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Multiple Regulatory Regions on the 5' Side of the Mouse Ea Gene

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Multiple Regulatory Regions on the 5' Side of the Mouse Ea Gene Proc. Nad. Acad. Sci. USA Vol. 85, pp. 3075-3079, May 1988 Immunology Multiple regulatory regions on the 5' side of the mouse Ea gene (major histocompatibility genes/y-interferon/y-interferon response region) DIMITRIS THANOS*, GEORGE MAVROTHALASSITIS*, AND JOSEPH PAPAMATHEAKIS*t *Institute of Molecular Biology and Biotechnology, Research Center of Crete, P.O. Box 1527, and tDepartment of Biology, University of Crete, 711 10 Heraklion, Crete, Greece Communicated by Fotis C. Kafatos, December 10, 1987 ABSTRACT The function of the 5'-flanking region of the motifs in the 5'-flanking region of all major histocompatibil- mouse major histocompatibility complex gene E4 has been ity complex class II genes (15, 16). We provide evidence that studied by deletion analysis with the chloramphenicol acetyl- the CS are elements necessary for the inducibility by IFN-y. transferase gene as a transient expression marker in various cell lines. This analysis reveals the presence of several control regions on the 5' side of the gene. Sequences between base pair MATERIALS AND METHODS (bp) -873 and bp -353 have a negative function in human Plasmid Constructions. The following vectors were used and mouse fibroblasts but not in the mouse macrophage line for cloning Ea and E1n gene fragments. pL51CAT was derived WEHI-3. Additional positive and negative elements have been from pSV2CAT (17) by replacing the 350-base-pair (bp) Acc mapped between bp - 353 and bp - 38. A V-interferon I-Sph I fragment by a pUC19 polylinker. pLSVOCAT was response region has been also identified within that sequence. derived from pL51CAT by elimination of the 150-bp Sma I The 5' and 3' boundaries of the -interferon response region (polylinker)-HindIII fragment. aGSCAT was produced by have been located between bp - 164 and bp -43. Inducible inserting the Pst I-Dde I fragment of a-globin (bp - 590 to bp human cell lines showed the same y-interferon response region + 20) in the Xba I site of the polylinker and subsequent endpoints with the mouse cell line WEHI-3. A DNA fragment removal of the Sma I fragment spanning bp -235 to bp - 85. spanning the equivalent region of the mouse E' gene confers aGXCAT was derived from aGSCAT by removing upstream yinterferon inducibility to the simian virus 40 and a-globin globin sequences with Xma I (bp -54), Sal I (polylinker) promoters in an orientation-independent manner. We further digestion, and religation. The 5', 3', and internal deletions provide evidence that the conserved sequence motifs on the 5' were generated on the Rsa I-Pvu I fragment (bp - 353 to bp side of all major histocompatibility complex class II genes are + 14) of the Ea gene by using convenient restriction sites or indispensable for -interferon induction. BAL-31 exonuclease, and their endpoints were determined by dideoxy sequencing. The sequence ACCCTCGACTC- Human and mouse major histocompatibility complex class II TAG links the original endpoints of internal deletions. genes are regulated by lymphokines including interferons Polylinker sequences between the original deletion end- and other cellular factors (1). Macrophages and B lympho- points and CAT sequences are as follows: ACCCCTAGAG- cytes are the main cellular targets for class II induction by GATCCCC (5' and internal deletions) and ACCC (3' dele- y-interferon (IFN-'y) and interleukin 4 (2-4), respectively. In tions). Deletion plasmids are named by their terminal base addition, other cell types such as fibroblasts and endothelial relative to the site cells (5) are induced to express class II genes in response to pairs, transcription initiation (position IFN-'y. Class II expression is critical for antigen presentation + 1). and hence the development of immune response (6, 7). The Cell Culture and Transfections. Leukemic cell lines were cell-surface density of class II antigens plays an important maintained in RPMI 1640 with 10% (vol/vol) fetal calf serum role in determining the strength of the immune response by and 5 x 10-6 M 2-mercaptoethanol. All other cell lines were generation of helper T cells (8, 9). Inappropriately high levels grown in Dulbecco's modified Eagle's medium with 10% of class II gene expression may be part of positive feedback (vol/vol) fetal calf serum. All media contained gentamycin at loops operating in certain autoimmune reactions (10, 11). At 50 Ag/ml. Transfections of 150,000 cells by the calcium the other extreme, lack of normal class II gene expression phosphate technique were performed essentially as described will produce severe immunodeficiency (12). by Graham and Van der Eb (18). For transfections with the Many gene-specific cis-acting regulatory elements that DEAE-dextran method, i07 exponentially growing cells were mediate responses to a variety of stimuli, including hor- incubated for 30 min in serum-free medium containing 50 mM mones, are known. The presence of both constitutive and Tris HCl (pH 7.4), DEAE-dextran at 300 ,tg/ml, and DNA at interferon-inducible regulatory sequences on the 5' side of 6 pug/ml. This medium was removed, and the cells were the promoter region of mouse class I genes has been shown further incubated in medium containing serum and 100 ,uM (13, 14). To study class II gene regulatory sequences we chloroquine diphosphate for 1.5 hr before washing the cells have used plasmid constructs carrying 5' sequences of the and incubating in regular medium. Cells were harvested mouse Ea and E,, genes fused to the chloramphenicol ace- usually 40-44 hr after initiation of the transfection. tyltransferase (CAT) gene. Various cell lines were transfect- Response to interferons (100 units/ml) was usually evalu- ed with these constructs, and CAT activities were deter- ated at the end of 22-24 hr of incubation. Recombinant mined in transient expression assays. We describe here the human a-interferon and IFN-y were produced by Genentech identification of several control regions on the 5' side of the (South San Francisco, CA) and supplied by Boeringher Ea gene, including a IFN-y response region. The IFN-y Ingelheim (Vienna, Austria). A mixture of mouse a- and response region encompasses the conserved sequence (CS) p-interferon (type I) was kindly provided by I. Gresser (Villejuif, France). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: IFN-'y, y-interferon; CS, conserved sequence(s); in accordance with 18 U.S.C. §1734 solely to indicate this fact. SV40, simian virus 40; CAT, chloramphenicol acetyltransferase. 3075 3076 Immunology: Thanos et al. Proc. Natl. Acad. Sci. USA 85 (1988) CAT and RNA Analysis. CAT assays were performed A +1 essentially as described by Gorman et al. (17). Enzyme S activities were normalized by assays (13) -1717 I- P3-galactosidase H through cotransfection of test plasmids with pCH110 (19). i R Isolation of total cellular RNA, agarose/formaldehyde - gels, and transfer to GeneScreen membranes were as de- -33-215p. scribed by Maniatis et al. (20). Hybridizations were per- x formed with nick-translated fragments. S1 protection analy- sis was performed with 5'-end-labeled DNA probes as de- - 630 scribed by Weaver and Weissman (21). B RESULTS * * * * * fiq.: V::; 0 Mouse Class 1 5' Genomic Sequences Confer IFN-y Induc- * *ft ,@O@ e ibility to the a-Globin Gene. Mouse class II-human a2-globin 40 0A hybrids were constructed (Fig. 1A) and transfected into _I* 6 , *- HeLa cells, which have an endogenous class II response to O'm.s _f. a do IFN-y (unpublished data). RNA expression and response to _ + -4_ + - + _9+ - + IFN-'y were studied by RNA gel blot analysis and S1 nuclease mapping. These hybrids showed increased levels of 1 717 8 73 353 215 97 630 RNA in response to IFN-y from the expected initiation sites ofthe E, and E, genes (Fig. 1B). Plasmid constructs with the simian virus 40 (SV40) enhancer-promoter region fused to the E. gene (bp 14 to bp 5390) or an intact a-globin gene did not respond to IFN-y (data not shown). These results suggest that sequences within bp -630 to bp + 50 and bp ..****P*, S.,. - 353 to bp + 14 of the E,, and E, genes, respectively, are _ + 8. + _ + _ + - + _ + involved in IFN--y control. Deletion Analysis of the 5' Region of E. Gene. To analyze FIG. 2. Structure of 5' deleted E.-CAT gene fusion constructs. in more detail the sequences involved in IFN-,y response, we (A) Position of transcription initiation site (arrow), class II- have studied deletions from bp - 1717 of the E, gene fused conserved sequences (solid box) and TATA box (triangle) are to the CAT gene (Fig. 2A). Transient expression assays were indicated. The bp - 630 to bp + 50 construct is an E fusion to CAT. performed by transfection in HeLa, xeroderma GM4429B, The rest are E. constructs fused at bp + 14 to CXT. S, Sal I; H, HincII; R, Rsa I; A, AcC I; X, Xmn I; B, BamHI. (B) Analysis of and the mouse macrophage-like line WEHI-3. Treatment with CAT activities of the deletions after transient expression into GM4429B or WEHI-3 cells (upper and lower sets, respectively) A following a 24-hr incubation in the absence (-) or presence (+) of human or mouse IFN-y. The 5' endpoints of each construct are -630 Ep T r shown between sets. +50 +36 IFN-y over the last 20-24 hr before termination of the -2451 +80 experiment showed stimulation of CAT activity for all plas- mids except the deletion at bp -97 (Fig. 2B). Heterologous - 353 Ea IVrl IFN-y showed marginal stimulation of CAT activity and a- or type I interferon was totally inactive (data not shown).
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