Regulation of expression of glial filament acidic protein

Regulation of expression of glial filament acidic protein SRIJATA SARKAR and NICHOLAS J. COWAN Department of Biochemistry, NYU Medical Center, 550 First Avenue, New York, NY 10016, USA Summary The regulation of cell type-specific expression of the gene encoding glial filament acidic protein (GFAP) was examined by introducing various deletion mutants of the gene into GFAP-expressing (U251 human astrocytoma) and non-expressing (HeLa) cell lines, and measuring their transcriptional activity in an RNAase protection assay. The expression of GFAP is influenced by a number of cis-acting elements. A domain that resides between nucleotides -1631 and 1479 can confer cell type-specific expression when coupled to a heterologous gene. We also present evidence for the existence of a negative regulatory element that resides within the first intron of the GFAP gene. Key words: glial filament acidic protein, U251 human astroma cells, HeLa cells, transcription, cis-acting regulation. Introduction In higher eucaryotes, the cytoskeleton is composed of three filamentous networks consisting of microtubules, actin filaments or intermediate filaments. Early in the developmental lineage leading to mature astrocytes, there is a switch from the synthesis of vimentin to the synthesis of glial filament acidic protein (GFAP) as the principal intermediate filament protein. The restricted expression of GFAP to astrocytes has made it a convenient marker for these cells, as well as for the study of gliomas. In addition, GFAP is a major component of the glial scars that occur following physical or biological trauma in the CNS. GFAP is therefore a major cell type-specific protein whose expression changes with development and responds to external cues. Several years ago, we cloned a GFAP-encoding cdna from mouse brain and determined the complete sequence of the corresponding single copy gene (Balcarek and Cowan, 1985). We have used a variety of constructs (described herein) to define two major cis-acting regulatory elements that contribute to the cell type-specific expression of this gene. One is a positive upstream regulatory element that resides about 1.5 kb 5' to the transcriptional initiation site (nucleotides 1631 to -1479). The second is a negative regulatory element that lies within the first intron of the gene. Materials and methods Plasmids The constructs used in this work are described in the figure legends. Details of the assembly of these constructs has been published elsewhere (Sarkar and Cowan, 1991). Journal of Cell Science, Supplement 15, 97-102 (1991) Printed in Great Britain The Company of Biologists Limited 1991 Ribonuclease protection assay Three antisense 32P-labelled probes were generated for quantitative analysis of expression in transfected cells (Fig. 1). The first extended from nucleotide -31 6 to +232 of the GFAP gene; the second spanned the cap site of the human /3-globin gene, and was used to detect expression of GFAP/human /3-globin chimeras; and the third extended from nucleotides -3 1 6 to +227 of a mouse /3- tubulin cdna, M/31 (Wang et al. 1986) and was used for the analysis of transcripts from GFAP/M/31 chimeras (see text). Cell culture and transfection HeLa cells, rat C6 gliomas and U251 human astrocytoma cells were grown in Dulbecco s modified Eagle s medium containing 10 % calf serum. Cells were cotransfected with the various GFAP promoter constructs and the psvneo plasmid (Mulligan and Berg, 1981) as described by Graham and Van der Eb (1973). 48h after the addition of DNA, cells were split and the aminoglycoside G418 was added to the medium (0.4-0.5 mg m l"1final concentration) so as to select for cells that had acquired the psvneo plasmid. 2-3 weeks later, G418-resistant cells were pooled, amplified and used for the isolation of RNA (Chirgwin et al. 1979). Results Two cell lines that express GFAP at high levels were chosen for these studies: rat C6 cells (Benda et al. 1971; Brissel et al. 1975) and human U251 cells (Westermark, 1973). HeLa cells were used as a model of a non-glial cell that expresses no detectable GFAP. In early experiments, we attempted to use the chloramphenicol acetyl transferase (CAT) gene as a reporter function to assay for the activity of a variety of GFAP- GFAP 1... _ l -3 1 6 + 232 (3-globin L_ -4 8 +473 131 -tubulin 1-3 1 6 + 227 Fig. 1. Fragments used for RNAase protection experiments. (A) Pstl-Sacl fragment (nucleotides 316 to +232) spanning the cap site of the GFAP gene. (B) Xbal-BarriHl fragment (nucleotides -4 8 to +473) spanning the cap site of the human /3-globin gene. (C) Pstl-Smal fragment from the fusion construct G / /31 spanning the cap site of the GFAP gene and the first 227 nucleotides of the mouse /31 tubulin cdna (Lewis et al. 1987). 97

derived constructs. However, we found that the CAT plasmid without any GFAP promoter sequences was expressed at a high level in U251 cells, and it was impossible to distinguish background CAT activity from activity due to cloned GFAP-derived sequences. Conse quently, we adopted an RNAase protection assay to define cis-acting elements in the mouse GFAP gene. This assay depends on the hybridization of a labelled DNA probe with specific RNA transcripts such that hybridised RNA molecules become resistant to digestion with ribonucleases. Differences in DNA sequence allowed us readily to distinguish between transcripts from the endogenous rat (C6) or human (U251) GFAP genes. There are two advantages to this approach. First, transcripts that initiate artifactually on vector sequences can be readily distinguished from those that are correctly initiated. Second, GFAP encoding sequences are retained. This turned out to be important, since sequences within the GFAP gene itself do indeed affect its level of expression. We first established that the GFAP gene is regulated in a cell type-specific fashion by transfecting the entire cloned GFAP gene (including 2 kb each of upstream and downstream flanking DNA) into U251, C6 and HeLa cells. Analysis by RNAase protection of RNA prepared from pooled G418-resistant transfected cells (see Materials and methods) showed the presence of a diagnostic 232nucleotide protected fragment derived from correctly initiated transcripts in U251 and C6 cells; no correspond ing fragment was detectable in parallel experiments with HeLa cells (data not shown). Thus, the GFAP gene fragments used in these experiments contain sequences that are required in cis for the expression of the mouse GFAP gene in glial cells. Upstream sequences that regulate the glial-specific expression o f GFAP A positive upstream regulatory element. We first exam ined the consequences of removing elements of the 5' flanking sequence by deleting a 501 bp segment extending from nucleotide -1980 to -1479. This led to the virtual abolition of expression in transfected U251 cells (Fig. 2) leaving the very low level of expression in HeLa cells unaffected. Therefore, a significant positive regulatory element (which we refer to as the distal element) resides within this 501 bp sequence. The distal element confers glial-specific expression on a heterologous promoter. To see whether the distal element could confer cell type-specific expression on a heterologous promoter, we assembled constructs (termed G.H/3gl and G'.H/Sgl, see Fig. 3) in which the distal element was coupled in either orientation in front of a human /?-globin gene fragment containing upstream sequences including the TATA box, but excluding the natural enhancer. Control experiments showed that this latter fragment did not express on its own in either U251 or HeLa cells (Fig. 3, tracks 1 and 6). A third chimeric plasmid was also constructed in which the distal element was located downstream from the human /3-globin promoter. Analysis of U251 cells by RNAase protection assays yielded two bands (Fig. 3): a 140 bp band that is derived from correctly initiated GFAP transcripts and a 213 bp fragment rep resenting transcripts initiated both at the authentic GFAP cap site and at the second exon of the human /3globin gene. The construct G.H/3gl is strongly expressed in U251 cells, in contrast to G'.H/igl or GOP.H/igl (Fig. 3, tracks 3-5 ). None of these constructs are expressed in HeLa cells (Fig. 3, tracks 8-10); a control construct in 98 S. Sarkar and N. J. Cowan,CAAT ( - 8 0 ) ATA (-2 5 ) +1 Sc Sm _I_ Sa B 1 2 3 4 Wr flh Fig. 2. (A) Diagramatic representation (not to scale) of the GFAP 5' upstream region and the first exon. The solid box represents the 5' untranslated region and the open area represents the first exon. E, ScoRI; B, BgllI; P, Pstl; Sm, SmaI; Sa, Sail', Sc, Sad. Arrow shows the extent of the distal element that contains a positive regulatory element (see text). (B) Transcriptional activity of 5' deletion mutants in stably transfected U251 and HeLa cell lines. 20 ng of total RNA extracted from cells stably transfected with two constructs (each cloned in puc) were hybridized with GFAP antisense RNA. Ribonuclease-resistant fragments were analyzed by electrophoresis on a 6% polyacrylamide urea gel. Track 1: RNA from HeLa cells transfected with the entire GFAP gene. Track 2: RNA from HeLa cells transfected with the GFAP gene lacking the distal element (see Fig. 2A). Track 3: RNA from U251 cells transfected with the entire GFAP gene. Track 4: RNA from U251 cells transfected with the GFAP gene lacking the distal element. Upper arrowhead shows location of the protected fragment corresponding to correctly initiated transcripts from the transfected mouse GFAP gene. Lower arrowhead shows protected fragment resulting from the endogenous expression of human GFAP. which the SV40 enhancer was linked to the human /3globin gene yielded high levels of correctly initiated transcripts in both U251 and HeLa cells (Fig. 3, tracks 2 and 7). Two conclusions may be drawn from these data: (1) the distal element is capable of driving a heterologous gene in a cell type-specific fashion when it is positioned upstream in the 5-3 ' orientation; (2) the distal element cannot function in either a distance or orientation independent manner, in contrast to the behavior of classical enhancers. Critical regions within the distal element. To define critical regulatory regions within the distal element in more detail, various subfragments were made and fused to the 5' end of the human /3-globin promoter. Each construct was assayed for its ability to express in U251 cells (see Materials and methods). The results of these experiments (Fig. 4) show that the positive regulatory element confer ring cell type specificity on the human /3-globin gene is abolished by the deletion of sequences between nucleotides -1631 and -1569, suggesting that the binding of positive regulatory factor(s) depends on sequences in this region, either directly or indirectly, and that the upstream regulatory element is contained within the region -1631 to -1479. Intragenic sequences influence the expression o f GFAP To assess the contribution of intragenic sequences to the

U251 HeLa I------------------------------ 1 I------------------------------- 1 1 2 3 4 5 6 7 8 9 10 m m ( < * regulation of the GFAP gene, experiments were conducted in which these sequences were substituted by an unrelated cdna (encoding a mouse /3-tubulin) which served as a reporter. Transfection of this construct (G//31) into U251 and HeLa cells resulted in expression in both cell types, though at a higher level in U251 cells (Fig. 5, tracks 1 and 3). This result contrasts with parallel experiments using the entire GFAP gene cloned in the same vector; in this case, the level of expression in U251 cells is vastly greater than in HeLa cells (Fig. 5, tracks 2 and 4). These data imply the existence of one or more regulatory elements 1 2 3 4 5 6 m ' Fig. 3. Transcriptional activity of distal element - H/i-globin chimeric constructs in stably transfected U251 and HeLa cells. 20,ug of total RNA isolated from transfected cells were hybridized to /3-globin specific antisense RNA generated by in vitro transcription of the template psp65-h/3gl (Fig. 1). After RNAase digestion, ribonuclease resistant fragments were analyzed as described in Fig. 2. Tracks 1-5, RNA from U251 cells. Tracks 6-1 0, RNA from HeLa cells. Cells are transfected with: tracks 1 and 6, H/3gl(-E), a negative control consisting of the /3-globin gene (nucleotides 48 to +2166) lacking transcriptional enhancers; tracks 2 and 7, SV40.H/3gl, a positive control consisting of the /3-globin gene preceeded by the SV40 enhancer; tracks 3 and 8, G.H/3gl, the distal element (nucleotides -1 9 8 0 to 1479 of the GFAP gene) cloned 5'-3' in front of the /3-globin gene; tracks 4 and 9, G'H/3gl, the distal element cloned 3'-5' in front of the /3-globin gene; tracks 5 and 10, GOP.H/igl, the distal element cloned downstream from the /3-globin gene. Lower arrow, 140 bp ribonuclease resistant fragment corresponding to the correctly initiated /3-globin transcript. Upper arrow, 213 bp transcript arising from spurious initiation within the second exon of the H/3gl gene. Fig. 4. Delineation of sequences within the distal element (nucleotides 1980 to 1479) that are responsible for conferring cell type-specific expression of GFAP. Successive sequential deletions of the distal element were coupled to the gene encoding human /3-globin (H/3gl) (see lower part of the figure) and stably transfected into U251 cells. RNA from cells transfected with different constructs was isolated and analyzed by RNAase protection as described in Fig. 2. Track 1, H/3gl (human /3-globin gene lacking any distal element sequences); track 2, the intact distal element coupled to H/3gl (G.H/3gl); track 3, ( 1844 to 1479)-H/3gl; track 4, ( 1736 to 1479)H/?gl; track 5, (-1 6 3 1 to - 1479)-H/3gl; track 6, (-1 5 6 9 to -1479)-H/3gl. Diagramatic representations of each construct are shown below. Open box, /3-globin gene. Hatched boxes, successively 5' truncated distal element. Upper and lower arrows indicated spurious and correctly initiated transcripts, respectively. -1 980-1479 2-1844 3 4 5 6-1736 \y//////////a r- t -1631-1569 W r C Regulation o f the GFAP gene 99

12 3 4 Fig. 5. Regulation of expression of the chimeric gene G//31 (in which the GFAP promoter, including ~ 2 kb of upstream sequences, is fused to a /)tubulin cdna sequence) in transfected U251 and HeLa cells. 20 fig of RNA extracted from stable transformants were hybridized to a ptubulin or GFAP antisense probe. Ribonuclease-resistant fragments were analyzed as described in Fig. 2. Track 1, RNA from HeLa cells transfected with G//31. Track 2, RNA from HeLa cells transfected with the entire GFAP gene cloned in the same vector as G//31 (i.e. puc18 with an SV40 terminator placed downstream). Track 3, RNA from U251 cells transfected with G//31. Track 4, RNA from U251 cells transfected with the entire GFAP gene. Top arrow, correctly initiated transcripts from G//31; lower arrow, correctly initiated transcripts from transfected mouse GFAP sequences. that lie 3' to the transcriptional start site of the GFAP gene. Note that whereas the entire upstream fragment leads to the expression of a chimeric gene (G//31) in both HeLa and glioma cells (Fig. 5), if only a portion of this fragment corresponding to the distal element is included, expression of another chimeric gene (G.H/3gl) is restricted to glioma cells (Fig. 3). The difference between these two results is unlikely to be due to the different reporter sequences used; rather it must be due to sequences between the distal element and the transcription start site (nucleotides 1479 to 1): sequences in this region direct the expression of some constructs in HeLa cells, but only in the absence of the GFAP gene itself. These data suggest a negative interaction between sequences within the GFAP gene and upstream promoter elements (see Discussion). To further define the intragenic regulatory sequences, we made a series of constructs all containing the same 2 kb of upstream sequences, but lacking varying portions of the 3' end of the GFAP gene (Fig. 6A -E ). The transcriptional activity of each of these constructs was measured by RNAase protection following transfection into U251 and 100 S. Sarkar and N. J. Cowan HeLa cells. Deletion constructs up to and including sequences downstream from nucleotide +2836 (located in the 4th intron) had little discernable effect on the level of expression in U251 cells and were expressed at a very low level (if at all) in HeLa cells (Fig. 6B, tracks 1-4 and 6-9). In contrast, however, removal of sequences downstream from nucleotide +897 (located in the 1st intron) resulted in a dramatic increase in the expression of correctly initiated transcripts in HeLa cells (Fig. 6B, tracks 5 and 10); this construct had therefore lost cell type specificity. This result implies the existence of a negative regulatory element that lies within the GFAP gene itself and is responsible, at least in part, for conferring cell typespecific expression by inhibiting expression in non-glial cells. We made several additional deletion constructs in an attempt to further delineate this negative intragenic element (Fig. 6C). There is a conspicuous increase in expression in HeLa cells when the 3' deletion of sequences progresses from nucleotides +1264 to +1084 (labelled 6 and 7 in Fig. 6C and D). This result suggests the existence of a negative regulatory element within the first intron of the GFAP gene. Discussion Our data, together with that of Miura et al. (1990), demonstrate that a number of elements act together to direct the regulated expression of the gene encoding GFAP. Miura et al. (1990) identified three trans-acting factor binding sites in the GFAP gene by DNAase I footprinting between nucleotides 163 and 82. These binding sites, termed GFIII, GFII and GFI, show homology with the camp response element, the NF1 binding site motif and the AP2 binding site motif, respectively. By sitedirected mutagenesis, NF1 was shown to be a positive regulator required for efficient expression of GFAP, while the two flanking sites are negative regulators of GFAP expression. In contrast, our work has uncovered two more distant regulatory regions, a strong, glial-specific positive regulat ory element (the distal element) contained between nucleotides 1631 and 1479, and a negative regulatory element located in the first intron of the GFAP gene (the intragenic element). The distal element alone can drive the expression of a heterologous gene in glioma cells (but not in HeLa cells), although not in the manner of a classical enhancer (Fig. 3). On the other hand, deletion of the intragenic element leads to the activation of GFAP expression in HeLa cells (Fig. 6). Whereas our results are for the most part compatible with those of Miura et al. there are two points of apparent conflict. (1) Muira et al. found that many of their constructs, all of which lack intragenic sequences, are expressed specifically in glioma cells, and not control cells, whereas we show that removal of intragenic sequences activates transcription in HeLa cells (Figs 5, 6). This difference can probably be ascribed to the different control cells used in the two studies. (2) Miura et al. found that what we call the distal element is not necessary for expression of their constructs in glioma cells, whereas we find that it is essential for expression in glioma cells (Fig. 2). This difference may be ascribed to the presence of intragenic sequences in our constructs: our data suggests that there is a negative interaction between intragenic sequences and proximal GFAP upstream promoter el-

C A a b -H I------ (+ -H- c 0 d i 1.0 2.0 kb i. ^.j e B U251 '1 2 3 4 5' HeLa '6 7 8 910' 12 3 4 5 6 7 8 9 Fig. 6. (A) 3' deletion constructs used for the analysis of transcriptional activity in cells permissive or non-permissive for GFAP expression, a, entire GFAP gene; b -e, progressive deletions from the 3' end. Solid regions represent exons; the open box represents the 3 ' untranslated region. (B) Transcriptional activity of 3 ' deletion mutants (shown in A) in cells permissive or non-permissive for the expression of GFAP. 20 ng of RNA from stable transformants expressing a range of 3' deleted GFAP sequences were hybridized to a labelled GFAP antisense probe and analyzed by RNAase protection (see Materials and methods). Tracks 1-5, RNA from transfected U251 cells. Tracks 6-10, RNA from transfected HeLa cells. Tracks 1 and 6, construct a. Tracks 2 and 7, construct b. Tracks 3 and 8, construct c. Tracks 4 and 9, construct d. Tracks 5 and 10, construct e. The 3' end points of constructs b -e were at nucleotides +4810, +3820, +2836 and +897, respectively. Arrow shows protected fragment corresponding to the correctly initiated transcript from the transfected mouse GFAP gene. (C) Schematic representation of the 5' half of a truncated GFAP gene in which the position of the 3' truncation points of different constructs is shown by numbered arrows. The 3' end points were at nucleotides +2836, +2330, +1644, +1384, +1314, +1264, -1084, +897 and +447 for constructs 1-9, respectively. Each construct was assayed by RNAse protection (shown in panel D). Solid regions represent exons 1-4. (D) HeLa cells were transfected with 3' truncated GFAP genes (shown in panel C) whose 5' boundary was the same as the constructs shown in Panel A. 20 fig of RNA from stable transformants were analyzed by RNAase protection. Track numbers correspond to transfections using constructs shows in panel C. The arrow indicates the position of the correctly initiated transcript. All constructs were cloned in puc18, immediately upstream from an SV40 terminator. ements (see above, and Sarkar and Cowan, 1991). This negative interaction might lead to the dominance of the distal element we observe in our study. Since the constructs of Miura et al. lack intragenic sequences, this may allow the more proximal GFAP promoter elements they identify to predominate over the effects of the distal element in their study. The implied interaction of the upstream and intragenic elements is intriguing and could play a role in the modulation of GFAP expression during development and in response to injury. References J. M. a n d C o w a n, N. J. (1985). Structure o f the mouse glial fibrillary acidic protein gene: implication for the evolution of the intermediate filament multigene family. Nucl. Acids Res. 5527-5543. B e n d a, P., S m e d a, K., M e s s e r, J. a n d S w e e t, W. H. (1971). Morphological and immunological studies of rat glial tumors and clonal strains propagated in culture. J. Neurosurg. 34, 310-323. B r i s s e l, M. G., E n g, L. F., H e r m a n, M. M., B e n s c h, K. G. a n d M i l e s, L. E. M. (1975). Quantitative increase in neuroglia-specific GFA protein in rat C6 glioma cells in vitro. Nature 255, 633-634. B a lc a r e k, 13, Regulation o f the GFAP gene 101

C h ir g w in, T. M., P r z y b y la, A. E., M a c D o n a ld, R. J. a n d R u t t e r, W. J. (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294-5299. G ra h a m, F. a n d V a n d e r E b, (1973). A n e w te ch n iq u e fo r th e a ssa y o f infectivity o f hum an adenovirus II DNA. Virology 52, 457-465. L e w is, S. A., Gu, W. a n d C o w a n, N. J. (1987). Free intermingling of mammalian /5-tubulin isotypes among functionally distinct microtubules. Cell 49, 539-548. M iu r a, M., T a m u r a, T. a n d M ik o s h ib a, K. (1990). Cell-specific expression of the mouse glial fibrillary acidic protein gene: Identification of the cis- and trans-acting promoter elements for astrocyte-specific expression. J. Neurochem. 55, 1180-1188. M u l li g a n, R. C. a n d B e r g, P. (1981). Selection for animal cells that express the Escherichia coli gene coding for xanthione guanine phosphoribosyltransferase. Proc. natn. Acad. Sci. U.S.A. 78, 2072-2076. S a r k a r, S. a n d C o w a n, N. J. (1991). Intragenic sequences influence the expression of the gene encoding GFAP. J. Neurochem. (in press). W a n g, D., V i l l a s a n t e, A., L e w is, S. A. a n d C o w a n, N. J. (1986). The mammalian /3-tubulin repertoir: hematopoeitic expression of a novel, heterologous /3-tubulin isotype. J. Cell Biol. 103, 1903-1910. W e s t e r m a r k, B. (1973). The deficient density-dependent growth of human malignant glioma cells and virus-transformed glia-like cells in culture. Int. J. Cancer 12, 438-451. 102 S. Sarkar and N. J. Cowan