John P. Quinn, Christine Morrison, Jane McAllister & Susan. Mendelson. MRC Brain Metabolism Unit, Royal Edinburgh. Hospital, Morningside Park, Edinburgh, ...
Biochemical SocietyTransactions (1 993) 21
Evolution of enhancer domains within the preprotachykinin promoter John P. Quinn, Christine Morrison, Jane McAllister & Susan Mendelson. MRC Brain Metabolism Unit, Royal Edinburgh Hospital, Morningside Park, Edinburgh, EH10 5HF. The rat preprotachykinin-A promoter (PPT) which regulates the gene encoding the neuropeptides substance P and neurokinin A [ l ] is not, or is only minimally, active in all of the established tissue culture cell lines which have been analysed [2]. To search for transcriptional regulators within the promoter, we have used small fragments from the PPT promoter linked to heterologous reporter constructs. These reporter constructs allow us to determine the ability of individual PPT DNA elements to stimulate transcription. This approach has identified two functional elements within the PPT promoter, both of which are related to AP1 elements. AP1 elements, in general, bind heterodimers of the jun, fos, CREB and ATF families of proteins [3,4], in addition to several less well characterised protein complexes [5] and tissue-specific complexes [6]. These elements within the PPT promoter are at positions -340 and -196 relative to the major start of transcription. The complexes binding to these elements have been confirmed by gel retardation and DNase I analysis. The AP1 element at -340 in HeLa cells will bind the c-jun/c-fos complex whereas the complex binding to the element at -196 is quite distinct from c-jun, c-fos or CREB. The elements are also functionally quite distinct when placed upstream of a heterologous promoter. The sequence of these two PPT AP1-like elements and the AP1 consensus sequence is shown in Figure 1. We propose that these elements will synergise with one another and other distinct elements within the PPT promoter. DNase 1 footprinting analysis of this region of the PPT promoter has determined many other specific protein/DNA interactions several of which are adjacent to these AP1 elements [7]. Interestingly,there is significant conservation between the regions which are flanking these AP1 elements. This extended homology is shown in Figure 2. These two regions are potentially recognised by members of the same family of transcription factors. As these transcription factors are regulated in response to a multiplicity of stimuli, we propose a mechanism for stimulus induced regulation of the PPT promoter via these two regions outlined in Figure 2. This extended homology can be found in other AP1 elements which have been associated with enhancer function. This is most clearly illustrated by the AP1 enhancer element of the Gibbon Ape Leukaemia Virus (GALV) enhancer [5,8]. This is outlined in Figure 2. The viral AP1 element is highly homologous to both of the PPT elements we have characterised over an extended region. This extended homology may define a subset of AP1 elements. We view this extended homology as an AP1-like element flanked by two 6bp boxes GlTCCC and C A G m which flank an AP1 element. These two boxes were previously noted to be important in retrovirus LTRs as they were conserved in regions associated with enhancer function [8], as is the case amongst the different GALV strains.
37 1S
Figure 1 AP1 consensus
TGAGTCA AGCA TGAGTCA ACT
PPT AP1 at -340
AAAT TGcGTCA TTT
PPT AP1 at -196
Figure 2
GTTCCCagAAAT
AGaATGAGTCA
aCag
AAt
GALV AP1. (SF)
CTCAGTTT
AP1 at -340
TGcGTCA TTtCGaaCcCAaTTT
AP1 at -196
GTTCCC TAAgtccgAAGcATGAGTCAcTT CG
cTTCCC TAA
CAGTTT
The GALV enhancer element from the San Francisco strain is aligned with the two PPT AP1 elements.
References Helke, C.J., Krause, J.E., Mantyh, P.N., Couture, R. & Bannon, M.J. (1990). FASEB Journal 4, 1606-1615. 2. Personal Observations 3. Hai, T. & Curran, T. (1991). Proceedings of the National Academy of Sciences, USA 88, 3720-3724. 4. Ryseck, R.P. & Bravo, R. (1991). Oncogene 6,533542. 5. Quinn, J.P., Farina, A.R., Gardiner, K., Krutzsch, H. & Levens, D. (1989). Molecular and Cellular Biology 9, 4713-4721, 6. Andrews, N.C., Erdjument-Bromage, H., Davidson, M.B., Tempst, P. & Orkin, S.H. (1993). Nature 362, 722-728. 7. Personal Observations 8. Quinn, J.P., Holbrook, N. & Levens, D. (1989) Molecular and Cellular Biology 7, 2735-2744 1.
