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 References This article cites 61 articles, 33 of which you can access for free at: http://www.jimmunol.org/content/176/11/6906.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on October 2, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 leu- kemia 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 pro- moter 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.