Journal of Neuroimmunology, 35 (1991) 273-278 © 1991 Elsevier Science Publishers B.V. 0165-5728/91/$03.50

273

JNI 01074

Short Communication

Identification of an interferon-7-responsive element of a class II major histocompatibility gene in rat type 1 astrocytes H a r o l d M o s e s 1, Atsushi Sasaki 2 , . a n d J e n n y P.-Y. T i n g 1,2,3 I Curriculum in Neurobiology, 2 Lineberger Cancer Research Center, and s Department of Microbiology-Immunology, University of North Carolina, Chapel Hill, NC 27599, U.S.A.

(Received 11 September 1990) (Revised, received 20 November 1990) (Accepted 21 November 1990)

Key words: Astrocyte, type 1; Transfection; Class II major histocompatibility complex; Interferon-'/; Gene regulation

Summary Interferon-3'(IFN-3') has been shown to induce class II major histocompatibility complex (MHC) antigens on several cell types. Previous analysis of cell lines including a glioblastoma multiforme line by our laboratory has mapped an IFN-3,-responsive element to the u p s t r e a m - 141 to - 1 0 9 base pair (bp) region of the D R A promoter. Using deletion mutants, this report shows that this same general region ( - 1 3 5 to - 1 0 9 bp) is important for IFN-3, induction in two other human glioma lines and more importantly in primary astrocytes. We have confirmed that this regulatory region of the H L A - D R A gene is necessary for IFN-3, inducibility in astrocytes using a substitution mutant. Sequences beyond - 135 bp do not appear to have any additional positive or negative elements.

Introduction

Class II major histocompatibility complex (MHC) genes encode for proteins that have a central role in the immune response. Due primarily to the presence of the blood-brain barrier, the central nervous system was believed to be an immunologically privileged site. However, recent studies have demonstrated class II M H C antigens are present within the brain (Hickey et al., 1985;

Address for correspondence: Harold Moses, Ran. 209 Lineberger Cancer Research Center CB 7295, University of North Carolina, Chapel Hill, NC 27599-7295, U.S.A. * Present address: Department of Pathology, Gunma University School of Medicine, Gunma, Japan.

Male et al., 1987). More importantly, M H C class II proteins on glial cells are elevated in a number of pathological conditions (McGeer et al., 1988). In vitro studies have shown that class II bearing astrocytes can act as antigen presenting cells for encephalitogenic T-cells (Fontana et al., 1984). In culture, interferon-3, (IFN-3,) is a potent inducer of class II antigen expression on type 1 astrocytes (Wong et al., 1984). Previous work from our laboratory had identified cis-acting D N A sequences responsible for IFN-3, inducibility of M H C class II gene expression (Basta et al., 1987, 1988). These experiments were done using a glioblastoma multiforme line. In this report, we extended previous work and determined the activity of this IFN-3'-inducible responsive region in two other glioma cell lines.

274 Most significantly, the IFN-),-responsive region was also delineated in type 1 astrocytes by DNAmediated gene transfer. Conserved upstream sequences have been well characterized in the DRA promoter (Fig. 1). They are responsible for the transcriptional regulation of this coordinately regulated gene in a tissue- and cell type-specific manner (reviewed in Sullivan et al., 1987; Benoist and Mathis, 1990). These conserved elements include W, X and Y from 5' to 3' respectively (reviewed in Benoist and Mathis, 1990). X and Y have been shown to be functionally important in transfection assays (Dorn et al., 1987; Sloan and Boss, 1988; Sherman et al., 1989). They appear to be positive control elements for class II expression in different cell types (Dedrick and Jones, 1990; Finn et al., 1990), and to play a role in IFN-3, inducibility in a HeLa cell line (Tsang et al., 1990). The W box is located from - 1 3 5 to - 1 1 8 bp of the DRA regulatory region. Several laboratories, including our own, have demonstrated that this element is important in IFN-y inducibility (Basta et al., 1988; Tsang et al., 1990). However, to date the analysis of IFN-7-responsive elements has not been performed using primary, nontransformed cells. This is the first report of such an analysis. Materials and methods

Primary glial cultures. Sprague-Dawley rats (2 days postnatal at the latest) were used to prepare mixed brain cell cultures. Cerebral cortical tissue was used as previously described (McCarthy and de Vellis, 1980). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with glucose (0.9%), L-glutamine (2 mM), sodium bicarbonate, penicillin (50 I U / m l ) , streptomycin (50 p g / m l ) plus fetal calf serum (FCS) (10% v / v , Gibco). The cultures became confluent on approximately day 14 and type 1 astrocytes were purified by a differential adhesion technique (Sasaki et al., 1989). Cells were prepared for transfection by trypsinization. Gfioma lines. U105-MG (Takiguchi et al., 1985) and D54-MG (Wikstrand et al., 1984) are two glioblastoma multiforme lines that are induced to express class II M H C molecules with IFN-y treatment. These cells were maintained in

McCoy's 5A media supplemented with 10% FCS, 2 mM L-glutamine, and 0.05 m g / m l gentamicin. Recombinant rat IFN-3, (rlFN-7). Rat, rIFN-3, was purchased from Amgen (Thousand Oaks, CA, U.S.A.). A stock solution was made in phosphatebuffered saline (PBS) at 2 × 10 4 U / m l . Astrocytes were treated with rIFN-~, (50-100 U / m l ) for 2 days prior to the CAT assay. Plasmids. Plasmids containing 5' deletions (p5'A-1028 and 5'A-267) were previously described (Basta et al., 1988). They contain D R A promoter sequences from - 1 0 2 8 and - 2 6 7 bp, respectively, to +27 bp linked to a chloramphenicol acetyltransferase (CAT) gene. The plasmids in the deletion series 5'A-210 to 5'A-109 were derived from pDRA1000 as described (Sherman et al., 1987). They contain 210 and 109 bp of DRA sequence 5' of the transcription start site respectively. A substitution mutant was made by randomly mutating the region of a p D R A - C A T construct from - 1 4 1 to - 1 0 9 bp. This construct is known as pMut-IRE. The wild-type control is denoted as pWT-IRE. These were previously described in detail (Basta et al., 1988). The negative (pD164-2) and positive (pSV2CAT) control plasmids have been described (Basta et al., 1987). Transfection. Transfections were performed by electroporation using a Bio-Rad gene pulser. 5 / t g of plasmid D N A were used per transfection. 3 x 10 6 cells in 300 #1 of medium were pulsed at 200 mV at a capacitance setting of 960 #F. The cells were plated into 10 ml of complete medium and allowed to adhere before the addition of IFN-y. CA T assay. CAT activity was assayed according to the method of G o r m a n et al. (1982). Quantitative measurements were obtained by cutting out both the acetylated and nonacetylated products on thin-layer chromatography (TLC) plates and measuring radioactivity in a scintillation counter. Percent acetylation is calculated as acetylated counts/(acetylated counts + nonacetylated counts) × 100%. Results and discussion

Description of a nested series of 5" p D R A - C A T deletion mutants. A series of deletion mutants encoding the 5' regulatory region of the DRA

275

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Fig. 1. Promoter region of the HLA-DRA gene and a map of the 5' deletion mutants used in transfection experiments. The DRA transcription start site is marked by the bent arrow. The hatched box represents the CAT gene. Solid boxes represents known cis-acting dements necessary for basal transcription (Sherman et al., 1987). The open box shows the location of the IFN-y-responsive element (IRE-y) transfected into gliomas and astrocytes.

gene were m a d e to c h a r a c t e r i z e i m p o r t a n t seq u e n c e for I F N - y i n d u c i b i l i t y (Fig. 1). These constructs v a r y in length f r o m - 1 0 2 8 to - 1 0 9 b p o f the D R A u p s t r e a m sequence a n d are p l a c e d 5 ' of the i n d i c a t o r gene C A T in the p a r e n t p l a s m i d , p D 164-2 ( B a s t a et al., 1987). A l l of the c o n s t r u c t s c o n t a i n a C A A T box, a T A T A b o x a n d a trans c r i p t i o n a l start site. T h e class II boxes (X + Y) are also p r e s e n t in these constructs.

DR.4 regulatory sequences important for rlFNy-induced gene expression in gliomas. T h e deletion m u t a n t s shown in Fig. 1 were used to transfeet two g l i o m a lines ( U 1 0 5 - M G a n d D 5 4 - M G ) that express class II M H C antigens u p o n I F N - y t r e a t m e n t . T h e s e e x p e r i m e n t s are i m p o r t a n t because p r e v i o u s l y we o n l y a n a l y z e d one g l i o m a line for an I F N - y - r e s p o n s i v e e l e m e n t in the D R A gene. C A T activity was d e t e r m i n e d b y m e a s u r i n g the a b i l i t y of t r a n s f e c t e d cell extracts to acetylate the s u b s t r a t e [14C]chloramphenicol. Fig. 2 shows a r e p r e s e n t a t i v e C A T assay using a g l i o b l a s t o m a m u l t i f o r m e line d e s i g n a t e d D 5 4 - M G . S i m i l a r results were o b t a i n e d with U 1 0 5 - M G ( d a t a n o t shown). I n b o t h cell lines, I F N - y i n d u c i b i l i t y was o n l y seen w h e n the c o n s t r u c t 5'A-135 was utilized. T h e 5'za-109 c o n s t r u c t s h o w e d little or n o i n d u c tion with I F N - 7 t r e a t m e n t . Sequences further u p s t r e a m of - 1 3 5 b p d i d n o t a p p e a r to c o n t a i n a n y a d d i t i o n a l I F N - y - r e s p o n s i v e elements. These d a t a i n d i c a t e that the region b e t w e e n - 1 3 5 a n d - 1 0 9 b p is necessary for I F N - y i n d u c t i o n in two g l i o m a

a s u m m a r y of t r a n s f e c t i o n e x p e r i m e n t s d o n e using the d e l e t i o n m u t a n t s in t y p e 1 astrocytes. D e l e t i o n c o n s t r u c t s of the h u m a n H L A - D R A p r o m o t e r were t r a n s f e c t e d i n t o rat a s t r o c y t e s b e c a u s e the class II M H C p r o m o t e r sequence in the r a t has n o t been characterized. F o r each c o n s t r u c t , at least four i n d e p e n d e n t e x p e r i m e n t s were done. T h e fold i n d u c t i o n u p o n I F N - - / t r e a t m e n t was c a l c u l a t e d b y d i v i d i n g the p e r c e n t a c e t y l a t i o n value of an I F N - y - t r e a t e d c u l t u r e b y the p e r c e n t acetyl a t i o n value of an u n t r e a t e d culture. T h e c o n s t r u c t c o n t a i n i n g o n l y the class II b o x e s a n d n o t a n y a d d i t i o n a l 5 ' sequences was n o t i n d u c i b l e with I F N - y t r e a t m e n t (5'A-109). H o w e v e r , the 5'A-135 p l a s m i d elicited a 4.33 times i n c r e a s e over b a s a l levels with I F N - y t r e a t m e n t . F u r t h e r m o r e , sequences u p s t r e a m of - 135 b p d i d n o t affect I F N - y

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Fig. 2. IFN-y induction of DRA 5' deletion mutants in a human glioma line, D54-MG. The deletion constructs were assayed for CAT activity with or without IFN-y-treatment. pD164-2 contains the CAT gene without any DRA promoter sequence. Transient transfection assays were performed. Each construct was transfected in the cell line at least 4 times and a representative result is shown. The symbol ( - ) denotes no IFN-y treatment. The symbol ( + ) indicates that cells were treated with IFN-y.

276 TABLE 1 IFN-T I N D U C T I O N OF P R O M O T E R A C T I V I T Y OF p D R A - C A T D E L E T I O N M U T A N T S IN TYPE 1 ASTROCYTES Percent acetylation was calculated by comparing the amount of radioactivity in the acetylated p r o d u c t / r a d i o a c t i v i t y in the acetylated + nonacetylated products. The values shown for percent acetylation are from a representative experiment. The fold induction values are from four experiments and are calculated as the ratio of percent treated vs. percent untreated. This ratio demonstrates the IFN-y inducibifity of the various deletion constructs. The negative control, pD164-2, contains the CAT gene without D R A promoter sequence and had no induction with IFN-y treatment. Another control was pSV2CAT which contains the strong SV40 promoter that is not IFN-y inducible. pDRA-CAT construct

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the sequence of interest is altered. These differences may contribute to false levels of transcription due to cryptic start sites. As seen in Fig. 3, pWT-IRE induced CAT expression to similar levels as observed with the deletion construct, 5%-135. However, mutations in p M U T - I R E abolished IFN-y-inducible CAT expression. This confirms the importance of this region in IFN-yregulated DRA gene expression. The mechanism by which IFN-y induces class II antigen expression is not clear. Different cell lines have been shown to use various second messengers to mediate IFN-y inducibility (Jones et al., 1988; Rosa et al., 1988). Therefore, we believe that it is important to examine primary cell types to understand the precise role of IFN-y induction in a more physiologically significant model. Since class II M H C antigen expression has been correlated to autoimmune and neurodegenerative events in the nervous system (McGeer et al., 1988), defin-

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