Latest revision as of 13:30, 2 October 2024

3xCre3xAP1-miniCMV Promoter

Usage and Biology

To be able to receive and detect the signal sent by PknB kinase activity/ATF2 phosphorylation and activation, we have developed an ATF2-responsive promoter. Based on observations made by Miller and colleagues (2010) showing similar kinase mechanisms between the prokaryotic PknB and eukaryotic MAPK towards ATF2, we generated a synthetic ATF2-responsive promoter construct with three Cre and three AP1 binding sites as well as a miniCMV promoter sequence.

The ATF2 transcription factor belongs to the ATF/CREB family and regulates genes involved in cell growth, stress responses, and apoptosis (Kirsch et al., 2020). Activated ATF2 binds to the cAMP-responsive element (CRE) with the consensus sequence 5'-GTGACGT[AC][AG]-3' (Miller et al., 2010; Hai et al.,1989). Additionally, ATF2 can form homo- or heterodimers together with members of its own protein family, as well as the Fos protein family or Jun protein family, and bind to AP1-binding sites TGAG/CTCA by their conserved basic region leucine zippers (bZIPs) motifs (Kim et al., 2021). Therefore, we generated a promoter combining CRE as well as AP1-bindin sites to increase the binding possibility of our ATF2-mRuby2 fusion protein. A further increase in promoter efficiency was achieved by not only including one but three of each binding motif, this enables signal amplification by increasing the possibility of interaction between ATF2 and our promoter. Finally, we constructed a miniCMV promoter, just containing the TATA-box and the Initiator-Sequence of the original CMV-promoter, downstream of our 3xCRE3xAP1-binding sites to ensure a functional and strong transcription when activated by ATF2, of the, in the composite part, downstream positioned reporter protein miRFP670.

Cloning

Theoretical Part Design

We generated a promoter sequence containing three CRE-binding motifs as well as three AP1-binding sites followed by the minimal CMV (miniCMV) promoter. The miniCMV promoter contains the TATA-box and the Initiator-Sequence of the original CMV-promoter, ensuring a successful transcription, but in parallel allowing for specific expression dependent on upstream laying individually chosen binding sites. The sequence was synthesized with approx. 20 bp-long overhangs to allow for correct orientation when integrated upstream of a reporter gene in a plasmid backbone.

Sequence and Features

Characterization

The characterization experiments described below were conducted using the composite part containing the 3xCre3xAP1-miniCMV promoter downstream of the reporter gene miRFP670 (K5317022) to evaluate promoter activity.

Transfection experiments in mammalian HEK293T cells assessed the promoter functionality, sensitivity, and specificity. The fully assembled cell-based beta-lactam-sensor requires the successful triple-transduction with the detecting unit, CMV-EGFP-PknB, the signal transmitting unit, CMV-ATF2-mRuby2, and the receiving and signal translating into a fluorescence signal unit, the promoter 3xCre3xAP1-miniCMV-miRFP670. However, before all three are introduced into the cell and tested with antibiotic stimulation, it is necessary to test them alone and control for any sensor-unrelated responses of the promoter.

Single-transfection experiments

The composite part carrying plasmid was introduced via transfection to establish a baseline of endogenous promoter activity before performing co-transfection experiments with the CMV-ATF2-mRuby2 (K5317021) and CMV-EGFP-PknB carrying plasmid (K5317018) for ampicillin stimulation. The EGFP, mRuby2, and miRFP650 fluorescence signals were analyzed for presence and localization by microscopy.

Figure 2: Representative microscopic images of HEK293T cells expressing 3xCre3xAP1-miniCMV-miRFP670. Fluorescence channels for EGFP, mRuby2, and miRFP670 are shown. The basal activity of the promoter is presented in the upper row. The promoter activity after induction with 100 µg/mL ampicillin for four hours is demonstrated in the lower row. Scale bar = 100 µm.

The single-transfection experiment with the promoter and with or without beta-lactams demonstrated no unspecific signals from the miRFP670 in the green or lower red channels. The specific miRFP670 signal did not increase after incubation with ampicillin as expected since the signal detecting and transmitting units are not present in the cell. Rather less cells demonstrated a miRFP670 signal with ampicillin incubation. But its important that this could result from different transfection efficiencies in the representative wells.

Triple-transfection experiments

To test the functionality of the cell-based beta-lactam-sensor, HEK293T cells were triple-transfected with all necessary parts for signal detection (EGFP-PknB), signal transmission (ATF2-mRuby2) and signal emittance (3xCre3xAP1-miniCMV-miRFP670) and the fluorescence signals analyzed via microscopy and later on FACS analysis.

Figure 3: Representative microscopy image of HEK293T cells expressing EGFP-PknB, ATF2-mRuby2 and 3xCre3xAP1-miniCMV-miRFP670. Shown are the fluorescence channels for EGFP, mRuby2, and miRFP670 (first three images from the left) and an overlay of the three channels (right). In a) is shown the basal activity of the promoter. In b) is shown the promoter activity after induction with 100 µg/mL ampicillin after four hours of incubation.

The representative images in figure 3 show nicely the correct localizations of all three sensor components with EGFP-PknB being mostly localized in the membrane regions, the mRuby2-ATF2 signal being emitted from the nucleus, and the miRFP670 from the nucleocytoplasm. The detectable miRFFP670 signal even without the presence of ampicillin could be explained by possible binding of ATF2 to the 3xCre3xAP1-sites after its activation via other mammalian mechanisms, since ATF2 is a mammalian transcription factor and possibly endogenously expressed and activated or other AP proteins are present, binding to our promoter. Nevertheless, the representative images of the miRFP670 channel indicate an increase in fluorescence intensity after ampicillin incubation suggesting a functional PASTA domain activity followed by ATF2 phosphorylation leading to miRFP670 expression.

FACS Analysis

FACS analysis enables the quantification of the miRFP670-positive cells. To exclude single- and double-transfected cells from the evaluation, the cells were pregated regarding their EGFP- and mRuby2-positivity, counting only cells that carry the CMV-EGFP-PknB-C2, CMV-ATF2-mRuby2-C2 and promoter plasmid. The cells were incubated with varying, increasing concentrations of ampicillin to stimulate the miRFP670 expression via our sensor cascade.

Figure 4: Quantitive validation of reporter activity by flow cytometry analysis. The percentage of cells expressing the fluorophore miRFP670 under the control of the tested 3xCre3xAP1-miniCMV promoter is displayed as a function of various concentrations of ampicillin. n=1.

The quantification of the miRFP670-positive cells dependent on the beta-lactam concentration present in the medium for four hours shows a high basal activity of the 3xCre3xAP1-miniCMV promoter (approx. 70 % miRFP670-positive cells), as mentioned, possibly caused by endogenously active ATF2 or other transcription factors interacting with Cre and/or AP1 proteins-binding sites, activating our promoter independent of the presence of ampicillin. Nevertheless, the percentage of miRFP670-expressing cells increased slightly with ampicillin supplementation of the culture media. The bars indicate the sensitivity of the sensor-cassette, depicting already approx. 79 % miRFP670-positive cells under stimulation with 2.5 µg/mL ampicillin and no further increase with rising ampicillin concentrations. In conclusion, the evaluation of the sensor is made more difficult by a high baseline, but it is also characterized by a high sensitivity to beta-lactams. However, it must be taken into account that the experiment could only be carried out once due to time constraints.

References

Hai, T. W., Liu, F., Coukos, W. J., & Green, M. R. (1989). Transcription factor ATF cDNA clones: An extensive family of leucine zipper proteins able to selectively form DNA-binding heterodimers. Genes & Development, 3(12b), 2083–2090. https://doi.org/10.1101/gad.3.12b.2083

Kim, E., Ahuja, A., Kim, M. Y., & Cho, J. Y. (2021). DNA or Protein Methylation-Dependent Regulation of Activator Protein-1 Function. Cells, 10(2), 461. https://doi.org/10.3390/cells10020461

Kirsch, K., Zeke, A., Tőke, O., Sok, P., Sethi, A., Sebő, A., Kumar, G. S., Egri, P., Póti, Á. L., Gooley, P., Peti, W., Bento, I., Alexa, A., & Reményi, A. (2020). Co-regulation of the transcription controlling ATF2 phosphoswitch by JNK and p38. Nature Communications, 11(1), 5769. https://doi.org/10.1038/s41467-020-19582-3

Miller, M., Donat, S., Rakette, S., Stehle, T., Kouwen, T. R. H. M., Diks, S. H., Dreisbach, A., Reilman, E., Gronau, K., Becher, D., Peppelenbosch, M. P., Van Dijl, J. M., & Ohlsen, K. (2010). Staphylococcal PknB as the First Prokaryotic Representative of the Proline-Directed Kinases. PLoS ONE, 5(2), e9057. https://doi.org/10.1371/journal.pone.0009057