In previous studies, increases in both mRNA and proteins derived from several nuclearly encoded genes of B. malayi
have been observed in response to treatment with tetracycline [17
]. The underlying rationale for these experiments was that the expression of nuclearly encoded proteins involved in the host-endosymbiont relationship would be perturbed by elimination of the endosymbiont. The data presented above confirm that transcription driven from the promoters of some genes whose expression has previously been shown to be affected by tetracycline treatment is up-regulated in response to tetracycline treatment. This finding suggests that at least part of the increased expression of some gene products in response to tetracycline is controlled at the level of transcription.
Previous studies have utilized transient transfection to identify the core promoter domains of B. malayi
]. In addition, this technique has been used to demonstrate the presence of an ecdysone responsive regulatory pathway in B. malayi
, employing a synthetic construct consisting of the BmRPS12 promoter modified to contain a synthetic ecdysone response element [23
]. The current report demonstrating up-regulation of the BmRPL13, BmRPS4 and BmHSP70 promoters in response to tetracycline represents the first direct demonstration of transcriptional regulation by a small molecule in a native B. malayi
Tetracycline treatment, which results in the elimination of the Wolbachia
endosymbiont, would be expected to be stressful to the host. This would in turn be expected to result in the up-regulation of transcription of proteins involved in the generalized stress response, including BmHSP70. In the heat shock proteins, up-regulation in response to stress is mediated by heat shock transcription factors, which act by binding to heat shock elements (HSEs) in the promoter, up-regulating transcription [28
]. The BmHSP70 promoter contains a functional HSE [20
], and if tetracycline treatment was causing up-regulation of transcription through the stress pathway, one would expect that mutation of the HSE in the BmHSP70 promoter would have eliminated the tetracycline response. However, mutation of the HSE did not result in any change in the response of the BmHSP70 promoter to tetracycline (c.f. construct −246 to −217, ). This suggests that the up-regulation of transcription by tetracycline involves a regulatory pathway that is distinct from the generalized stress response pathway.
Previous studies have identified a TATAA box-like sequence present in the BmHSP70 promoter [19
]. Initially, it was believed that this motif would represent part of the core of the BmHSP70 promoter. However, mutation of this motif has little effect on basal promoter activity (, Panel A, construct −216 to −187 and [20
]), demonstrating that the motif is not part of the core promoter. However, the mapping studies presented above demonstrate that the TATAA box-like motif is necessary for tetracycline-mediated up-regulation of the BmHSP70 promoter. There are two possible explanations for this finding. First, it is possible that the TATAA-box-like element actually represents an enhancer-like sequence that binds a particular transcription factor, up-regulating transcription from the core promoter domain surrounding the SL addition site in the BmHSP70 promoter. Alternatively, a global analysis of mammalian promoter structure has brought into question the paradigm that most eucaryotic promoters utilize TATAA and CAAT boxes as their core promoter domains [30
]. Such prototypical core promoter domains are now known to be lacking in many mammalian promoters. Promoters lacking such prototypical core domains are generally found in constitutively expressed mammalian genes. In these genes, transcription start sites are diffused over a relatively wide area, and the promoters of such genes are characterized by CpG islands [31
]. TATAA-box containing promoters are generally found in mammalian genes whose transcription is regulated, and transcription start sites in such genes are confined to a single nucleotide or a small number of nucleotides in a confined area [31
]. Previous studies have demonstrated that the transcription start sites of the BmHSP70 gene are diffuse in nature [19
]. It is thus logical to hypothesize that transcription of the BmHSP70 gene might be regulated by the interaction of two distinct types of core promoter domains. The first of these would be diffuse, and would occur during basal transcription of the BmHSP70 gene, or in response to generalized stress. The second domain would involve transcription induced by tetracycline and would involve a specific transcriptional start mediated by the TATAA box. This latter hypothesis could be tested by analyzing the transcriptional start points of transgenic mRNAs produced from the BmHSP70 promoter in the presence of tetracycline.
The experiments described above provide direct evidence that the TATAA box is necessary for up-regulation of promoter activity in the presence of tetracycline, but the experiments do not directly address the question if the TATAA box alone is sufficient to mediate this response. However, tetracycline responsiveness was altered when only two regions of the BmHSP70 promoter were mutated. These included a region formerly shown to be essential for basal promoter activity and the TATAA box. Mutation of the former domain resulted in a complete loss of promoter activity; since the promoter in this construct was completely inactivated, it is likely that the lack of tetracycline responsiveness seen with this mutant was a result of the overall inactivation of the promoter. Thus, the TATAA box was found to be the only region which when mutated resulted in a construct that retained basal promoter activity but which lost its ability to respond to tetracycline. This finding provides indirect evidence suggesting that the TATAA box is both necessary and sufficient to mediate the tetracycline response. Direct confirmation of this hypothesis will require introducing the TATAA box into a promoter that is not responsive to tetracycline and demonstrating that transcription from this mutant is now up-regulated in response to tetracycline.
Tetracycline treatment of B. malayi
results in the clearance of the w
Bm endosymbiont and concurrent changes in the mRNA pools of several nuclearly encoded genes [17
]. The data presented above suggest that at least some of this change is affected at least in part through changes in transcription from the affected genes. It is tempting to speculate that the changes in gene expression associated with tetracycline treatment are a result of the parasite’s response to the loss of its endosymbiont. However, the data presented above do not rule out the possibility that tetracycline is having an effect on these genes through a mechanism that is independent of its effect on the w
Bm endosymbiont. More work dissecting how tetracycline effects transcription of these genes will be needed to differentiate these possibilities.
Tetracycline has been used in conjunction with the Tn10 tetracycline resistance operon of Escherichia coli
to develop a very tightly controlled conditional expression system for eucaryotic cells [32
]. The data presented above suggest that tetracycline may induce gene expression of some B. malayi
genes, making it tempting to speculate that a similar system might be developed for B. malayi that could be based upon endogenous tetracycline responsive promoters. Such a conditional expression system, when coupled with the stable transfection method recently reported for B. malayi
] might prove to be a powerful approach to studying gene function in this parasite. However, tetracycline generally has no effect on mammalian cells. In contrast, tetracycline treatment of B. malayi
results in elimination of the Wolbachia
endosymbiont, resulting in sterilization and eventually death of the parasite. This would complicate the interpretation of any phenotype resulting from tetracycline-mediated induction of transgene expression by such a conditional expression system. Exploring the use of tetracycline analogs [34
], which might induce expression of the appropriate genes without affecting the viability of the Wolbachia
endosymbiont could be a way to overcome this difficulty.