferase activity was determined using a luciferase assay kit (Promega) with a ML3000 Microtiter Plate Luminometer (Dynatech Laboratories, Chantilly, VA). Results were expressed as relative light units (RLU) luciferase activity per OD420 bgalactosidase. DNase I footprinting. DNA probes for footprinting studies were prepared by restriction digestion of MPO promoter plasmids so as to create 50 -overhangs at one end and 30 -overhangs at the other end. Probes were labeled with 32 P by fill-
in reaction at the site of the 50 -overhangs using avian myeloblastosis virus reverse transcriptase. DNase I footprinting was performed according to the method of Henninghausen and Lubon [12]. Mobility shift assays. Complementary oligonucleotides with 50 -overhangs for mobility shift assays were synthesized and annealed. The double-stranded DNA segments were then labeled with [a-32P]dCTP (7500 Ci/mmol) by a fill-in reaction with DNA polymerase 1, Klenow fragment. The incorporated label was purified using Sephadex G50 spin columns. Nuclear extracts of cultured cells were prepared essentially according to the method of Dignam et al. [13]. Gel shift assays were carried out as described by Ausubel et al. [14]. In competition experiments 100-fold molar excess of cold competitor was added. DNA-protein complexes were visualized on 6% nondenaturing polyacrylamide gels. Supershift experiments were performed by preincubating the oligonucleotides with appropriate antibodies prior to electrophoresis.
Results A highly sensitive reporter system confirmed the activity of the MPO enhancer in myeloid cells Our initial studies of enhancer activity employed a relatively insensitive reporter system which resulted in rather low reporter activities, particularly in cell lines such as HL-60 which are relatively difficult to transfect. We have now repeated these observations using an improved transfection protocol, a more sensitive transfection system, a shorter basal MPO promoter (270 to +15 bp), and a more easily transfected cell line, U937. Fig. 1 illustrates representative data showing that a segment of the 50 -flanking region of the MPO gene, between 4100 and 3844 bp, enhances the activity of a 285 bp minimal MPO promoter regardless of its orientation in U937 cells. DNase I footprinting reveals three additional footprints within the MPO upstream enhancer In our previous communication, we described a footprint site (now termed as ‘‘F4”) at the 50 -end of the MPO enhancer region, between 4100 and 4040 bp, and showed that an AML1 site is the principal active element located within that footprint. To detect and localize other possible elements within the upstream enhancer
Relative Luc expression 0
5
10 15
20
25
U937 cell only phRG-Basic
LUC
phRG-TK
LUC
phRG/MPO(-270/+15)
LUC
phRG/MPO(-3844/+15) LUC
phRG/MPO(-4100/+15) LUC
phRG/MPO(-4100/-3844, -270/+15) LUC
phRG/MPO(-3844/-4100, -270/+15) LUC
Untreated TPA Treated Fig. 1. Identification of promoter activity in an upstream enhancer spanning between 4100 and 3844 of the human MPO gene. Left panel shows schematic representation of the structures of the 50 -flanking region of human MPO gene constructs. Luciferase reporter plasmids containing the indicated portions of the human MPO gene inserted into the pGL-3 Basic vector were assayed in transiently transfected cells. Luciferase activities represent mean ± SE for five experiments. The negative control plasmid (phRG-Basic) lacks a promoter; the positive control plasmid (phRG-TK) contains herpes simplex virus thymidine kinase (HSV-TK) promoter. U937 cell was treated by TPA for 24 h before measurement of luciferase activities.
C. Yao et al. / Biochemical and Biophysical Research Communications 371 (2008) 309–314
which contribute to its activity, a series of additional DNase I footprinting experiments were carried out using segments of enhancer DNA and nuclear extracts of myeloid cell lines HL-60 and K562. Fig. 2(A–C) depicts autoradiograms demonstrating three additional footprints within the upstream enhancer DNA segment between 4100 and 3844 bp. The first of these footprints (termed as ‘‘F1”) lies between 3864 and 3844 bp (Fig. 2A). The second footprint (termed as ‘‘F2”) lies between 3914 and 3892 bp (Fig. 2B), while the third footprint (termed as ‘‘F3”) lies between 3998 and 3942 bp (Fig. 2C). Footprints F1 and F2 appear similar in size and position using either nuclear extracts of HL-60 cells (which express MPO RNA) or nuclear extracts of K562 cells (in which the MPO gene is minimally transcribed). On the other hand, the pattern of the F3 footprint differs according to the source of the nuclear extract employed. When nuclear extracts derived from HL-60 cells are used, a single footprint spanning the entire F3 region is seen, whereas when extracts of K562 cells are used, two smaller, discrete footprints are seen, F3A (3972 to 3938 bp) and F3B (3998 to 3976 bp) (Fig. 2D). The difference in footprint F3 made with HL-60 nuclear extracts and that made with K562 nuclear extracts suggests a correlation between patterns of protein binding to the F3 site and physiologic enhancement of MPO transcription. Furthermore, the presence of three newly defined footprints within the enhancer DNA segment suggests that each of these sites may be important for the regulation or enhancement of MPO transcription. The F1 footprint contains sequences essential for enhancer function which bind transcription factors c-Myb and C/EBP Gel mobility shift assays were performed to characterize the specific DNA-protein interactions responsible for the footprints noted above. We focused our studies on sites F1 and F2, since in our previous communication [10], we had shown by transfection studies using deletion mutants that footprints F1 and F2 lie within regions that are essential for full enhancer activity. To investigate the F1 site, a 29 bp probe with the sequence 50 -ATAGTCTTGAGATGTAGCAGTCCCAGCAG-30 (corresponding to 3868 to 3840 bp and encompassing the entire F1 footprint) was employed. The F1 segment contains (in its sense strand at bases 3847 to 3851) the sequence GACTG which shows homology with a c-Myb consensus sequence (AACTG). In addition, it includes the sequence TTGAGATGTAGCAGT which resembles a consensus C/EBP site (T[T/G]NNGNAA[G/T]) plus a half site ([G/C]AAT). Fig. 3A illustrates a gel shift experiment demonstrating the retarded bands produced by incubating the F1 probe with nuclear extracts prepared from HL-60 or K562 cells. Incubation of the F1 oligonucleotide with HL-60 nuclear extract produces two retarded bands, which are competed by an excess of the homologous probe, but not by a heterologous oligonucleotide corresponding to a GABP site. Preincubation of the nuclear extract with a probe mutated from 3853 to 3846 bp, including the c-Myb site (GCAGTCCC replaced by ATATTTCT), produces little competition of the retarded bands. This suggests that c-Myb is involved in binding to this sequence. By contrast with the results obtained using HL-60 nuclear extracts, incubation of the F1 probe with nuclear extracts prepared from K562 cells produces only a single major retarded band. This band is competable by the homologous oligonucleotide, but not by the probe mutated at the c-Myb site or by the heterologous oligonucleotide corresponding to the GABP site (Fig. 3A). The 8-bp sequence (GCAGTCCC) within the F1 site appears to be involved in the binding of c-Myb and perhaps other nuclear proteins. To determine if c-Myb protein does, in fact, bind to the F1 segment of enhancer DNA, we screened this oligonucleotide by gel shift and supershift experiments using antibodies to c-Myb and to a number of other transcription factors, looking for proteins
311
which form functionally important complexes with F1 DNA. Fig. 3B (top panel) shows that the single retarded band produced by K562 nuclear extracts is eliminated by an antibody directed against cMyb but not by other antibodies such as anti-C/EBPb and antiAML1 as illustrated in same figure. In addition, as shown in Fig. 3B (bottom panel), the upper (higher molecular weight) of the two bands seen with HL-60 nuclear extracts is also partially eliminated by the antibody against c-Myb. These results strongly suggest that c-Myb is one of the proteins which bind to the F1 site. To determine if this c-Myb sequence is critical for enhancer activity, a pGL3 plasmid containing the entire distal enhancer with a mutation at this 8-bp c-Myb site, linked to a minimal 128 bp MPO promoter and a luciferase reporter, was created by site-directed mutagenesis. Table 1 illustrates data comparing the activity of the basal promoter and the intact enhancer with a number of distal enhancer mutants. Mutation of the c-Myb site of the enhancer (plasmid pGL3-128-Enh-MutF1-Myb) eliminates enhancer function in HL-60 cells or K562 cells almost completely, while reducing the activity in HeLa cells by 55%. These results strongly suggest that the c-Myb sequence within the F1 site is critically important for enhancer activity. Since the F1 oligonucleotide also contains a sequence which closely resembles a consensus C/EBP site, we wished to determine if a member of the C/EBP family of transcription factors binds to the F1 site and enhances MPO activity. An antibody raised against C/EBP b and cross-reactive against C/EBP a, C/EBP d, and CRP1, was used to determine whether the supershift to the more rapidly retarded band can be observed when F1 is incubated with HL-60 nuclear extracts. Fig. 3B (bottom panel) demonstrates that preincubation of the extract with this antibody eliminates this more rapidly migrating band and produces the expected supershifted band, suggesting that a member of the C/EBP family of transcription factors binds to this sequence. Since the supershifted band, while reproducible, is quite weak, it is possible that the DNA-protein interaction at this site involves a C/EBP family member distinct from C/EBP a, b, or d. These experiments are specific, because gel shift experiments using an F1 oligonucleotide mutated at this site show an absence of the C/EBP band (Fig. 3C), confirming that this is the site within F1 to which C/EBP binds. To determine if C/EBP binding to the F1 site also contributes to enhancer activity, a plasmid in which the enhancer was mutated in the C/EBP-like sequence was prepared and its activity compared with that of the wild-type enhancer. As shown in Table 1 this enhancer mutant (pGL3-128-Enh-MutF1-C/EBP) showed 70% reduction in activity in HL-60 cells and small reductions in activity in minimal MPO-expressing cells such as HeLa and K562. Also, gel shift experiments using an oligonucleotide mutated at the C/EBP sequence, show an absence of the C/EBP band, confirming that this is the site within F1 to which C/EBP binds (Fig. 3C). Site F2 binds nuclear proteins of leukemic cells and contributes to enhancer activity The 24 bp probe for site F2 has the sequence 50 -GTGGGGTG TGAGTGTGTTGGAGGC-30 . This sequence contains three adjacent, slightly overlapping AML1 consensus-like sequences (3913 to 3897 bp). Incubation of this probe with nuclear extracts prepared from K562 cells produces two major, slowly migrating retarded bands and one, more rapidly migrating retarded band. Preincubation of the F2 oligonucleotide with an antibody against AML1a results in total elimination of the most slowly migrating of the retarded bands and reduces the intensity of the middle band (Fig. 3D), suggesting that AML1 binds to this site. Preincubation with antibodies against other transcription factors fails to eliminate these bands. Comparable results are observed with HL-60 cells (data not shown).
312
C. Yao et al. / Biochemical and Biophysical Research Communications 371 (2008) 309–314
Fig. 2. DNase I footprinting experiments showing the four major nuclear protein binding sites located within the upstream enhancer region. (A) Autoradiogram of acrylamide gel showing location of footprint F1. Aliquots of 22,000 cpm (20 fmol) 32P-end-labeled DNA probe containing 3800 to 3900 bp of MPO DNA (in the presence or absence of 5 mg HL-60 nuclear extract) were digested for 2 min at room temperature with DNase I (0.01 U for probe alone, 2.5 U for probe plus nuclear extract), and analyzed on a 6% polyacrylamide sequencing gel. (B) Autoradiogram of gel showing the location of footprint F2. Aliquots of a 32P-end-labeled DNA containing the MPO DNA segment from 3850 to 3950 bp were incubated in the presence or absence of nuclear extract of K562 cells, treated with various levels of DNase I, and then analyzed on a 6% polyacrylamide sequencing gel. The four lanes on the left indicate the sequence of this part of the enhancer region. (C) Autoradiogram of gel showing the location of footprint F3 obtained using HL-60 nuclear extract. Note that with HL-60 nuclear extracts F3 appears as a single region. (D) Autoradiogram of gel showing that with K562 nuclear extracts the F3 footprint is split into two smaller footprints, labeled F3A and F3B. This difference may relate to the fact that the MPO gene is actively transcribed in HL-60 cells whereas in K562 cells it is not.
313
C. Yao et al. / Biochemical and Biophysical Research Communications 371 (2008) 309–314
Fig. 3. (A) Gel shift with F1 probe showing the patterns of retarded bands produced by incubating this probe with nuclear extract of HL-60 or K562 cells. The retarded bands are competed by the homologous probe but much less so by a probe mutated at the c-Myb site. Heterologous probes do not compete the bands. (B) Gel shift experiments showing the effect of preincubation of the F1 probe with an antibody against c-Myb upon retarded bands produced by extracts of myeloid cells. Top panel: Elimination of the retarded band seen with k562 nuclear extracts. Bottom panel: Reduction in the upper band seen with HL-60 nuclear extracts. (C) Gel shift experiments with F1 probe showing binding of HL-60 nuclear extracts to probe corresponding to C/EBP site. The retarded bands are competed by the homologous probe and C/EBP consensus probe but not by the mutant probe or by a heterologous probe. showing that an antibody against c-Myb supershifts a retarded band produced by HL-60 extract. (D) Gel shift experiments showing binding of K562 nuclear extracts to a probe corresponding to probe F2. Supershift experiment showing that an antibody against AML1 supershifts a retarded band produced by nuclear extract.
Table 1 Site-directed mutation analysis of the human distal MPO enhancer Plasmida
pGL3-128 pGL3-128-Enh-Wild Type pGL3-128-Enh-MutF1-MYB pGL3-128-Enh-MutF1-C/EBP pGL3-128-Enh-MutF2-AML1 pGL3-128-Enh-MutF3A pGL3-128-Enh-MutF3B pGL3-Basic (no promoter) a b c
Site of mutation in MPO gene (bp)
(3853 (3862 (3908 (3966 (3989
to to to to to
3846) 3855) 3903) 3961) 3984)
Base substitution
GCAGTCCC-ATATTTCA TTGAGATG-GGAGCCTA TGTGAG-AAGCTT CACCCT-AAGCTT TTATCC-AAGCTT
Promoter activity (Luciferase u/b-gal u) HeLa
K562
135 ± 26b 348 ± 41b(100%)c 230 ± 13 (45%) 311 ± 25 (91%) 414 ± 16 (131%) 442 ± 39 (144%) 358 ± 61 (105%) 6±2
138 ± 32 340 ± 34 137 ± 30 238 ± 41 293 ± 21 348 ± 48 325 ± 31 3±1
HL-60 (100%) (0%) (70%) (71%) (105%) (91%)
13 ± 4 189 ± 23 (100%) 41 ± 7 (16%) 57 ± 10 (30%) 78 ± 13 (37%) 162 ± 22 (85%) 201 ± 33 (107%) 2±1
Constructed from pGL3 plasmids (Promega). Mean ± SD (n = 5). Enhancer activity (Percent of wild type).
The functional promoter activity of a pGL3 plasmid containing a 139 bp MPO promoter linked to an enhancer with a mutation in the middle of the AML1 consensus region of the F2 site, 3908 to 3903 bp (TGTGAG ? AAGCTT), was compared with that of a plasmid containing the wild-type enhancer (Table 1). This mutation reduces enhancer activity in the MPO-producing cell line
HL-60 by 63% but does not reduce activity in HeLa cells and reduces activity only marginally in K562 cells, suggesting that AML1 binding to this site contributes to enhancer activity in a tissue-specific manner. Screening the F2 oligonucleotide with a variety of other transcription factors failed to identify the additional proteins which bind to this site.
314
C. Yao et al. / Biochemical and Biophysical Research Communications 371 (2008) 309–314
Site F3 contains two protein binding sites but lacks functional enhancing activity Enhancer site F3 contains two distinct regions which bind to HL-60 nuclear extracts. Oligonucleotides corresponding to these two footprints were prepared and shown to bind to HL-60 nuclear proteins in gel shift experiments (data not shown). However, mutation of the F3 site failed to significantly affect enhancer activity. Representative examples are shown in Table 1. While it remains possible that further work might reveal a functionally important element within F3, this footprint was not further examined in the present study.
patterns formed using HL-60 nuclear extracts and those seen with K562 or HeLa nuclear extracts may also reflect differences in the functional activity of the human MPO enhancer in MPO-expressing and minimal MPO-expressing cells. Patterns of nuclear protein binding to sites F1, F3, and F4 in gel shift experiments all show differences between MPO-expressing cells such as HL-60 and minimal MPO-expressing cells such as K562 or HeLa. We presume that these differences may relate in most cases to differences in enhancer activity among these cells. Surprisingly, although the F3 footprints differed in HL-60 and K562 cells, we were unable to demonstrate that F3 was important for enhancer activity. The importance of other individual differences in protein binding must remain a subject for further study.
Discussion References It is important to elucidate the factors which regulate tissuespecific and maturation stage-specific gene expression in developing myeloid cells. The MPO gene remains a useful model for studying the control of gene expression at the myeloblast and promyelocyte stages of myeloid maturation. We now describe additional features of the structural and functional aspects of the human upstream MPO enhancer. By DNase I footprinting studies we have demonstrated three additional footprints within the 365 bp enhancer segment. Mutation studies suggest that two of these footprints appear to be involved in enhancer activity. A C/ EBP site and a c-Myb site, as well as an additional AML1 site appear to be important elements within the enhancer. The transfection studies described in this communication suggest that the human upstream enhancer contributes significantly to the tissue- and maturation stage-specificity of MPO transcription. This enhancer stimulates the activity of a minimal human MPO promoter by a greater extent in HL-60 cells (which express MPO) than in non-leukemic cells or leukemic cells which only minimally express MPO RNA or protein. Furthermore, treatment of HL60 cells with chemical inducers of myeloid maturation such as TPA or DMSO, which result in the arrest of MPO transcription in vitro, virtually eliminates enhancer-stimulated MPO promoter activity by 24 h after induction [10]. This suggests that the activity of this enhancer is dependent upon the maturational stage of the leukemic cells. Interestingly, as we showed in our previous communication [10], the MPO enhancer can also confer TPA- or DMSOresponsiveness upon a heterologous SV40 promoter, providing further evidence that enhancer activity is maturation stage-specific. (By contrast, promoter activity stimulated by an SV40 enhancer increased rather than decreased following TPA or DMSO treatment, and hence did not mimic the effects that these agents have on MPO expression in vitro.) In addition, the differences of gel shift
[1] S.J. Klebanoff, Myeloperoxidase—occurrence and biological function, in: J. Everse, M. Grisham (Eds.), Peroxidases in Chemistry and Biology, CRC Press, Boca Raton, FL, 1990, pp. 180–192. [2] J.M. Bennett, D. Katowski, M.T. Daniel, G. Flandrin, D.A. Galton, H.R. Gralnick, C. Sultan, Proposed revised criteria for the classification of acute myeloid leukemia: a report of the French-American-British Cooperative Group, Ann. Intern. Med. 103 (1985) 620–624. [3] G.E. Austin, W.-G. Zhao, W. Zhang, E.D. Austin, H.W. Findley, J.J. Murtagh Jr., Identification and characterization of the human myeloperoxidase promoter, Leukemia 7 (1995) 848–857. [4] W.-G. Zhao, A. Regmi, E.D. Austin, J.E. Braun, M. Racine, G.E. Austin, Ciselements in the promoter region of the human myeloperoxidase gene, Leukemia 10 (1996) 1089–1103. [5] W.-G. Zhao, J.-P. Lu, G.E. Austin, Enhancement of myeloperoxidase promoter function by PEBP2/CBF or inhibition by an Sp1-containing repressor requires the participation of a third element, DP4, Blood 88 (Suppl. 1) (1996) 47a. [6] W.-G. Zhao, A. Regmi, J.-P. Lu, G.E. Austin, Identification and functional analysis of multiple murine myeloperoxidase (MPO) promoters and comparison with the human MPO promoter region, Leukemia 11 (1997) 97–105. [7] K.-W.M. Lin, G.E. Austin, Functional activity of three distinct myeloperoxidase (MPO) promoters in myeloid cells, Leukemia 16 (2002) 1143–1153. [8] M. Lübbert, C.W. Miller, H.P. Koeffler, Changes in DNA methylation and chromatin structure in the human MPO gene during myeloid differentiation, Blood 78 (1991) 345–356. [9] K.F. Jorgenson, G.R. Antoun, T.F. Zipf, Chromatin structural analysis of the 50 end and contiguous flanking region of the myeloperoxidase gene, Blood 77 (1991) 159–164. [10] G.E. Austin, W.-G. Zhao, A. Regmi, J.-P. Lu, J. Braun, Identification of an upstream enhancer containing an AML1 site in the human myeloperoxidase (MPO) gene, Leuk. Res. 22 (1998) 1037–1048. [11] K. Lim, C.B. Chae, A simple assay for DNA transfection by incubation of the cells in culture dishes with substrates for b-galactosidase, Biotechniques 7 (1989) 576–579. [12] L. Hennighausen, H. Lubon, Interaction of protein with DNA in vitro, Methods Enzymol. 152 (1987) 721–735. [13] J.D. Dignam, R.M. Labovitz, R.G. Roeder, Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei, Nucleic Acids Res. 11 (1983) 1481–1489. [14] F.M. Ausubel, R. Brent, R.E. Kingston, Current Protocols in Molecular Biology, Greene, New York, NY, 1988.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
C/EBP and C-Myb sites are important for the functional activity of the human myeloperoxidase upstream enhancer Congjun Yao a,b, Zhenyu Qin c,1, Kimberly N. Works a, Garth E. Austin a,b,2, Andrew N. Young a,b,* a
Pathology and Laboratory Medicine Service, Veterans Affairs Medical Center, Decatur, GA 30033, USA Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA c Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA b
a r t i c l e
i n f o
Article history: Received 8 April 2008 Available online 22 April 2008
Keywords: Myeloperoxidase Enhancer Promoter C/EBP c-Myb
a b s t r a c t Myeloperoxidase (MPO), an enzyme active against bacterial and fungal infections, is expressed specifically in myeloblasts and promyelocytes and minimal in other cell types. We recently identified and partially characterized an upstream enhancer located between 4100 and 3844 bp of the MPO gene. We showed that an AML1 site contributes to enhancer activity and specificity. We now demonstrate three additional footprints within the MPO enhancer and provide evidence that C/EBP and c-Myb sites contribute to its functional, tissue-specific activity. This distal enhancer appears to play an important role in the control of MPO transcription during differentiation of myeloid cells. Ó 2008 Elsevier Inc. All rights reserved.
Tissue-specific and maturation stage-specific gene expression in myeloid cells are dependent in large part upon the specific interactions of transcription factors with particular cis-elements within or near the promoter regions of the genes whose expression they control. We have been examining the mechanisms responsible for tissue-specific gene expression in immature myeloid cells by determining the regulation of myeloperoxidase (MPO) gene expression during differentiation of granulocytes and monocytes. MPO (donor: H2O2 oxidoreductase, EC1.11.1.7) is an enzyme which plays an important role in the defense against bacteria and fungi [1]. Since the MPO gene is transcribed only during the myeloblast and promyelocyte stages of myeloid maturation and MPO is expressed in myeloid cells but generally minimal in lymphoid cells, MPO enzyme activity has been widely used in diagnosis and classification of acute leukemias [2]. We previously identified and characterized a basal promoter in the 50 -flanking region of the human MPO gene, lying between 128 and +11 bp. This promoter appears to be responsible for initiation of most full length MPO transcripts [3]. We also described a series of cis-elements in the proximal 1 kb of 50 -flank* Corresponding author. Address: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA. Fax: +1 404 6169913. E-mail address: [email protected] (A.N. Young). 1 Present address:Department of Medicine, College of Medicine, University of Cincinnati, Cincinnati, OH 45267, USA. 2 Deceased. 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.04.065
ing MPO gene which appear to modulate the function of this basal promoter [4,5]. More recently, we described two additional human MPO promoters (P2 and P3) lying within the proximal 1 kb of DNA directly upstream from the major (P1) MPO promoter [6], although the P2 and P3 promoters may play only a minor role in the physiologic, tissue-specific transcription of the human MPO gene [7]. The possible existence of an upstream enhancer in the MPO gene was first suggested by early studies of the chromatin structure of the MPO gene in HL-60 cells by Lubbert et al. [8] and Jorgenson et al. [9]. These studies demonstrated the existence of a DNase I hypersensitive site in the human MPO gene approximately 4 kb upstream from the transcription start site. The DNase I hypersensitivity of this site was reduced following induction of terminal differentiation of these cells by DMSO or 1,25-dihydroxyvitamin D3, suggesting that this region might contain regulatory elements involved in the control of MPO gene expression. In response to these studies, we investigated an MPO enhancer, and reported its identification and partial characterization at this location in the human MPO gene, approximately 4000 bp upstream from the transcription initiation site [10]. We showed that this enhancer increases the activity of the minimal human MPO P1 promoter in leukemic cells and is involved in the decrease in MPO promoter activity in HL-60 cells after treated with chemical inducers of myeloid maturation. We also demonstrated that an AML1 binding site contributes substantially to the activity of the upstream enhancer. However, it is unknown whether other binding sites in the human
310
C. Yao et al. / Biochemical and Biophysical Research Communications 371 (2008) 309–314
upstream MPO enhancer could contribute to its activity and specificity. We now report results of further analysis of the human MPO upstream enhancer. We demonstrate the existence of three additional footprints within the upstream enhancer, and we show that C/EBP and c-Myb sites within one of these footprints and a second AML1 site within another footprint contribute to the tissue-specific and maturation stage-specific activity of the human MPO upstream enhancer. Materials and methods Cells. The human cell lines HL-60 (myeloblastic), K562 (erythroleukemic), U937 (histiocytic), and HeLa (squamous carcinoma) were cultured as previously described [7]. Construction of luciferase reporter plasmids containing normal or mutated segments of the 50 -flanking sequence of the human MPO gene. To prepare luciferase reporter plasmids containing portions of MPO promoter DNA, selected segments of the MPO gene were amplified using human genomic DNA as template. Promoter segments were inserted into the multiple cloning site of pGL3-Basic or phRG-Basic vectors (Promega). Enhancer segments were amplified by PCR and inserted into the TA cloning vector pCR2.1 (Invitrogen). The segments were digested and inserted into upstream from the MPO ‘‘minimal” promoter in the Basic vectors. Site directed mutants of the upstream enhancer were prepared using the Altered SitesÒ II in vitro mutagenesis system (Promega) as described previously [10]. The selection of sites to mutate was guided by DNase I footprinting and gel shift experiments and by computerized analysis of DNA motifs and of sites of sequence homology between human and murine upstream enhancers. The enhancer DNA fragments containing mutated sites were restricted by EcoRI and ligated to the pGL3-128 plasmid vector at its EcoRI site. Dideoxy terminator sequencing was performed to check the correct sequence of all mutations. Transient transfection by electroporation or using Lipofectamine reagentÒ. Leukemic cell lines were transfected by electroporation as previously described [3,4], using a Gene Pulser System (BioRad, Richmond, CA), at 250 V, 960 lF, and Tau of 39 ms. HeLa cells were transfected using Lipofectamine Reagent (Gibco BRL). To normalize for efficiency of transfection, cells were co-transfected with plasmid pSV-b-galactosidase control vector (Promega) [11]. After overnight incubation, luciferase activity was determined using a luciferase assay kit (Promega) with a ML3000 Microtiter Plate Luminometer (Dynatech Laboratories, Chantilly, VA). Results were expressed as relative light units (RLU) luciferase activity per OD420 bgalactosidase. DNase I footprinting. DNA probes for footprinting studies were prepared by restriction digestion of MPO promoter plasmids so as to create 50 -overhangs at one end and 30 -overhangs at the other end. Probes were labeled with 32 P by fill-
in reaction at the site of the 50 -overhangs using avian myeloblastosis virus reverse transcriptase. DNase I footprinting was performed according to the method of Henninghausen and Lubon [12]. Mobility shift assays. Complementary oligonucleotides with 50 -overhangs for mobility shift assays were synthesized and annealed. The double-stranded DNA segments were then labeled with [a-32P]dCTP (7500 Ci/mmol) by a fill-in reaction with DNA polymerase 1, Klenow fragment. The incorporated label was purified using Sephadex G50 spin columns. Nuclear extracts of cultured cells were prepared essentially according to the method of Dignam et al. [13]. Gel shift assays were carried out as described by Ausubel et al. [14]. In competition experiments 100-fold molar excess of cold competitor was added. DNA-protein complexes were visualized on 6% nondenaturing polyacrylamide gels. Supershift experiments were performed by preincubating the oligonucleotides with appropriate antibodies prior to electrophoresis.
Results A highly sensitive reporter system confirmed the activity of the MPO enhancer in myeloid cells Our initial studies of enhancer activity employed a relatively insensitive reporter system which resulted in rather low reporter activities, particularly in cell lines such as HL-60 which are relatively difficult to transfect. We have now repeated these observations using an improved transfection protocol, a more sensitive transfection system, a shorter basal MPO promoter (270 to +15 bp), and a more easily transfected cell line, U937. Fig. 1 illustrates representative data showing that a segment of the 50 -flanking region of the MPO gene, between 4100 and 3844 bp, enhances the activity of a 285 bp minimal MPO promoter regardless of its orientation in U937 cells. DNase I footprinting reveals three additional footprints within the MPO upstream enhancer In our previous communication, we described a footprint site (now termed as ‘‘F4”) at the 50 -end of the MPO enhancer region, between 4100 and 4040 bp, and showed that an AML1 site is the principal active element located within that footprint. To detect and localize other possible elements within the upstream enhancer
Relative Luc expression 0
5
10 15
20
25
U937 cell only phRG-Basic
LUC
phRG-TK
LUC
phRG/MPO(-270/+15)
LUC
phRG/MPO(-3844/+15) LUC
phRG/MPO(-4100/+15) LUC
phRG/MPO(-4100/-3844, -270/+15) LUC
phRG/MPO(-3844/-4100, -270/+15) LUC
Untreated TPA Treated Fig. 1. Identification of promoter activity in an upstream enhancer spanning between 4100 and 3844 of the human MPO gene. Left panel shows schematic representation of the structures of the 50 -flanking region of human MPO gene constructs. Luciferase reporter plasmids containing the indicated portions of the human MPO gene inserted into the pGL-3 Basic vector were assayed in transiently transfected cells. Luciferase activities represent mean ± SE for five experiments. The negative control plasmid (phRG-Basic) lacks a promoter; the positive control plasmid (phRG-TK) contains herpes simplex virus thymidine kinase (HSV-TK) promoter. U937 cell was treated by TPA for 24 h before measurement of luciferase activities.
C. Yao et al. / Biochemical and Biophysical Research Communications 371 (2008) 309–314
which contribute to its activity, a series of additional DNase I footprinting experiments were carried out using segments of enhancer DNA and nuclear extracts of myeloid cell lines HL-60 and K562. Fig. 2(A–C) depicts autoradiograms demonstrating three additional footprints within the upstream enhancer DNA segment between 4100 and 3844 bp. The first of these footprints (termed as ‘‘F1”) lies between 3864 and 3844 bp (Fig. 2A). The second footprint (termed as ‘‘F2”) lies between 3914 and 3892 bp (Fig. 2B), while the third footprint (termed as ‘‘F3”) lies between 3998 and 3942 bp (Fig. 2C). Footprints F1 and F2 appear similar in size and position using either nuclear extracts of HL-60 cells (which express MPO RNA) or nuclear extracts of K562 cells (in which the MPO gene is minimally transcribed). On the other hand, the pattern of the F3 footprint differs according to the source of the nuclear extract employed. When nuclear extracts derived from HL-60 cells are used, a single footprint spanning the entire F3 region is seen, whereas when extracts of K562 cells are used, two smaller, discrete footprints are seen, F3A (3972 to 3938 bp) and F3B (3998 to 3976 bp) (Fig. 2D). The difference in footprint F3 made with HL-60 nuclear extracts and that made with K562 nuclear extracts suggests a correlation between patterns of protein binding to the F3 site and physiologic enhancement of MPO transcription. Furthermore, the presence of three newly defined footprints within the enhancer DNA segment suggests that each of these sites may be important for the regulation or enhancement of MPO transcription. The F1 footprint contains sequences essential for enhancer function which bind transcription factors c-Myb and C/EBP Gel mobility shift assays were performed to characterize the specific DNA-protein interactions responsible for the footprints noted above. We focused our studies on sites F1 and F2, since in our previous communication [10], we had shown by transfection studies using deletion mutants that footprints F1 and F2 lie within regions that are essential for full enhancer activity. To investigate the F1 site, a 29 bp probe with the sequence 50 -ATAGTCTTGAGATGTAGCAGTCCCAGCAG-30 (corresponding to 3868 to 3840 bp and encompassing the entire F1 footprint) was employed. The F1 segment contains (in its sense strand at bases 3847 to 3851) the sequence GACTG which shows homology with a c-Myb consensus sequence (AACTG). In addition, it includes the sequence TTGAGATGTAGCAGT which resembles a consensus C/EBP site (T[T/G]NNGNAA[G/T]) plus a half site ([G/C]AAT). Fig. 3A illustrates a gel shift experiment demonstrating the retarded bands produced by incubating the F1 probe with nuclear extracts prepared from HL-60 or K562 cells. Incubation of the F1 oligonucleotide with HL-60 nuclear extract produces two retarded bands, which are competed by an excess of the homologous probe, but not by a heterologous oligonucleotide corresponding to a GABP site. Preincubation of the nuclear extract with a probe mutated from 3853 to 3846 bp, including the c-Myb site (GCAGTCCC replaced by ATATTTCT), produces little competition of the retarded bands. This suggests that c-Myb is involved in binding to this sequence. By contrast with the results obtained using HL-60 nuclear extracts, incubation of the F1 probe with nuclear extracts prepared from K562 cells produces only a single major retarded band. This band is competable by the homologous oligonucleotide, but not by the probe mutated at the c-Myb site or by the heterologous oligonucleotide corresponding to the GABP site (Fig. 3A). The 8-bp sequence (GCAGTCCC) within the F1 site appears to be involved in the binding of c-Myb and perhaps other nuclear proteins. To determine if c-Myb protein does, in fact, bind to the F1 segment of enhancer DNA, we screened this oligonucleotide by gel shift and supershift experiments using antibodies to c-Myb and to a number of other transcription factors, looking for proteins
311
which form functionally important complexes with F1 DNA. Fig. 3B (top panel) shows that the single retarded band produced by K562 nuclear extracts is eliminated by an antibody directed against cMyb but not by other antibodies such as anti-C/EBPb and antiAML1 as illustrated in same figure. In addition, as shown in Fig. 3B (bottom panel), the upper (higher molecular weight) of the two bands seen with HL-60 nuclear extracts is also partially eliminated by the antibody against c-Myb. These results strongly suggest that c-Myb is one of the proteins which bind to the F1 site. To determine if this c-Myb sequence is critical for enhancer activity, a pGL3 plasmid containing the entire distal enhancer with a mutation at this 8-bp c-Myb site, linked to a minimal 128 bp MPO promoter and a luciferase reporter, was created by site-directed mutagenesis. Table 1 illustrates data comparing the activity of the basal promoter and the intact enhancer with a number of distal enhancer mutants. Mutation of the c-Myb site of the enhancer (plasmid pGL3-128-Enh-MutF1-Myb) eliminates enhancer function in HL-60 cells or K562 cells almost completely, while reducing the activity in HeLa cells by 55%. These results strongly suggest that the c-Myb sequence within the F1 site is critically important for enhancer activity. Since the F1 oligonucleotide also contains a sequence which closely resembles a consensus C/EBP site, we wished to determine if a member of the C/EBP family of transcription factors binds to the F1 site and enhances MPO activity. An antibody raised against C/EBP b and cross-reactive against C/EBP a, C/EBP d, and CRP1, was used to determine whether the supershift to the more rapidly retarded band can be observed when F1 is incubated with HL-60 nuclear extracts. Fig. 3B (bottom panel) demonstrates that preincubation of the extract with this antibody eliminates this more rapidly migrating band and produces the expected supershifted band, suggesting that a member of the C/EBP family of transcription factors binds to this sequence. Since the supershifted band, while reproducible, is quite weak, it is possible that the DNA-protein interaction at this site involves a C/EBP family member distinct from C/EBP a, b, or d. These experiments are specific, because gel shift experiments using an F1 oligonucleotide mutated at this site show an absence of the C/EBP band (Fig. 3C), confirming that this is the site within F1 to which C/EBP binds. To determine if C/EBP binding to the F1 site also contributes to enhancer activity, a plasmid in which the enhancer was mutated in the C/EBP-like sequence was prepared and its activity compared with that of the wild-type enhancer. As shown in Table 1 this enhancer mutant (pGL3-128-Enh-MutF1-C/EBP) showed 70% reduction in activity in HL-60 cells and small reductions in activity in minimal MPO-expressing cells such as HeLa and K562. Also, gel shift experiments using an oligonucleotide mutated at the C/EBP sequence, show an absence of the C/EBP band, confirming that this is the site within F1 to which C/EBP binds (Fig. 3C). Site F2 binds nuclear proteins of leukemic cells and contributes to enhancer activity The 24 bp probe for site F2 has the sequence 50 -GTGGGGTG TGAGTGTGTTGGAGGC-30 . This sequence contains three adjacent, slightly overlapping AML1 consensus-like sequences (3913 to 3897 bp). Incubation of this probe with nuclear extracts prepared from K562 cells produces two major, slowly migrating retarded bands and one, more rapidly migrating retarded band. Preincubation of the F2 oligonucleotide with an antibody against AML1a results in total elimination of the most slowly migrating of the retarded bands and reduces the intensity of the middle band (Fig. 3D), suggesting that AML1 binds to this site. Preincubation with antibodies against other transcription factors fails to eliminate these bands. Comparable results are observed with HL-60 cells (data not shown).
312
C. Yao et al. / Biochemical and Biophysical Research Communications 371 (2008) 309–314
Fig. 2. DNase I footprinting experiments showing the four major nuclear protein binding sites located within the upstream enhancer region. (A) Autoradiogram of acrylamide gel showing location of footprint F1. Aliquots of 22,000 cpm (20 fmol) 32P-end-labeled DNA probe containing 3800 to 3900 bp of MPO DNA (in the presence or absence of 5 mg HL-60 nuclear extract) were digested for 2 min at room temperature with DNase I (0.01 U for probe alone, 2.5 U for probe plus nuclear extract), and analyzed on a 6% polyacrylamide sequencing gel. (B) Autoradiogram of gel showing the location of footprint F2. Aliquots of a 32P-end-labeled DNA containing the MPO DNA segment from 3850 to 3950 bp were incubated in the presence or absence of nuclear extract of K562 cells, treated with various levels of DNase I, and then analyzed on a 6% polyacrylamide sequencing gel. The four lanes on the left indicate the sequence of this part of the enhancer region. (C) Autoradiogram of gel showing the location of footprint F3 obtained using HL-60 nuclear extract. Note that with HL-60 nuclear extracts F3 appears as a single region. (D) Autoradiogram of gel showing that with K562 nuclear extracts the F3 footprint is split into two smaller footprints, labeled F3A and F3B. This difference may relate to the fact that the MPO gene is actively transcribed in HL-60 cells whereas in K562 cells it is not.
313
C. Yao et al. / Biochemical and Biophysical Research Communications 371 (2008) 309–314
Fig. 3. (A) Gel shift with F1 probe showing the patterns of retarded bands produced by incubating this probe with nuclear extract of HL-60 or K562 cells. The retarded bands are competed by the homologous probe but much less so by a probe mutated at the c-Myb site. Heterologous probes do not compete the bands. (B) Gel shift experiments showing the effect of preincubation of the F1 probe with an antibody against c-Myb upon retarded bands produced by extracts of myeloid cells. Top panel: Elimination of the retarded band seen with k562 nuclear extracts. Bottom panel: Reduction in the upper band seen with HL-60 nuclear extracts. (C) Gel shift experiments with F1 probe showing binding of HL-60 nuclear extracts to probe corresponding to C/EBP site. The retarded bands are competed by the homologous probe and C/EBP consensus probe but not by the mutant probe or by a heterologous probe. showing that an antibody against c-Myb supershifts a retarded band produced by HL-60 extract. (D) Gel shift experiments showing binding of K562 nuclear extracts to a probe corresponding to probe F2. Supershift experiment showing that an antibody against AML1 supershifts a retarded band produced by nuclear extract.
Table 1 Site-directed mutation analysis of the human distal MPO enhancer Plasmida
pGL3-128 pGL3-128-Enh-Wild Type pGL3-128-Enh-MutF1-MYB pGL3-128-Enh-MutF1-C/EBP pGL3-128-Enh-MutF2-AML1 pGL3-128-Enh-MutF3A pGL3-128-Enh-MutF3B pGL3-Basic (no promoter) a b c
Site of mutation in MPO gene (bp)
(3853 (3862 (3908 (3966 (3989
to to to to to
3846) 3855) 3903) 3961) 3984)
Base substitution
GCAGTCCC-ATATTTCA TTGAGATG-GGAGCCTA TGTGAG-AAGCTT CACCCT-AAGCTT TTATCC-AAGCTT
Promoter activity (Luciferase u/b-gal u) HeLa
K562
135 ± 26b 348 ± 41b(100%)c 230 ± 13 (45%) 311 ± 25 (91%) 414 ± 16 (131%) 442 ± 39 (144%) 358 ± 61 (105%) 6±2
138 ± 32 340 ± 34 137 ± 30 238 ± 41 293 ± 21 348 ± 48 325 ± 31 3±1
HL-60 (100%) (0%) (70%) (71%) (105%) (91%)
13 ± 4 189 ± 23 (100%) 41 ± 7 (16%) 57 ± 10 (30%) 78 ± 13 (37%) 162 ± 22 (85%) 201 ± 33 (107%) 2±1
Constructed from pGL3 plasmids (Promega). Mean ± SD (n = 5). Enhancer activity (Percent of wild type).
The functional promoter activity of a pGL3 plasmid containing a 139 bp MPO promoter linked to an enhancer with a mutation in the middle of the AML1 consensus region of the F2 site, 3908 to 3903 bp (TGTGAG ? AAGCTT), was compared with that of a plasmid containing the wild-type enhancer (Table 1). This mutation reduces enhancer activity in the MPO-producing cell line
HL-60 by 63% but does not reduce activity in HeLa cells and reduces activity only marginally in K562 cells, suggesting that AML1 binding to this site contributes to enhancer activity in a tissue-specific manner. Screening the F2 oligonucleotide with a variety of other transcription factors failed to identify the additional proteins which bind to this site.
314
C. Yao et al. / Biochemical and Biophysical Research Communications 371 (2008) 309–314
Site F3 contains two protein binding sites but lacks functional enhancing activity Enhancer site F3 contains two distinct regions which bind to HL-60 nuclear extracts. Oligonucleotides corresponding to these two footprints were prepared and shown to bind to HL-60 nuclear proteins in gel shift experiments (data not shown). However, mutation of the F3 site failed to significantly affect enhancer activity. Representative examples are shown in Table 1. While it remains possible that further work might reveal a functionally important element within F3, this footprint was not further examined in the present study.
patterns formed using HL-60 nuclear extracts and those seen with K562 or HeLa nuclear extracts may also reflect differences in the functional activity of the human MPO enhancer in MPO-expressing and minimal MPO-expressing cells. Patterns of nuclear protein binding to sites F1, F3, and F4 in gel shift experiments all show differences between MPO-expressing cells such as HL-60 and minimal MPO-expressing cells such as K562 or HeLa. We presume that these differences may relate in most cases to differences in enhancer activity among these cells. Surprisingly, although the F3 footprints differed in HL-60 and K562 cells, we were unable to demonstrate that F3 was important for enhancer activity. The importance of other individual differences in protein binding must remain a subject for further study.
Discussion References It is important to elucidate the factors which regulate tissuespecific and maturation stage-specific gene expression in developing myeloid cells. The MPO gene remains a useful model for studying the control of gene expression at the myeloblast and promyelocyte stages of myeloid maturation. We now describe additional features of the structural and functional aspects of the human upstream MPO enhancer. By DNase I footprinting studies we have demonstrated three additional footprints within the 365 bp enhancer segment. Mutation studies suggest that two of these footprints appear to be involved in enhancer activity. A C/ EBP site and a c-Myb site, as well as an additional AML1 site appear to be important elements within the enhancer. The transfection studies described in this communication suggest that the human upstream enhancer contributes significantly to the tissue- and maturation stage-specificity of MPO transcription. This enhancer stimulates the activity of a minimal human MPO promoter by a greater extent in HL-60 cells (which express MPO) than in non-leukemic cells or leukemic cells which only minimally express MPO RNA or protein. Furthermore, treatment of HL60 cells with chemical inducers of myeloid maturation such as TPA or DMSO, which result in the arrest of MPO transcription in vitro, virtually eliminates enhancer-stimulated MPO promoter activity by 24 h after induction [10]. This suggests that the activity of this enhancer is dependent upon the maturational stage of the leukemic cells. Interestingly, as we showed in our previous communication [10], the MPO enhancer can also confer TPA- or DMSOresponsiveness upon a heterologous SV40 promoter, providing further evidence that enhancer activity is maturation stage-specific. (By contrast, promoter activity stimulated by an SV40 enhancer increased rather than decreased following TPA or DMSO treatment, and hence did not mimic the effects that these agents have on MPO expression in vitro.) In addition, the differences of gel shift
[1] S.J. Klebanoff, Myeloperoxidase—occurrence and biological function, in: J. Everse, M. Grisham (Eds.), Peroxidases in Chemistry and Biology, CRC Press, Boca Raton, FL, 1990, pp. 180–192. [2] J.M. Bennett, D. Katowski, M.T. Daniel, G. Flandrin, D.A. Galton, H.R. Gralnick, C. Sultan, Proposed revised criteria for the classification of acute myeloid leukemia: a report of the French-American-British Cooperative Group, Ann. Intern. Med. 103 (1985) 620–624. [3] G.E. Austin, W.-G. Zhao, W. Zhang, E.D. Austin, H.W. Findley, J.J. Murtagh Jr., Identification and characterization of the human myeloperoxidase promoter, Leukemia 7 (1995) 848–857. [4] W.-G. Zhao, A. Regmi, E.D. Austin, J.E. Braun, M. Racine, G.E. Austin, Ciselements in the promoter region of the human myeloperoxidase gene, Leukemia 10 (1996) 1089–1103. [5] W.-G. Zhao, J.-P. Lu, G.E. Austin, Enhancement of myeloperoxidase promoter function by PEBP2/CBF or inhibition by an Sp1-containing repressor requires the participation of a third element, DP4, Blood 88 (Suppl. 1) (1996) 47a. [6] W.-G. Zhao, A. Regmi, J.-P. Lu, G.E. Austin, Identification and functional analysis of multiple murine myeloperoxidase (MPO) promoters and comparison with the human MPO promoter region, Leukemia 11 (1997) 97–105. [7] K.-W.M. Lin, G.E. Austin, Functional activity of three distinct myeloperoxidase (MPO) promoters in myeloid cells, Leukemia 16 (2002) 1143–1153. [8] M. Lübbert, C.W. Miller, H.P. Koeffler, Changes in DNA methylation and chromatin structure in the human MPO gene during myeloid differentiation, Blood 78 (1991) 345–356. [9] K.F. Jorgenson, G.R. Antoun, T.F. Zipf, Chromatin structural analysis of the 50 end and contiguous flanking region of the myeloperoxidase gene, Blood 77 (1991) 159–164. [10] G.E. Austin, W.-G. Zhao, A. Regmi, J.-P. Lu, J. Braun, Identification of an upstream enhancer containing an AML1 site in the human myeloperoxidase (MPO) gene, Leuk. Res. 22 (1998) 1037–1048. [11] K. Lim, C.B. Chae, A simple assay for DNA transfection by incubation of the cells in culture dishes with substrates for b-galactosidase, Biotechniques 7 (1989) 576–579. [12] L. Hennighausen, H. Lubon, Interaction of protein with DNA in vitro, Methods Enzymol. 152 (1987) 721–735. [13] J.D. Dignam, R.M. Labovitz, R.G. Roeder, Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei, Nucleic Acids Res. 11 (1983) 1481–1489. [14] F.M. Ausubel, R. Brent, R.E. Kingston, Current Protocols in Molecular Biology, Greene, New York, NY, 1988.