The EMBO Journal vol. 11 no.8 pp.2971 - 2980, 1992

Cell-specific transcription of the peripherin gene neuronal cell lines involves a cis-acting element surrounding the TATA box Danielle Desmarais1, Mario Filion', Line Lapointe' and Andre Royal1'2 1Institut du Cancer de Montreal, 1560, rue Sherbrooke est, Montreal, Quebec, H2L 4M1 and 2D6partement de Pathologie, Universite de Montreal, C.P. 6128, Succursale A, Montreal, Qu6bec, Canada, H3C 3J7 Communicated by J.C.Mandel

Peripherin is a neurone-specific intermediate filament protein expressed mostly in the peripheral nervous system. To localize sequences that are important for the regulation of peripherin gene transcription, we have functionally dissected its promoter. Transfection into different cell lines and deletion mapping of peripherinlacZ hybrid constructs indicated that the first 98 bp preceding the transcription start site of the gene were sufficient to confer cell-type specific expression. DNase I footprinting experiments revealed three protected sequences in this region, that were named PER1, PER2 and PER3. The PER2 and PER3 elements, localized between -98 to -46, interact with proteins that seem widely distributed. Deletion of these elements severely decreased the level of reporter gene activity. The PER1 element, which overlaps the TATA box, interacts with a DNA-binding protein prevailing in peripherin expressing cell lines. However, the core promoter, which contains the PER1 element, was inefficient in driving gene expression. Experiments designed to test the contribution of each element showed that PER2 and PER3 were important in determiniing the level of expression, while PER1 was important for cell-type specificity. In fact the polyoma virus enhancer linked to the peripherin gene core promoter was found to limit reporter gene activity to peripherin expressing cell lines. Together, these experiments indicate that co-operative interactions between different regions of the promoter are necessary for efficient and cell-type specific transcription of the peripherin gene in a subset of neuronal cells. Key words: intermediate filament/peripherin/promoter/ TATA box/transcription

Introduction Development is a complex process involving cellular differentiation and the sequential activation of specific genes. Although a large number of developmentally important regulatory genes have now been identified, comparatively little is known about their targets and their roles in differentiation of specific cell types. By contrast, recent studies have demonstrated that in specific systems, like muscle cell differentiation (Weintraub et al., 1991) and erythrocyte differentiation (Pevny et al., 1991) transcription factors that control tissue specific genes have an important Oxford University Press

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role to play in cellular differentiation. As a model system to identify genes which may play a determiining role in neural differentiation, we have focused on the peripherin gene, a member of the intermediate filament protein family (Leonard et al., 1988; Parysek et al., 1988). Intermediate filaments (IF) are a heterogeneous family of cytoskeletal proteins classified according to sequence homologies and intron-exon organization of the genes. In most cells, a single type of cytoplasmic IF protein predominates; e.g. keratins are found in epithelial cells, vimentin and desmin in mesenchymal and muscle cells respectively, and glial fibrillary acidic protein (GFAP) in neuroglial cells (Steinert and Roop, 1988). However, in neurones, the IF network is more complex. In addition to the neurofilament triplet proteins that are expressed in all neurones, three other IF proteins are expressed in different subsets of neurones. These include nestin in neuroepithelial stem cells (Lendahl et al., 1990), a-internexin in the central nervous system (CNS) (Fliegner et al., 1990; Ching and Liem, 1991), and peripherin in the peripheral nervous system (PNS) as well as in some CNS neurones (for a review see Greene, 1989). In the group of neuronal IF proteins, peripherin is distinguished by being the only type HI IF protein (homologous to vimentin, desmin and GFAP). The functional basis of the diversity of IF proteins is not understood, but changes in their expression occur sequentially, coincidentally with changes in cellular differentiation states. IF cell-type specificity and differentiation are so tightly integrated that the IF phenotype constitutes a fundamental property of differentiated cells. Peripherin was first identified in cultures of mouse neuronal cells, in differentiating neuroblastoma cells and in PC12 cells (Portier et al., 1982, 1984a; Parysek and Goldman, 1987). PC12 cells are tumour cells with a chromaffin-like phenotype that can be induced to differentiate into sympathetic neurone-like cells by nerve growth factor (NGF) (Greene and Tischler, 1976). NGF also induces an increase in peripherin mRNA by a transcriptional mechanism (Thompson and Ziff, 1989). Studies in vivo using in situ hybridization and immunohistochemical methods have shown that peripherin is found abundantly in PNS neurones, most cranial nerves, ventral horn motor neurones and a few other nuclei in the CNS, localized in the hindbrain and midbrain (Portier et al., 1984b; Leonard et al., 1988; Parysek and Goldman, 1988; Brody et al., 1989). Thus, neuronal populations which express peripherin belong to distinct lineages (neural tube, neural crest and placodes) and have different functions (motor and sensory). During development, peripherin is induced after the low molecular mass neurofilament (NF-L) protein (Escurat et al., 1990) and is first detected in the myelencephalon. In subsequent stages, positive cells localized at the outer border of the spinal cord migrate towards the center as differentiation proceeds. Peripherin-expressing cells are then localized only in the ventral horn of the spinal cord (Escurat et al., 1990). While 2971

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NF-L induction is rapid and occurs progressively in rostral and caudal directions, peripherin remains localized in the motor neurones. The fact that peripherin expression closely follows that of NF-L demonstrates that it is expressed postmitotically, as neurofilament proteins are (Tapscott et al., 1981; Bennett and DiLullo, 1985). However, the mechanisms by which specific neuronal cells are committed to express peripherin are not known. As a first-step to approach this question and identify regulatory molecules that play a role in the control of neural cell-type specificity, we have cloned the mouse peripherin gene and dissected its flanking 5' region. Here, we report that the proximal peripherin gene promoter is sufficient to control cell-type specific expression and that cell-type specificity relies on one element overlapping the TATA box. Our results also demonstrate that co-operative interactions between two regions of the promoter are essential for peripherin gene expression.

Results Transcnptonal regulation of the peripherin gene relies on multiple elements Recombinant phages containing the peripherin gene were isolated. The sequence of the 5' end of the gene and 425 bp of flanking sequence were determined and found to be 95 % homologous to the rat peripherin sequence (Thompson and Ziff, 1989) up to position -200 from the transcription initiation site (data not shown). To delineate sequences essential for efficient transcription of the mouse peripherin gene, a DNA segment extending from +28 to -3450 bp was fused to the Escherichia coli lacZ gene in p46D. The pe*pherin promoter

construct named pGAL3450, was used to produce a series of 5' end deletions (Figure 1). The constructs are referred to as pGAL(X). For each, X represents the 5' end position in nucleotides. Each construct was then transfected into peripherin-expressing PC12 cells. Deletion mutants pGALA25 and pGAL256 showed the highest activity and, thereafter, all results were expressed relative to pGAL256. The (3galactosidase (3-gal) activity of pGAL3450 was 30% of that obtained with pGAL256. Transfection of a number of constructs deleted from position -3450 to -46 suggested the presence of at least three regions containing negative regulatory sequences, between -3450/-3200, -2400/-2150 and -1250/1100, and two regions with positively acting sequences between -2800/-2400 and -425/-46. Deletion to -46 and -33 decreased (-gal activity to the level of the promoterless plasmid p46D. To determine if the peripherin-lacZ hybrid constructs were correctly initiated, primer extension experiments were performed with RNA from PC12 cells transfected with pGAL256. Initiation occurred at a cytosine residue located 30 nucleotides (nt) downstream from the TATA box, exactly at the transcription startpoint of the mouse peripherin gene (Figure 2). The transcription startpoint determined for the mouse gene is 3 bp downstream from that determined for the rat gene (Thompson and Ziff, 1989). To determine if the 3.4 kb promoter region used in these experiments was sufficient to confer cell-type specificity, some hybrid constructs were transfected into N18TG2, another peripherin-expressing cell line, and into OBL24 and KLN205 cells which do not express the peripherin gene as determined by Northern analysis (data not shown). Transfection into N18TG2 cells resulted in relative ,8-gal activities -

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Fig. 1. Expression of lacZ fusion gene constructs containing 5' deleted peripherin promoter sequences. The mouse peripherin promoter extending from +28 to -3450 (black box) was placed upstream of the E.coli-lacZ gene (open box). The transcriptional start site (+1) is indicated by the arrow. The deletion endpoints are indicated in bp relative to the +1 position. The constructions were transiently transfected into PC12, Nl8TG2 (N18), OBL24 (OBL), and KLN205 (KLN) cells. Activities are expressed relative to pGAL256 arbitrarily set to 100%. Hybrid constructs transfected into OBL24 and KLN205 cells never showed detectable /3-gal activity that exceeded the level of the promoterless plasmid (p46D) or of the untransfected control cells, even when the /-gal assays were incubated overnight. /3-gal activities have been normalized to the internal pSV2CAT control in each extract. (-), Not determined.

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similar to those obtained with PC12 cells (Figure 1). By contrast, none of the constructs were active after transfection into OBL24 or KLN205 cells (Figure 1). Thus, the peripherin promoter can be divided into three regions: a distal upstream region extending up to -425 containing mainly negative regulatory elements; the region -425/-46 which contains an unknown number of positive elements; and finally, a region downstream of position -46 that had no promoter activity in any of the cell lines tested. At least three protein complexes bind to the peripherin gene proximal promoter Peripherin-lacZ hybrid constructs were not expressed in peripherin-negative cells, suggesting that cell-type specific cis-acting elements were localized in the shortest active construct (pGAL98). To map the DNA region involved in conferring cell-type specific expression of the peripherin gene, we used a 127 bp PstI DNA fragment extending from nt -130 to -3 of ihe promoter as a probe in DNase I footprinting experiments. We limited our analysis to the region aT5G

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Cell-specific transcription of the peripherin gene in neuronal cell lines involves a cis-acting element surrounding the TATA box.

Peripherin is a neurone-specific intermediate filament protein expressed mostly in the peripheral nervous system. To localize sequences that are impor...
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