Onto TFIID Function TATA Box That Can Impose Cell Specificity

PU.1 and a TTTAAA Element in the Myeloid Defensin-1 Promoter Create an Operational TATA Box That Can Impose Cell Specificity onto TFIID Function This information is current as of October 2, 2021. Mariana Yaneva, Serena Kippenberger, Nan Wang, Qin Su, Margaret McGarvey, Arpi Nazarian, Lynne Lacomis, Hediye Erdjument-Bromage and Paul Tempst J Immunol 2006; 176:6906-6917; ;

doi: 10.4049/jimmunol.176.11.6906 Downloaded from http://www.jimmunol.org/content/176/11/6906

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

PU.1 and a TTTAAA Element in the Myeloid Defensin-1 Promoter Create an Operational TATA Box That Can Impose Cell Speciﬁcity onto TFIID Function1

Mariana Yaneva, Serena Kippenberger, Nan Wang, Qin Su, Margaret McGarvey, Arpi Nazarian, Lynne Lacomis, Hediye Erdjument-Bromage, and Paul Tempst2

Defensins are major components of a peptide-based, antimicrobial system in human neutrophils. While packed with peptide, circulating cells contain no defensin-1 (def1) transcripts, except in some leukemia patients and in derivative promyelocytic leukemia cell lines. Expression is modulated by serum factors, mediators of inflammation, and kinase activators and inhibitors, but the underlying mechanisms are not fully understood. A minimal def1 promoter drives transcription in HL-60 cells under control of PU.1 and a def1-binding protein (“D1BP”), acting through, respectively, proximal (؊22/؊19) and distal (؊62/؊59) GGAA elements. In this study, we identify D1BP, biochemically and functionally, as GA-binding protein (GABP)␣/GABP␤. Whereas Downloaded from GABP operates as an essential upstream activator, PU.1 assists the flanking “TTTAAA” element (؊32/؊27), a “weak” but essential TATA box, to bring TBP/TFIID to the transcription start site. PU.1 thus imparts a degree of cell specificity to the minimal promoter and provides a potential link between a number of signaling pathways and TFIID. However, a “strong” TATA box (“TATAAA”) eliminates the need for the PU.1 binding site and for PU.1, but not for GABP. As GABP is widely expressed, a strong TATA box thus alleviates promyelocytic cell specificity of the def1 promoter. These findings suggest how the myeloid def1 promoter may have evolutionarily acquired its current properties. The Journal of Immunology, 2006, 176: 6906–6917. http://www.jimmunol.org/

ell differentiation is genetically encoded and consists of a concomitant increase in def1 expression, thereby providing a series of specific molecular events at the individual gene model system for study of differentiation-specific gene regulation C and protein levels. Among those is the synthesis of key (13–15). It should be recognized, however, that some molecular components that enable fully matured cells to function properly. events during drug-induced differentiation may perhaps not en- For instance, in the case of human neutrophils, short-lived myeloid tirely reflect those occurring during normal granulopoiesis. In con- blood cells, one such substance is a peptide antibiotic, known as trast, def1 transcripts have never been found in the related myelo-

defensin-1 or human neutrophil peptide-1 (1–3). Defensins are the blastic (KG-1), monoblastic (U-937), myeloblastic/erythroblastic by guest on October 2, 2021 major components of an oxygen-independent system used by scav- (K-562), or lymphoid B and T cell lines, not even after extensive enger cells to eliminate invading microorganisms. Among several RA treatment (8, 12). Studies aimed at understanding this unique others, corticostatic, chemotaxic, immunostimulatory, and anti- granulocytic expression of defensin genes must converge, eventu- HIV-1 activities have also been ascribed to defensins (3–5). Interest- ally, at the identification of cis-regulatory control elements and the ingly, synthesis of the peptide occurs in precursor cells, while still cognate transactivating factors (16, 17). residing in the bone marrow, and it gets stockpiled in granules for later Instead of being strictly myeloid specific, many transcription use when confronting microbes in the blood stream (6, 7). Circulating 3 factors involved in regulation of “myeloid” genes are more com- cells have no measurable levels of defensin-1 (def1) transcripts, ex- monly expressed (18). Lineage specificity is controlled, in most of cept in a subset of myelogenous leukemia patients (6–8). these cases, through unique factor combinations or related mech- Consistent with these findings is the presence of def1 mRNA, anisms (17, 19). A case in point is PU.1, a member of the ETS albeit at relatively low levels, in the HL-60 human promyelocytic family of transcription factors, known to physically interact with leukemia cell line (6, 8–12). HL-60 cells can be induced by reti- other regulatory proteins or to otherwise function in activating, noic acid (RA) to mature along the granulocytic pathway, with combinatorial arrangements (20, 21). Some factors implicated in the process also gain, or gain further, transactivating potential

Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, and Weill through posttranslational modifications (17, 22), which may either Graduate School of Medical Sciences, Cornell University, New York, NY 10021 impart another layer of cell specificity and/or tie gene expression Received for publication August 17, 2005. Accepted for publication March 13, 2006. to a signaling pathway (22). The costs of publication of this article were defrayed in part by the payment of page Previous studies have indicated that lineage- and stage-specific charges. This article must therefore be hereby marked advertisement in accordance elements control myeloid def1 expression, a process modulated by with 18 U.S.C. Section 1734 solely to indicate this fact. serum factors, kinase activators and inhibitors, and mediators of 1 The Sloan-Kettering Microchemistry and Proteomics Core Facility is supported by National Institutes of Health Cancer Center Support Grant P30 CA08748. inflammation (12, 23, 24), but the underlying mechanisms of basal and induced expression in promyelocytic cells are not yet fully 2 Address correspondence and reprint requests to Dr. Paul Tempst, Memorial Sloan- Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. E-mail address: understood. A minimal core promoter (Ϫ83 to ϩ82), containing [email protected] two functionally essential, ETS-like elements, can drive transcrip- 3 Abbreviations used in this paper: def1, defensin-1; RA, retinoic acid; GABP, GA- tion in a quasi cell-specific manner (24, 25), perhaps in conjunc- binding protein; D1BP, D1-binding protein; MS, mass spectrometry; ChIP, chromatin immunoprecipitation; RT, room temperature; MALDI-reTOF, MALDI reflectron tion with an upstream C/EBP-like element of as yet unproven ca- TOF; hGH, human growth hormone. pacity (26, 27). The proximal GGAA element (Ϫ22/Ϫ19),

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 6907 downstream of a TTTAAA sequence, and the related distal one attached to a 6-carbon spacer at the 5Ј end; the complementary strand was (Ϫ62/Ϫ59), each bind a different factor in vitro (24, 25). def1 without a biotin moiety. induction is also directed through both elements and factors. Using a specific Ab in an in vitro DNA-binding assay (EMSA), it has EMSA previously been suggested that PU.1 was the leading candidate for Prebinding of nuclear extract (5–10 ␮g) or respective column or other specific interaction with the proximal GGAA site (25). PU.1 was fractions to poly(dI:dC) was conducted at 25°C for 10 min in buffer con- subsequently purified and positively identified as the binding ac- taining 4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 25 mM NaCl, 10 mM Tris-HCl (pH 7.5) and 0.05 mg/ml poly(dI:dC) (Amersham tivity (24). As the next step to further dissect and reconstitute this Biosciences). When indicated, unlabeled competitor oligonucleotides (200- system, we sought to purify and characterize the postulated ETS fold molar excess) were included in the incubation mixture at this point. factor binding to the distal GGAA regulatory sequence (“D1 Radiolabeled probe (3.5 fM; ϳ2 ϫ 104 cpm) was then added to the reaction box”), termed D1-binding protein or D1BP. above, mixed and incubated at 25°C for 20 min. One microliter of 10ϫ In this study, we report identification of D1BP, biochemically gel-loading buffer, containing 250 mM Tris-HCl (pH 7.5), 0.2% of brom- phenol blue, 0.2% xylene cyanol, and 40% glycerol, was added to the and functionally, as the heterodimeric factor GA-binding protein reaction and then loaded onto a 6% native gel (which was prerun for 90 (GABP). While GABP functions as an essential, typical upstream min at 100 V) in 0.5ϫ nondenaturing Tris-borate-EDTA (TBE) buffer. The activator, PU.1 assists the flanking TTTAAA element, a weak yet electrophoresis was run at 25°C and 100 V for ϳ3.5 h. The gel was then essential TATA box, to bring TBP/TFIID to the def1 promoter transferred onto Whatman paper, vacuum-dried, and exposed to Hyperfilm (Amersham Biosciences) for the desired period of time at Ϫ80°C and with near the transcriptional start site. As a result, PU.1 imparts a degree an intensifier screen. For Ab supershift experiments, Abs (0.1 ␮g/␮l; 1 ␮l of cell specificity to the minimal promoter and provides a potential volume) were added to the solutions after protein-DNA incubation for 20 link between a number of signaling pathways and TFIID. How- min at 25°C, and the DNA-protein complexes were also resolved in 6% ever, PU.1 is incapable of functioning in such a capacity in a polyacrylamide gels. Abs recognizing PU.1, Ets-1, Ets-2, Elk-1, Elf-1, Downloaded from TATA-less (TGTAAA) def1 promoter. Conversely, creating a Egr-2, and Etv1 were purchased from Santa Cruz Biotechnology. strong TATA box (TATAAA) in the def1 promoter eliminates the Preparation of DNA concatamers and attachment to magnetic need for the PU.1 binding site and for PU.1, but not for GABP. beads Because GABP is more ubiquitously expressed, a strong TATA box mitigates promyelocytic cell specificity of the def1 promoter. Multimers of the wild-type (D1) and the mutant (D1M3) DNA binding Thus, depending on the switch, mutation of the same nucleotide sites were generated by a PCR-based method (29) using complementary, http://www.jimmunol.org/ single-stranded oligonucleotides of two direct repeats of D1 or D1M3, can either eliminate or boost minimal promoter activity, thereby respectively. The forward single-stranded oligonucleotides were biotinyl- alleviating cell specificity in the latter case. This finding suggests ated at the 5Ј end. PCRs (50 ␮l vol) contained 460 ng of each primer, 8 ␮M a mechanism of how the myeloid def1 promoter may have evolu- dNTPs (Roche Molecular Biochemicals), and2UofVent polymerase tionarily acquired its current properties. (New England Biolabs) in the following buffer: 10 mM KCl, 10 mM (NH4)2SO4, 3.5 mM MgSO4, 0.1% Triton X-100, and 20 mM Tris-HCl (pH 8.8; at 25°C). The cycling conditions were: 95°C for 2 min followed Materials and Methods by 14 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 3 min. PCR Cell lines and culture conditions products were purified using QiaQwick kit (Qiagen), and analyzed by agarose gel electrophoresis in TBE buffer. Each PCR yielded ϳ3–5 ␮gof Human promyelocytic leukemia HL-60 and myeloblastic leukemia KG-1 DNA with sizes between 200 bp and 10 kb. The 5Ј-biotinylated, concate- by guest on October 2, 2021 cells (American Type Culture Collection (ATCC)), and acute promyelo- merized DNA was attached to M280 streptavidin-coated magnetic beads cytic leukemia NB4 cells (28) were grown at 37°C in RPMI 1640 medium (Dynal Biotech) using KilobaseBINDER kit (Dynal Biotech) according to (Invitrogen Life Technologies) supplemented with 10% heat-inactivated the manufacturer’s instructions. Approximately 10–50% of DNA con- FCS (HyClone Laboratories), nonessential amino acids, and penicillin and ␮ catamers in the incubation mixture were attached to the beads (up to 8 streptomycin at 5 g/ml. HL-60 and NB4 cell cultures were passaged twice ␮g/mg beads). a week to maintain the cell density between 0.5–2 ϫ 106 cells/ml. SL2 embryonic epithelial cells from Drosophila melanogaster Schneider line 2 (ATCC) were grown at 25°C in Schneider medium (Invitrogen Life Tech- Protein purifications nologies) supplemented with 10% FCS. SL2 cell cultures were passaged D1 DNA-binding activity was purified from 1.4 ϫ 109 exponentially grow- ϫ 6 every 3–4 days to maintain the cell density at 0.5–5 10 cells/ml. All ing NB4 cells, following established protocol (30). All procedures were cells were counted in hemocytometer chamber, and the viability was as- performed at 4°C. Seventy-five milligrams of nuclear extract (31) was frac- sessed by exclusion of 0.1% trypan blue. tionated on a P11 phosphocellulose (Whatman) column, with 22-ml bed Synthetic oligonucleotides volume, equilibrated with buffer D (20 mM HEPES (pH 7.9), 0.2 mM EDTA, 0.5 mM DTT, 0.01% Nonidet P-40, 0.2 mM PMSF, 10% glycerol) Complementary, single-stranded oligonucleotides were custom synthe- containing 0.075 M KCl. Bound proteins were eluted using a 200-ml linear sized by Integrated DNA Technologies (IDT) and annealed before use as gradient of 0.075–0.85 M KCl in the same buffer. Fractions containing D1 probes and/or as competitors in EMSAs. Only the sense sequences of each DNA-binding activity (as monitored by EMSA) eluted at ϳ0.1 M KCl, and pair are listed here: D1, (5Ј-GACCCAACAGAAAGTAACCCCGGAAATT were pooled and dialyzed overnight against 50 vol of buffer D containing AG-3Ј); D1M2, (5Ј-GACCCAACAGAAAGTAACCCCAAGGATTAG-3Ј); 0.1 M KCl. All protein concentrations were determined using the Bradford D1M3, (5Ј-GACCCAACAGAAAGTAACCCCTGAAATTAG-3Ј); TA, assay (Bio-Rad) and BSA standards (Sigma-Aldrich). Affinity capture of (5Ј-CAAGACCTTTAAATAGGGGAAGTCCACTTG-3Ј); TAM2, (5Ј-CAA D1-binding protein(s) on DNA-magnetic beads was then conducted (30). GACCTTTAAATAGGGCCCGTCCACTTG-3Ј); TA(T-31G), (5Ј-CAAGA One-milligram beads with attached D1-concatamerized DNA (5 ␮g/mg; CCTGTAAATAGGGGAAGTCCACTTG-3Ј); TA(T-31A), (5Ј-CAAGACC dsDNA/beads) were first washed in DNA-binding solution (20 mM TATAAATAGGGGAAGTCCACTTG-3Ј); TAM2(T-31A), (5Ј-CAAGACC HEPES (pH 7.9), 0.1M KCl, 0.2 mM EDTA, 0.5 mM DTT, 0.01% Nonidet TATAAATAGGGCCCGTCCACTTG-3Ј); PU.1(SV40), (5Ј-TGAAATAAC P-40, 10% glycerol), mixed with 3.5 mg of the dialyzed P11 (0.1 M) CTCTGAAAGAGGAACTTGGTTAGGTA-3Ј); GABP␣ (thrombopoietin), protein fraction, and incubated for 3 h with rotation. Binding buffers always (5Ј-GTGAAGGCCCCCGGAAGT5ACGCCT-3Ј); Ets-1, (5Ј-CGGCCAA contained oligo(dI:dC) (30 bp in length; custom synthesized by IDT) and ACCGGAAGTATGTGC-3Ј); Elk-1, (5Ј-TCCTGATCATCCACCGGAA poly(dI:dC) (Amersham Biosciences) at 0.1 mg/ml each, except where GTGAG-3Ј); Elf-1, (5Ј-TAAACCCGGAAGTGTAGTACATC-3Ј); ER81, noted. After binding, the beads were washed once with 1.2 ml of complete (5Ј-AACCCCCGCCGGAAGTACTGATCT-3Ј); Erg-2, (5Ј-CCCTGAG binding buffer, and three times with binding buffer in the presence of Esch- ACCGGAAGTATTAGGCT-3Ј). erichia coli double-stranded and ssDNA, at 0.1 mg/ml each, as nonspecific Underlined nucleotides have been changed from the wild-type se- competitor. The beads were then eluted with 50 ␮l of binding buffer con- quences. D1 and D1M3 (both sense and antisense) were also used for taining 0.5 M KCl, for 15 min on ice, and stored. The eluate was diluted concatamerization by PCR to generate the surface ligand for affinity puri- in 1.2 ml of fresh, complete binding buffer and incubated for 3 h with 1-mg fication of the D1BP. For coupling to streptavidin-derivatized magnetic beads derivatized with attached mutant D1M3-concatamerized DNA to beads, the sense oligonucleotide of each pair was synthesized with biotin remove nonspecific nucleic acid-binding proteins by negative selection. 6908 MYELOID DEFENSIN-1 PROMOTER

The nonbound fraction was then taken for another round of positive se- creasing concentrations of 5 M, 3 M, 1 M, and no urea. Recombinant lection on D1-concatamerized DNA beads, followed by salt elution of proteins were analyzed by EMSA, and also fractionated by SDS PAGE and bound proteins, as described above. The final eluate was taken for EMSA identities confirmed by MS. analysis, SDS gel electrophoresis, Western blotting, and mass spectrometric identification. In addition, beads stored after each round of positive or Production of anti-GABP (␣ and ␤) Abs negative selection were suspended in 50 ␮l of Laemmli sample buffer and also taken for gel analysis. Peptides corresponding to residues 421–433 of GABP␣ (CEQKKLAK MQLHG) and residues 365–381 of GABP␤ (RQQLLKKEQEAEAYRQK; Mass spectrometry (MS) with an additional Cys residue at the N terminus) were synthesized by S. S. Yi in the Microchemistry and Proteomics Core Facility (Memorial Sloan- Gel-resolved proteins were stained with Coomassie Blue R250 (Bio-Rad), Kettering Cancer Center), and were conjugated to keyhole lympet hemo- bands excised and digested with trypsin, the mixtures fractionated on a cyanin using the Imject Maleimide-Activated Conjugation kit (Pierce). Poros 50 R2 RP microtip, and resulting peptide pools analyzed by MALDI Polyclonal anti-GABP␣ and anti-GABP␤ Abs were individually produced reflectron TOF (MALDI-reTOF) MS using a Bruker UltraFlex TOF/TOF by Pocono Rabbit Farm and Laboratory. Specific Abs were purified from instrument (Bruker Daltonics), as described (32, 33). Selected experimental rabbit sera after the first bleedings using prepacked Econo-Pac Protein A masses (m/z) were taken to search the human segment of a nonredundant ϳ Cartridge (Bio-Rad) as per the manufacturer’s procedure. Purified Ig frac- protein database (NR; 108,000 entries; National Center for Biotechnol- tions (at 1 mg/ml) were stored at 4°C. ogy Information, Bethesda, MD), using the MASCOT Peptide Mass Fin- gerprint (PMF) program, version 2.0.04 for Windows (Matrix Science), Western blotting with a mass accuracy restriction better than 30 ppm, and maximum one missed cleavage site allowed per peptide. To confirm PMF results with Protein solutions were separated by electrophoresis in SDS gels, and the scores Ն40, mass spectrometric sequencing of selected peptides was done proteins were transferred to a polyvinylidene difluoride Immobilon-P by MALDI-TOF/TOF (MS/MS) analysis on the same prepared samples, membrane (Millipore) in Tris-glycine-methanol buffer as described previ-

using the UltraFlex instrument in LIFT mode. Fragment ion spectra were ously (30). The respective Abs were added to the membranes at 1 ␮g/ml in Downloaded from taken to search NR using the MASCOT MS/MS Ion Search program (Ma- PBS/0.05% Tween 20 for2hatroom temperature (RT). Anti-mouse or trix Science). Any tentative confirmation (Mascot score Ն30) of a PMF anti-rabbit-HRP-conjugated Abs (Santa Cruz Biotechnology) and ECL kit result thus obtained was verified by comparing the computer-generated (Pierce) were used for visualization of the immune complexes. fragment ion series of the predicted tryptic peptide with the experimental MS/MS data. Chromatin immunoprecipitation (ChIP) Plasmids ChIP was conducted using the Upstate Biotechnology Chromatin Immu- Ј noprecipitation Assay kit as per the manufacturer’s instructions. Briefly, http://www.jimmunol.org/ Luciferase reporter constructs were all derived from the pGL3 series of ϫ 6 vectors (Promega). pGL3B-def1(Ϫ83/ϩ82), (Ϫ34/ϩ82) and (ϩ11/ϩ83), 5 10 NB4 cells were treated with 1% formaldehyde for 15 min at RT. Cells were washed twice with PBS containing protease inhibitors (Roche), are basic (B) vectors containing human defensin 1 promoter inserts that ␮ span sequences from nucleotide Ϫ83 (Sau96I site) and, respectively, Ϫ34 and lysed in 200 l of SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM ϩ ϩ Tris-HCl (pH 8.1), and protease inhibitors). After incubation on ice for 10 and 11 (ExoIII truncations), to 82 (ScaI site), as described (25). These ϫ three constructs will be further referred to as, respectively, a, b, and c. min, the samples were sonicated on ice (14 10 s, at 15% amplitude; Mutants derived from pGL3B-def1(Ϫ83/ϩ82) were constructed using the Fisher Sonic Dismembrator, Model 500) to an average DNA length of QuikChange (Stratagene) site-directed mutagenesis kit as per the manu- 200-3000 bp. Samples were the centrifuged for 10 min at maximum speed facturer’s instructions. Appropriate sense and antisense mutation primers (Eppendorf centrifuge) at 4°C and the sonicated cell supernatant was di- (35-nt long) were designed to introduce the following modifications (sin- luted 10-fold with ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl (pH 8.1), 167 mM NaCl, and protease gle, double, or triple sites): construct d, GAA to CCC (Ϫ21/Ϫ19); e, T to by guest on October 2, 2021 Ϫ ϭ ϩ Ϫ ϭ ϩ inhibitors). To reduce nonspecific background, each sample was precleared G( 31); f d e;g,TtoA( 31); h d g; i, GGAA to AAGG ␮ ␮ (Ϫ62/Ϫ59); j ϭ d ϩi; k ϭ d ϩ e ϩ i; l ϭ d ϩ g ϩ i. Mutant plasmid with 50 l of protein A agarose (50% slurry) in the presence of 500 g constructs were sequenced to confirm the desired alterations; sequence salmon sperm DNA for 30 min at 4°C with agitation. Incubations with analysis was done at the DNA Service Laboratory, Biotechnology Center, specific Abs, and appropriate negative controls, were performed overnight at 4°C. The immune complexes were then collected with 60 ␮l of protein Utah State University (Logan, UT). The expression plasmid pCMV-hGH ␮ (human growth hormone gene under control of a cytomegalovirus pro- A agarose-50% slurry, again in the presence of 500 g of salmon sperm moter) was obtained from Invitrogen Life Technologies and used through- DNA. After a 1-h rotation at 4°C, the beads were collected and washed out as an internal control for transfection efficiency (25). For the construc- once for 5 min with each of the following buffers: Low Salt Immune Com- tion of expression plasmid pCDNA3.1/PU.1, mouse PU.1 cDNA (34) was plex Wash Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM excised with EcoRI from the PJ6 plasmid (gift from Dr. M. Klemsz, In- Tris-HCl (pH 8.1), 150 mM NaCl), high salt immune complex wash buffer diana University School of Medicine, Indianapolis, IN), and inserted into (0.1% SDS, 1%Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), the corresponding site of plasmid pCDNA3.1ϩ (Invitrogen Life Technol- 500 mM NaCl), LiCl immune complex wash buffer (0.25 mM LiCl, 1% ogies). Human GABP␣ and GABP␤ cDNAs (35) were similarly excised Nonidet P40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl (pH 8.1)), and twice with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8). The from pBluescript plasmids (gift from Dr. T. Brown, Pzifer, Groton, CT) ␮ and inserted into pCDNA3.1ϩ. Final constructs were confirmed by washed resin was resuspended in 250 l of freshly prepared elution buffer sequencing. (1% SDS, 0.1 M NaHCO3). Cross-linking was reversed by incubation at 65°C overnight. DNA fragments were purified by phenol/chloroform ex- Recombinant proteins traction, amplified by PCR using primers that covered the Ϫ160/ϩ11 region of def1 promoter (25), the Ϫ143/ϩ8 region of the CD89 promoter Using PCR, BglII or BamH1 sites were introduced at the 5Ј and 3Ј ends of (forward: 5Ј-GCTGTGAGGCAATAGATGTGGAA-3Ј; reverse: 5Ј- the open reading frames of GABP␣, GABP␤, and PU.1-containing plas- GGGTCCACTGTGCTGACAC-3Ј), or the Ϫ162/ϩ137 region of the mids (see above). The cDNAs were inserted into the BamH1 site of the p40phox promoter (forward: 5Ј-GCGCCAAGGACTGACATC-3Ј; reverse: His-tag expression vector pET-19b (Novagen), and transformed into E. coli 5Ј-GAGCAGGTGGTGCGTCTC-3Ј), and analyzed by electrophoresis in strain BL21-Gold (DE3)p LysS (Stratagene). Cultures (250 ml) were 1% agarose gel/TBE. grown at 27°C in Luria Broth containing 50 ␮g/ml ampicillin and 20 ␮g/ml chloramphenicol until the A600 reached 0.4. Expression was induced by the Transient transfections addition of isopropyl-␤-D-thiogalactopyranoside to 1 mM. Cells were grown for a further4hat27°C, then harvested by centrifugation and stored Transfections of HL-60 and KG-1 cells were performed as previously de- at Ϫ80°C until use. Cells were lysed at 4°C for 30 min in 10 mM sodium scribed (25). In brief, cells were diluted into the corresponding growth phosphate buffer (pH 7.4), containing 0.2 mg/ml lysozyme and 0.1% Non- medium (see cell culture section), at densities of 4 ϫ 105/ml, the day before idet P-40, and centrifuged at 12,000 ϫ g for 20 min. His-tagged proteins transfection. After 18 h, cells (1 ϫ 107 per transfection) were pelleted and were purified from cell lysates supernatants on 1-ml HisTrap columns washed with prewarmed (37°C) IMDM, centrifuged at 500 ϫ g for 5 min (Amersham Biosciences) according to the manufacturer’s instructions. Pro- at RT, resuspended at a density of 1 ϫ 107 cells in 0.4 ml of warm IMDM teins that eluted with a 300–500 mM gradient of imidazole were precipi- containing 2.5 ␮g of pCMV-hGH plasmids. This suspension was added tated with 20% ammonium sulfate, and dissolved in 6 M urea, 50 mM into the electroporation cuvette already containing the luciferase expres- Ͻ ␮ K3PO4, 100 mM KCl, 5 mM DTT. They were then renatured by stepwise sion DNA constructs ( 20 l). Cells and plasmids were then mixed with dialysis, for 4 h each at 4°C, against 50 vol of the same buffer with de- a pipetter, incubated for 5 min at RT, followed by electroporation at 975 The Journal of Immunology 6909

␮F capacitance and 280 V using a Gene Pulser II (Bio-Rad), unless oth- functional, ETS-like elements, TA and D1, in the minimal pro- erwise indicated. The cells were then transferred to 10 ml of warm IMDM moter (Ϫ83/ϩ82). The proximal TA element binds PU.1 in vitro, with 10% FCS, the dishes swirled and incubated at 37°C for 5 h, and the cells harvested in 15-ml tubes by centrifugation at 500 ϫ g for 5 min at RT. and was successfully used for specific DNA affinity purification of One milliliter of supernatant from each experiment was stored in an Ep- that same factor from HL-60 cells (24). Next, identification of the pendorf tube for human growth hormone (hGH) assay. Pellets were washed D1BP was a key step to further resolve the mechanism of def1 ␮ with 5 ml of PBS at RT, 300 l of lysis buffer (1% Triton X-100, 25 mM minimal promoter activity. EMSA using a probe corresponding to Gly-Gly (pH 7.8), 15 mM MgSO4, 4 mM EGTA (pH 7.8), 1 mM DTT) was added, pellets were then resuspended, transferred to Eppendorf tubes, vor- the D1 box (Fig. 1) suggested highest levels of D1BP expression texed for 5 s and spun at full speed for 3 min at RT. Fifteen microliters of in promyelocytic NB4 cells (data not shown), which were there- the above lysate was mixed with 300 ␮l of freshly made assay buffer, fore taken as the source for isolation of this factor. The same probe which contained 25 mM Gly-Gly (pH 7.8), 15 mM KPO (pH 7.8), 15 mM 4 was used to assay for the D1BP during various steps of purifica- MgSO4, 4 mM EGTA (pH 7.8), 2 mM ATP (pH 7.8), 1 mM DTT. Relative light units were measured for 20 s in a model Monolight 2010 luminometer tion, as outlined in Fig. 2A. D1BP was retained on a P11 phos- (Analytical Luminescence Laboratory) after addition of 100 ␮lof1mM phocellulose column and eluted with 0.1 M KCl. TA-binding ac- D-luciferin potassium salt (Analytical Luminescence). The hGH was mea- tivity, containing PU.1 as assayed by Western blotting, eluted from sured with the ELISA kit from the Nichols Institute as per the manufac- the P11 column with 0.85 M KCl (data not shown). The subse- turer’s instructions. Briefly, 100 ␮l of supernatant was mixed with an equal volume of Ab solution; the latter is a mixture of two mAbs, each one quent procedure of positive/negative/positive affinity selection, in- specific for a different and distinct epitope on the hGH molecule, to form volving wild-type (D1) and mutant (D1M3) fragments immobi- a soluble sandwich complex in the presence of hGH. One of the Abs was lized to magnetic particles, as previously developed in our 125I-labeled for detection while the other Ab is coupled to biotin. The reaction was then mixed with an avidin coated plastic bead and incubated laboratory (30), was applied for straightforward purification of the for 90 min at RT while shaking (180 rpm). After two washes, the bead was D1-binding activity. P11 fractions containing the desired activity Downloaded from counted in a gamma counter (LKB 1272 Clinigamma; Wallac) for 1 min. were further enriched in D1BP by binding to wild-type (D1)n- Cotransfections of Drosophila SL2 cells affinity beads. Proteins eluted from the beads with 0.5 M KCl (Fig. 2B, lane 4; and 2C, lane 3) were then incubated with magnetic ϫ 5 Exponentially growing SL2 cells were diluted to 5 10 /ml and plated beads carrying mutant (D1M3)-DNA, differing in just a single into 6-well tissue-culture dishes (1 ml/well). Cells were transfected at a ratio of 1:3 nucleic acid/DOSPER Liposomal Transfection Reagent base-pair from the wild-type sequence. The nonbound proteins (Roche) according to the manufacturer‘s instructions. One microgram of (Fig. 2C, lane 4) were once more incubated with D1 beads for a http://www.jimmunol.org/ D-luciferase-containing pGL3B-def1 (Ϫ83/ϩ82) wild-type or mutant re- final round of positive selection (Fig. 2B, lanes 5 and 6; and 2C, porter construct was cotransfected with equal amounts of the expression ϩ lanes 6 and 7). We consistently observed two shifted bands in vector pcDNA3.1 (Invitrogen Life Technologies) containing PU.1, GABP␣, and/or GABP␤ and with 1 ␮g of plasmid expressing ␤-galacto- EMSA throughout the purification procedure (Fig. 2B), as origi- sidase under control of a Drosophila actin promoter (gift from Dr. M. nally reported (25). Four bands observed after SDS gel electro- Baylies, Memorial Sloan-Kettering Cancer Center, New York, NY) as in- phoresis of the eluted fraction (indicated with closed or open cir- ternal control for transfection efficiency (36). The DNA concentration for each transfection was equalized by addition of salmon sperm DNA. Twen- cles in Fig. 2C, lane 7) were excised and the proteins identified by ty-four hours after transfection, cells were resuspended in 2 ml of fresh a combination of PMF using MALDI-TOF MS, and MS-based

medium and incubated further for another 24 h. Transfected cells were sequencing using MALDI-TOF/TOF MS/MS. by guest on October 2, 2021 harvested by centrifugation, washed once with PBS and resuspended in 100 As indicated in Fig. 2C (lane 7), two of the bands (F) contained ␮l of lysis buffer (25 mM Gly-Gly (pH 7.8), 15 mM MgSO , 4 mM EGTA 4 ␣ ␣ ␤ (pH 7.8), 1% Triton X-100, 1 mM DTT). Cells were then vortexed for 30 s, GABP , the DNA-binding subunit of the GABP / heterodimer, lysed by freezing and thawing and incubated on ice for 20 min. After a well-characterized member of the ETS-family of transcription centrifugation for 5 min at full speed in a table centrifuge, 50 ␮lofthe factors (18, 37, 38). No data were obtained to identify any of the ␮ supernatant was mixed with 300 l of freshly made assay buffer (25 mM visualized bands as GABP␤. However, the protein was detected by Gly-Gly (pH 7.8), 15 mM KPO4 (pH 7.8), 15 mM MgSO4, 4 mM EGTA, 2 mM ATP, 1 mM DTT). Luciferase activity was measured in a luminom- Western blot analysis of an aliquot of the same fraction (data not eter as described above. Five microliters of lysate was used to determine shown). In addition, the DNA-binding activity was intact (Fig. 2B, ␤-galactosidase activity with the ␤-Gal-Assay kit (Invitrogen Life Tech- lane 6), although some faster migrating bands, most likely indi- nologies) as per the manufacturer‘s instruction, and was used to normalize cating protein degradation, were also observed. In a comparable luciferase activity. The data shown are the mean values of at least three independent transfections, each in triplicate, and are expressed in percent- analysis of the prolactin gene promoter, the ␣ subunit of GABP age compared with the activity generated by the reporter construct alone. was also identified by MS, while the GABP␤ was not detected either (39). We do not have a ready explanation for these obser- Results vations. The two other bands on the gel (E) were both identified as Purification and identification of GABP as a D1 box-binding PARP1, an abundant nuclear protein that has frequently been ob- protein served to nonspecifically bind to nucleic acids (Ref. 30; M. def1 expression in promyelocytic leukemia cells is controlled by a Yaneva, H. Erdjument-Bromage, and P. Tempst, unpublished ob- combination of at least two nuclear factors, acting through two servations). In a parallel analysis, the P11 fractions (0.85 M salt

FIGURE 1. Human myeloid def1 minimal promoter (Ϫ83/ϩ82). The numbering is relative to the site of transcriptional initiation (ϩ1) (25); the sequence is shown down to position ϩ3. The positions of distal and proximal ETS-like sites are indicated by black dots under the GGAA motifs. The heavy bar underneath nucleotides Ϫ32/Ϫ25 indicates the position of the TATA-like box. The sequences of the two dsDNA oligonucleotides (D1 and TA), used as EMSA probes and for protein puriﬁcations, are indicated by the arrows. Site-speciﬁc mutations are indicated with the respective nucleotide changes above or under the sequence. 6910 MYELOID DEFENSIN-1 PROMOTER Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 2. Purification of the proteins binding to the distal GGAA element (D1 box) in the def1 promoter. A, Purification scheme. Nuclear extract (NE) from NB4 cells was fractionated using phosphocellulose (P11) chromatography, followed by stepwise, batch affinity capture. Double-stranded (ds), wild-type D1- and mutant D1M3-oligonucleotides (F and E, respectively) were attached to magnetic beads for the affinity purifications. B, EMSA of the protein fractions (shown in C) using a ds D1 oligonucleotide as probe. Lane 1, Free D1-probe; lane 2, binding of proteins in the starting nuclear extract; lane 3, fraction from P11 column eluted with 0.1M KCl; lane 4, DNA binding of proteins eluted from the first binding to wild-type D1 beads; lanes 5 and 6, activities of the nonbound (NB) and eluted (B) with 0.5 M salt from the second round of binding to D1-magnetic beads. The arrows point to the position of the complexes that were formed consistently throughout the purification. C, Protein profiles of fractions containing D1-binding activity. The proteins were analyzed by electrophoresis on 4–15% SDS gel, and stained with Coomassie Blue R250. Lane 1, Initial nuclear extract; lane 2, proteins from P11 column eluted with 0.1 M KCl; lane 3, proteins eluted from the first binding to wild-type D1 beads; lanes 4 and 5, nonbound (NB) and eluted (B) with 0.5 M KCl fractions from magnetic beads with mutant D1M3-DNA; lanes 6 and 7, nonbound (NB) and eluted (B) with 0.5 M NaCl fractions from the second binding to magnetic beads with wild-type D1-DNA. Arrows on the right indicate the bands excised for identification by MS; E correspond to PARP1 and F to GABP␣.

elution) containing TA-binding activity were pooled and also pu- Ni-NTA columns, renatured and tested for DNA binding in rified on DNA-magnetic beads as previously described for HL-60 EMSA. The results showed that rGABP ␣ but not ␤ bound to the cell nuclear extracts (24). Mass spectrometric analysis confirmed D1 probe, forming the faster migrating band (Fig. 3A, lanes 2 and that the TA-binding protein in NB4 cells was PU.1 as well (data 3), consistent with earlier observations of GABP subunit binding not shown). to DNA (18). Presence of both subunits resulted in formation of an additional, slower migrating band, formed presumably by a com- Binding specificities of recombinant and native (endogenous) plex of both rGABP ␣ and ␤ (Fig. 3A, lane 4). Neither protein ␣ ␤ GABP /GABP and PU.1 to the distal and proximal ETS bound to the TA probe (Fig. 3A, lane 8), whereas rPU.1 did so elements specifically (Fig. 3A, lane 9). Addition of both rGABP subunits did To demonstrate that GABP␣ (ϩGABP␤) and PU.1 bind specifi- not affect this binding (Fig. 3A, lane 10). Binding of rPU.1 to the cally to, respectively, D1 and TA sequences, His-tagged versions D1 sequence had already been ruled out in earlier studies (25), of these proteins were expressed in E. coli, and affinity purified on which was confirmed here (Fig. 3A, lanes 5 and 6). The results The Journal of Immunology 6911 Downloaded from

FIGURE 3. Speciﬁc binding of recombinant and native GABP ␣ and ␤ and PU.1 proteins to def1 promoter sequences. A, EMSA of bacterially expressed GABP␣, GABP␤, and PU.1. The ds D1 oligonucleotide (see Fig. 1) was used as a probe in lanes 1–4; TA oligonucleotide was used as a probe in lanes

5–8. Where applicable (lanes 4, 6, 9, and 10), mixed recombinant factors were present in equimolar amounts. B, Effect of anti-GABP Abs on D1-binding http://www.jimmunol.org/ activity. Nuclear extract from HL-60 cells was preincubated with 1 ␮g/ml puriﬁed anti-GABP Abs, and subsequently tested for binding to a D1 probe in EMSA. Key: ␣, anti-GABP␣ Ab; ␤, anti-GABP␤ Ab; ␣ϩ␤, mixture of anti-GABP␣ and anti-GABP␤ Abs (1:1); pre, preimmune rabbit serum. C, Western blot analysis of GABPs and PU.1 binding to def1 promoter sequences. Biotinylated DNA fragments representing Ϫ83/Ϫ54 (D1), or Ϫ83/Ϫ23 (D10) or Ϫ83/ϩ11 (D11) were bound to streptavidin magnetic beads and incubated with nuclear extract as described in Materials and Methods. After washing, the bound proteins were assayed by Western blotting using speciﬁc Abs to PU.1 (lanes 1–5) or to GABP␣ (lanes 6–8). Lanes 3 and 8, A concatamerized D1,

(D1)n, oligonucleotide was bound to the beads for pull-down. indicated that the rGABP␣/rGABP␤ binds to the distal but not to beads were incubated with HL-60 nuclear extracts, washed and the by guest on October 2, 2021 the proximal ETS site, whereas rPU.1 clearly showed the opposite eluted proteins analyzed for the presence of GABP␣/GABP␤ and binding preference. PU.1 by Western blotting. The results showed that PU.1 protein Binding of native proteins from HL-60 nuclear extracts to D1 was only bound to D11 and not to D10 or D1 (Fig. 3C, lanes 4 and DNA probes also resulted in formation of two major shifted bands 5), i.e., this protein occupied the proximal but not to the distal ETS (Fig. 3B, lane 2). We then used purified polyclonal Abs raised site. GABP␣ was detected on the beads with D1 DNA, i.e., bound against recombinant GABP ␣ and ␤ (see Materials and Methods) to the upstream, distal ETS site (Fig. 3C, lane 7). GABP␣ binding in EMSA to specifically test for the presence of these proteins in to this site was significantly enhanced when a concatamerized D1 the D1 DNA-binding complexes. Both anti-GABP␣ and anti- oligonucleotide was used for the pull-down (Fig. 3C, lane 8), re- GABP␤ Abs, either alone or as an equimolar mixture, interfered flecting its low level of expression in the nuclear extract. In sep- with the formation of D1-protein complexes (Fig. 3B, lanes 3, 4, arate experiments, GABP ␣, as well as ␤, could be detected as and 6), whereas purified preimmune Igs from the same animals did bound to multimer D10 and D11 fragments by Western blotting not have an effect (Fig. 3B, lane 5). Abs specific for other ETS- (data not shown). Thus, three lines of evidence clearly established family proteins: PU.1, Elk-1, Ets-1, Ets-2, Elf-1, Etv-1, and Erg-1 that, in vitro, GABP specifically binds to the upstream GGAA site did not have any effect on D1-binding activity (data not shown). (Ϫ32/Ϫ25), whereas the PU.1 protein binds to the proximal Furthermore, dsDNA probes containing known binding sites for all GGAA site (Ϫ22/Ϫ19). these other factors did not effectively compete the D1 probe in an EMSA either, whereas a probe containing the GABP binding site PU.1 and GABP bind to the def1 promoter in vivo of the thrombopoietin promoter (40) did so quite well (data not To verify in vivo occupancy of the def1 promoter by PU.1 and shown). Comparable EMSA results in all cases were observed us- GABP␣/␤, in the context of chromatin in intact promyelocytic ing nuclear extract from NB4 cells as well (data not shown). These leukemia NB4 cells, we performed chromatin immuno-precipita- observations provide further proof that the endogenous GABP pro- tion analyses. Abs against PU.1 and GABP did efficiently and spe- teins are directly involved in binding to the distal ETS site in the cifically precipitate def1 promoter DNA (Ϫ160/ϩ11), whereas def1 promoter in vitro. preimmune sera could not, suggesting stable in vivo association of To further analyze factor occupancy, we prepared biotinylated these two factors with the promoter (Fig. 4A, lanes 3 and 4, re- DNA fragments spanning different overlapping sequences of the spectively). The proteins remained bound to the promoter in NB4 core promoter region, either as monomers or concatamers, that cells treated with 1 ␮M all-trans RA for 72 h (data not shown). were immobilized on streptavidin-coated magnetic beads as illus- Positive control ChIP analyses were conducted using the same trated in Fig. 3C (upper panel). The D1 and D10 DNA fragments cells as well as the anti-PU.1 and anti-GABP␣/GABP␤ Abs, and contained the distal ETS binding site; the D11 DNA fragment different sets of primers (see Materials and Methods and Fig. 4 contains both the distal and the proximal ETS sites. These DNA legend), corresponding to well-documented target genes of these 6912 MYELOID DEFENSIN-1 PROMOTER

Functional cooperativity between a weak TATA box and the adjacent PU.1 binding site in region (Ϫ33/Ϫ15) To gain further mechanistic insights into defensin-1 core promoter function, it was necessary to dissect the proximal control region into its individual, constituent elements, and to explore the possi- bility of physical and functional interaction between their cognate binding proteins. The transcription start site (ϩ1) was previously determined by primer extension and S1 nuclease protection (25), and the surrounding sequence is very similar to the initiator element consensus (43). Thus, the slightly upstream TTTAAATA sequence (Ϫ32/Ϫ25; underlined in Fig. 1) fulﬁlls the requirements of a vertebrate TATA box by established criteria of weighted consensus sequence and location (44). Mutation of this sequence ab- rogates minimal promoter (Ϫ83/ϩ82) activity in HL-60 cells, consistent with a functional role (25). The PU.1-binding, GGAA core sequence (Ϫ22/Ϫ19) just downstream from the TA-rich site is also functionally important, as its disruption resulted in reduced promoter efﬁcacy in vivo.

We compared the sequence of the Ϫ33/Ϫ15 region of the def1 Downloaded from promoter with known eukaryotic TATA box and the PU.1 binding site base frequencies, either derived from 389 unrelated promoter sequences (44) or, respectively, established by PCR-mediated random site selection (45). Two observations can readily be made from inspecting the alignment in Table I. First, the two 15-nt long

sites overlap by 11 bp. Second, the def1 promoter sequence is in http://www.jimmunol.org/ FIGURE 4. PU.1 and GABP bind to the def1 promoter in vivo. A, ChIP full accordance with the conserved core elements of each site (i.e., analyses were performed in NB4 cells using Abs against GABP␣ and ␤ TATAAA and GGAA), except that the second base of the TATA (lane 3) and PU.1 (lane 4), or in the absence of Abs (lane 5). Negative (the underlined nucleotide has been mutated) box is about 10 times controls were done with respective preimmune sera or nonspecific Abs more frequently an A in eukaryotic promoters than the T (91 A vs (data not shown). PCR primers were designed to amplify the def1 promoter 9% T; see Table I) that occurs in this particular position (at Ϫ31; Ϫ ϩ sequence 160 to 10 (25); the amplified DNA fragment is 170-bp long. see Fig. 1) in the def1 promoter. We therefore investigated the role B, ChIP analyses using NB4 cells and anti-GABP␣ and ␤ Abs (lane 4). this nucleotide might play in def1 minimal promoter activity. PCR primers (see Materials and Methods) were designed to amplify the Synthetic TA oligonucleotides were prepared that carried a mu- CD89 promoter sequence Ϫ143 to ϩ8 (42); the amplified DNA fragment by guest on October 2, 2021 is 152-bp long. C, ChIP analyses using NB4 cells and anti-PU.1 Abs (lane tation of T-31 to either G (0% frequency in the second position of 4). PCR primers (see Materials and Methods) were designed to amplify the a TATA box; Table I) or to A (91% frequency), and were used to p47phox promoter sequence Ϫ162 to ϩ137 (41); the amplified DNA frag- compete with the binding of nuclear proteins to the wild-type TA ment is 299-bp long. Analysis in A–C: lane 1, 100-bp ladder DNA-mark- probe (Fig. 1) in EMSA. Neither change had much noticeable ef- ers; lane 2, PCR on total input DNA. fect; both mutant oligonucleotides competed as effectively as the wild-type TA competitor (Fig. 5A, lanes 3, 6, and 7). As expected, mutations of the PU.1 binding site (GGAA to GCCC; TAM2 in factors, namely, the p40phox promoter (41) and the CD89 promoter Fig. 1 and Table I), alone or in combination with the aforemen- (42). The results shown in Fig. 4, B and C, indicate that our anal- tioned nucleotide changes, did not compete (Fig. 5A, lanes 4 and yses have been done properly and using adequate reagents. 5). The same trinucleotide (Ϫ21/Ϫ19) change is unlikely to affect

Table I. Putative location of a TATA box and PU.1 binding site in myeloid def1 promoter sequence (Ϫ33/Ϫ15)a

Base Frequency

TATA box, (n ϭ 389) A 16 4 91 1 91 69 93 57 40 14 21 21 21 17 20 C371203001 0 11 35 8 33 30 28 26 G 39500105124039 33 33 33 36 36 T 8 79 9 96 831 131 9 12 8 13 16 19 18

Ϫ33 cTTTAAA t a g gGGA A g t c cϪ15 PU.1 binding site, % (n ϭ 26–52) A 47486579 45 14 27 0 0 100 100 2 4 41 16 C 1514602253700 0 0341412 26 G 19 21 10 3 21 62 35 100 100 0062 93242 T 19 17 19 18 320100 0 0273 15 16

a The core elements of the putative TATA box (TTTAAA) and PU.1-binding site (GGAA) in the def1 promoter sequence (center panel) are in capitals; the rest of the sequence is in lower case. TATA-box base frequencies (in percent; upper panel) were derived from 389 unrelated promoter sequences (44); PU.1 binding site base frequencies (lower panel) were established by PCR-mediated random site selection (45). Frequencies of those nucleotides that occur in the def1 promoter in a particular position are shown in bold in the frequency tables. The Journal of Immunology 6913 Downloaded from http://www.jimmunol.org/

FIGURE 5. Sequence specificity of PU.1 binding to the def1 TA- probe (Ϫ39/Ϫ10). A, Nuclear extract from HL-60 cells was used in EMSA for binding to the wild-type TA-probe in the presence of 200-fold molar excess of mutant oligonucleotides (B). Lane 1, Free TA probe; lane 2, TA probe Ϫ Ϫ incubated with 10 ␮g of nuclear extract; lane 3, competition by a cold TA FIGURE 6. Function of a TA-rich sequence ( 32/ 27) and of proxi- Ϫ Ϫ Ϫ Ϫ probe; lanes 4–7, competition with mutant oligonucleotides as indicated mal ( 22/ 19) and distal ( 62/ 59) GGAA regulatory sequences in a Ϫ ϩ by guest on October 2, 2021 above the lanes. B, Wild-type and mutant oligonucleotides used in EMSA, def1 minimal promoter ( 83/ 82) for promyelocytic expression in vivo. representing the Ϫ39/Ϫ10 upstream sequence of the def1 promoter. Mu- The effect of specific mutations in these elements on def1 promoter activity tated nucleotides are presented in bold and underlined characters. was studied in transient transfection experiments in HL-60 (A) and KG-1 (B) cells as described under Materials and Methods. The wild-type construct (a), two truncations (b, c) and combinations of variously mutated promoter sequences (d-I; see Materials and Methods, Plasmids for details) putative TBP/TFIID binding to the TA region in terms of base were inserted upstream of the luciferase (Luc) gene. The numbering is preferences, as GAA and CCC occur with similar frequencies in relative to the transcriptional start site ϩ1ofthedef1 gene. Cells were those three positions of the TATA box (Table I). However, this transfected with these constructs by electroporation, and assayed for lucif- could not be validated by EMSA. erase activity after 5-h incubation at 37°C. All measurements were nor- These and previous (25) in vitro and in silico protein-binding malized per nanogram of secreted hGH, coexpressed under CMV promoter control. The results represent the mean of at least three different experi- data guided introduction of pinpoint mutations in the proximal and ments and are shown in each graph separately, plotted as a fraction of the distal regulatory regions for in vivo functional evaluation, using activity of the wild-type construct (arbitrary 100%). Note that the hGH- transient transfections of HL-60 cells. Thus, a series of reporter normalized, luciferase activity of the wild-type construct differed in the two constructs containing a luciferase gene fused to the def1 minimal cell types; on average 5- to 10-fold higher in HL-60 cells. Symbols: open promoter (Ϫ83/ϩ82) were created with targeted mutations in the boxes labeled D1 and PU.1 indicate binding sites for GABP and PU.1; the TTTAAA box, the PU.1 binding site and the distal GABP binding open box labeled TA is a TA-rich sequence (TTTAAA in the wild-type site, either alone or in various combinations (Fig. 6A). Analysis of promoter); a large X through any box indicates functional disruption (see the intact minimal promoter (construct a) and two truncated ver- Materials and Methods and Results for details); the enlarged (shaded) TA sions (b and c) recapitulated earlier experiments (25); normalized box represents a strong TATA box (TATAAA). activity of construct a was assigned an arbitrary value of 100%. Removal of the D1 box and of the entire ϽD1 ϩ TA ϩ Pu.1Ͼ containing region resulted in reduction of activity by 90 and 97%, respectively. Specific elimination of the functional PU.1 binding created a bona fide TATA box (T-31 to A; construct g) increased site (construct d) or the TTTA (the underlined nucleotide is T-31 minimal promoter activity to 160%. In combination with a PU.1 in defl promoter) box (T-31 to G; construct e), or both (construct binding site knockout (construct h), the improved (or strong) f), reduced promoter activity to, respectively, 45, 18, or 10%. In TATA box increased activity still further, up to ϳ250%. Similarly, addition, inactivation of the GABP binding site (construct i) cut the a strong TATA box could also boost activity of a minimal pro- activity to a mere 10% just by itself. A combination of distal and moter lacking both GABP- and PU.1 binding sites (construct l) by proximal site disruptions (constructs j and k) virtually eliminated a factor of 10; from 5 to 50% of wild-type promoter. This, how- all activity (ϳ3% of wild type). Interestingly, a point mutation that ever, was still five times lower than the most active combination 6914 MYELOID DEFENSIN-1 PROMOTER consisting of a strong TATA box, GABPϩ and PU.1Ϫ. Introduc- tion of a strong TATA box in a def1/Luc reporter construct analyzed in myeloblastic KG-1 cells also had a stimulatory effect on an otherwise much lower activity (ϳ5–10% of HL-60 cells), which was not further enhanced by disruption of the PU.1 binding site (Fig. 6B; constructs g and h). PU.1-site disruption had, by itself, little effect on reporter activity (construct d). These findings were, in fact, in perfect agreement with the above results obtained in HL-60 cells, because KG-1 cells do not express measurable levels of PU.1 protein (Ref. 25; data not shown), which makes the presence or absence of a functional binding site irrelevant. All the above observations suggest some intriguing mechanisms of activator and general transcription factor binding and cooperativity, which we will elaborate on in the discussion. Still, we first wanted to further establish in vivo function of each of the implicated factors, in combination with the effects of either a weak (TTTAAA) or strong (TATAAA) TATA box.

Functional reconstitution of human myeloid defensin-1 core Downloaded from promoter activity in Drosophila SL2 cells PU.1 expression is restricted to myeloid and B cells (46, 47), but GABP is rather ubiquitously expressed in human cells (18), which could potentially complicate coexpression analyses in HeLa or other type of heterologous cells. We therefore selected Drosophila SL2 cells for these experiments because they lack all the factors http://www.jimmunol.org/ under study. SL2 cells were transfected with either wild-type or mutant (T-31 to A) def1 minimal promoter luciferase constructs along with expression vectors carrying cDNAs for either GABP ␣, ␤, PU.1, or equimolar mixtures of any of those. Expression of these proteins was under control of the CMV promoter which had previously been found to function in SL2 cells (36). Cotransfec- tions with either GABP␣ alone or GABP␤ alone did not result in any measurable activity over background. However, cotransfection ␣ ␤ by guest on October 2, 2021 of two cDNAs expressing both and subunits resulted in a FIGURE 7. GABP␣/GABP␤ and PU.1 have different roles in activation 2.5-fold enhanced activation of the wild-type def1 reporter con- of the def1 minimal promoter in Drosophila cells. SL2 cells were trans- struct (Fig. 7A). The effect of PU.1 cDNA expression alone was fected with 1 ␮g of minimal def1 promoter (Ϫ83/ϩ82)-driven reporter quite low. In contrast, cotransfections with all three cDNAs, ex- construct in pGL3 as described in Materials and Methods. For cotransfec- pressing GABP ␣, ␤, and PU.1, resulted in a signiﬁcant, close to tions, 2 ␮g of pcDNA expressing GABP␣, GABP␤, and PU.1 alone or in 10-fold activation of the wild-type construct (Fig. 7A). Doubling combinations were added as indicated. A, Effects of expression of GABP the amount of cDNA for either GABP␣/GABP␤ or PU.1 could not and PU.1 on the wild-type luciferase construct. B, Effect of expression of compensate for the absence of the other factor (data not shown). GABP and PU.1 on a construct with a T-31A mutation in the TATA-like box (i.e., to create a strong TATA box). For normalizations, each sample These results strongly suggest that GABP␣/GABP␤ and PU.1 co- was cotransfected also with 1 ␮g of plasmid expressing ␤-galactosidase. operate in def1 minimal promoter activation. Luciferase activity was measured after 48 h; the results represent the mean In additional experiments, we tested the activation in SL2 cells (ϮSD) data from three separate experiments. of a mutant reporter construct, containing a strong TATA box (T-31 to A; construct g in Fig. 6), by the two ETS-family proteins. By itself, this particular construct showed almost double the activity of the wild-type version in ﬂy cells. Coexpression together Discussion with PU.1 cDNA did not appreciably increase transcriptional ac- A minimal def1 promoter (Ϫ83/ϩ82) can drive basal transcription tivity (ϳ1.2ϫ), whereas coexpression with both GABP subunits in HL-60 promyelocytic leukemia cells, and perhaps also during led to robust, 5-fold activation (Fig. 7B). This is also 4-fold over granulopoiesis, under direct control of at least two nuclear factors, the normalized activity of a wild-type reporter in the presence of PU.1 and a def1 D1BP of unknown identity at the onset of this GABP␣/GABP␤. Again, these results revealed an intricate rela- study. Each factor, plus various accessory proteins, uniquely func- tionship between the ETS-factors and the protein(s) binding the tions through one of two ETS-like, regulatory elements; PU.1 TATA box, likely TBP/TFIID. Apparently, PU.1 operates in a pos- through a proximal and D1BP through a distal site (D1 box), po- itive or helper role when positioned on the wild-type promoter in sitioned some 30 bp apart (24, 25). Inducers of granulocytic dif- close proximity to the weak TTTA box or to an inactive TGTA ferentiation, such as all-trans RA and 9Cis-RA, greatly enhance box but, conversely, has an appreciable negative effect when delayed late def1 expression through both ETS elements and each bound adjacent to a strong TATA box on an otherwise unaltered factor (24). In contrast, some agents that activate def1 expression promoter. No such effects were observed for GABP binding to the but fail to induce cell maturation appear to function through a distal control element, which consistently resulted in a 5- to 10- single site/factor only; for instance, PGE2 acts through the D1 box, fold relative transcriptional activation in the presence of either a and LPS and TNF-␣ through a proximal PU.1 binding site (24). weak or a strong TATA box. Furthermore, PGE2 treatments greatly augment the RA response The Journal of Immunology 6915

(23), whereas the inflammatory regulators do not (24), all suggesting a cAMP-stimulated, enhancer-like capacity for the D1 box and its binding protein, and supporting a model that D1BP and PU.1 are the endpoints of separate signaling pathways (24). As RA- dependent and -independent def1 regulation is strictly lineage specific, even within the class of myeloid blood cells (12, 23), trans- duction may be modulated by additional cis-acting regions outside the minimal promoter, or by the levels, modification states or interaction patterns of any of the binding proteins. As the next necessary step to further dissect and reconstitute this system, we have now identified D1BP biochemically and functionally as the heterodimeric factor GABP␣/GABP␤. GABP (18, 37) and particularly PU.1 (34, 48) have been frequently implicated in myeloid and myeloid-specific gene regulation, most often in combinations with various other factors (18, 49–51) but in a few cases in this specific pairing as well (38, 52). FIGURE 8. Proposed interactions between wild-type and mutant my- Through a series of in vitro and in vivo experiments, we have now eloid def1 promoter proximal region (Ϫ35/Ϫ15), and PU.1 and TBP/ conclusively established that, despite substantial sequence similar- TFIID. TTTAAA (Ϫ32/Ϫ27) and GGAA (Ϫ22/Ϫ19) sequences are ities between the two respective binding sites, there exists 1) a present in the wild-type promoter sequence (see Fig. 1); TGTAAA is an strict functional requirement for the presence of both factors and 2) inactive TATA box; TATAAA is a strong TATA box; PU.1 cannot bind to Downloaded from mutually exclusive site-occupancies in the minimal promoter. This the GCCC sequence. Activity to drive transcription of a reporter construct could be the result of critical flanking sequences influencing bind- in HL-60 cells increases from the situation depicted in A (no activity) to F ing specificities of the respective factors, as well as requirements (most activity). Note that the sequence in D is the one occurring in the of specific protein-protein interactions with other nuclear factors wild-type def1 promoter. Key: TBP, TATA-binding protein. and/or general transcription factors to initiate formation of an activating complex. In fact, we have obtained compelling evidence http://www.jimmunol.org/ that, at least in the case of PU.1 binding to the proximal site, the vious contrast to its proposed role in the human Fc␥R1b promoter second postulation may actually be the more important. Specifi- (54). In contrast, putting a strong TATA box in the def1 promoter cally, we propose here that the proximal ETS-site operates in con- eliminates the need for a PU.1 binding site and by extension for junction with the adjacent TATA-like box to assemble a func- PU.1, but not for GABP, a conclusion that was duly validated by tional, primarily myeloid-specific, basal transcription complex. functional analysis in fly cells. In fact, PU.1 is even less than The experimental observations supporting this model derive optional in the presence of a strong TATA box as it appears it from a mutational analysis of the promoter region that contains might interfere with optimal TFIID binding and function. As both a TTTAAAT sequence (Ϫ32/Ϫ26) and a flanking PU.1 bind- GABP is rather ubiquitously expressed, conversion to a bona fide by guest on October 2, 2021 ing site (GGAA; Ϫ22/Ϫ19). While the TA rich element meets all TATA box could mitigate strict promyelocytic cell specificity of criteria of a vertebrate TATA box (44), its precise sequence in the the def1 promoter, as shown in KG-1 myeloblastic cells. def1 promoter, in particular the T in the second position, only A unique feature about the TTTA element-plus-helper-site motif occurs in fewer than 10% of other genes (44). Selected mutations in the def1 promoter is that it could have originated by a single of this nucleotide, either alone or in combination with a disrupted base pair change from a prototypical TATA box (A to T). Possibly, PU.1-site, were evaluated for possible effects on PU.1-binding in the existing wild-type promoter may have evolved in this way to vitro and on transcriptional activity in vivo. A summary of the become increasingly reliant on nearby positioning of PU.1 for op- results is presented in cartoon format in Fig. 8 to illustrate the timal function, thereby resulting in a de facto myeloid-restricted protein-protein-DNA interactions that we believe are taking place activity. Furthermore, PU.1-binding affinity is regulated by phos- at this locus in each configuration, and how those affect transcrip- phorylation in response to extracellular stimuli and cytokines, most tional activity (measured to increase from scenario A (lowest) to F notably by casein kinase II and p38 MAPK-dependent pathways (highest)). Panel D depicts the wild-type promoter. Functional dis- (24, 55–58). Such signals can now be readily, albeit indirectly, ruption of the TTTA element, through a T-31 to G (as present in transduced to the general transcriptional machinery, thereby pro- 0% of vertebrate TATA boxes) mutation, results in near compete viding another layer of control in addition to activator-dependent loss of transcriptional activity (A and B). By contrast, changing mechanisms operating through GABP at the distal D1 box. Bind- T-31 to A (as present in ϳ90% of vertebrate TATA boxes) results ing of active GABP, itself the endpoint of various signaling path- in a moderate increase (1.5ϫ) of activity (E). The activity is fur- ways and posttranslational modification events (18, 24, 59, 60), is ther enhanced (2.5ϫ) when the same mutation is paired with an also essential for transactivation, even in the presence of a strong inactive PU.1 binding site (F), whereas a PU.1-site knockout by TATA box. itself results in considerable loss of activity (C), as anticipated. Finally, it should be noted that our past observations on factor Protein-protein interactions depicted in Fig. 8 are based on re- binding to specific GABP(D1)- and PU.1(TA)-probes following ports that PU.1 can bind TBP/TFIID in vitro (53), and by doing so RA-treatment (25) correlate well with the reported changes in is also capable of forming a functional preinitiation, transcription GABP and PU.1 expression (at the transcript and protein levels) complex in the context of TATA-less gene promoters (54). It ap- during myeloid differentiation along the granulocytic pathway. In- pears that the human def1 promoter contains a weak TATA box, deed, nuclear extracts from HL-60 cells showed increased PU.1- relying to some extent on the proven capacity of nearby bound binding activity (by EMSA) and PU.1 protein levels (by immuno- PU.1 to tether more TBP and TFIID, and/or at higher affinities, and blot analysis) 2 days after induction (41). Reportedly, expression assemble a basal transcription factor complex within reasonable of GABP␣ does not change during granulocytic differentiation but distance from the cap site. However, PU.1 is incapable of func- those of certain splicing forms of GABP␤, in contrast, are up- tioning in such a capacity in a TATA-less def1 promoter, in ob- regulated (18, 61). In conjunction with differentiation-dependent 6916 MYELOID DEFENSIN-1 PROMOTER phosphorylation of those same factors (24), these changes may 22. Holmberg, C. I., S. E. Tran, J. E. Eriksson, and L. Sistonen. 2002. Multisite fully account for the increased def1 transcription. The latter phosphorylation provides sophisticated regulation of transcription factors. Trends Biochem. Sci. 27: 619–627. changes may very well be the more important as overexpression of 23. Herwig, S., Q. Su, and P. Tempst. 1998. Drug-activated multiple pathways of cDNAs for either one factor, or both, did not affect def1 transcrip- defensin mRNA regulation in HL-60 cells are defined by reversed roles of par- tion significantly (data not shown). ticipating protein kinases. Leukemia Res. 22: 913–925. 24. Wang, N., S. Boeckh-Herwig, M. Yaneva, and P. Tempst. 2004. Delayed late In summary, the myeloid def1 promoter contains a weak TATA activation of a myeloid defensin minimal promoter by retinoids and inflammatory box plus one requisite and one helper site that specifically recruit mediators. Leukemia Res. 28: 879–889. GABP and PU.1 into an active transcription initiation complex. 25. Ma, Y., Q. Su, and P. Tempst. 1998. Differentiation-stimulated activity binds an ETS-like, essential regulatory element in the human promyelocytic defensin-1 The need for a nearby PU.1 binding site for optimal TBP binding promoter. J. Biol. Chem. 273: 8727–8740. imparts a degree of cell specificity to the minimal promoter and 26. 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Michael Klemsz and 29. Hemat, F., and K. McEntee. 1994. A rapid and efficient PCR-based method for synthesizing high-molecular-weight multimers of oligonucleotides. Biochem. Thomas Brown for the gift of cDNAs, and Tony Riley and John Philip for Biophys. Res. Commun. 205: 475–481. Downloaded from help with the artwork. 30. Yaneva, M., and P. Tempst. 2003. Affinity capture of specific DNA-binding proteins for mass spectrometric identification. Anal. Chem. 75: 6437–6448. Disclosures 31. Dignam, J. D., P. L. Martin, B. S. Shastry, and R. G. Roeder. 1983. Eukaryotic gene transcription with purified components. Methods Enzymol. 101: 582–598. The authors have no financial conflict of interest. 32. Erdjument-Bromage, H., M. Lui, L. Lacomis, A. Grewal, R. S. Annan, D. McNulty, S. A. Carr, and P. Tempst. 1998. Examination of micro-tip reversed- References phase liquid chromatographic extraction of peptide pools for mass spectrometric analysis. J. Chromatogr. 826: 167–181.

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