Part:BBa_K5267044

P_5xNFAT->IgK->Nluc->bGH_polyA

Transpose and respond to calcium ion signals

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 312
  
• 1000
  COMPATIBLE WITH RFC[1000]

Profile

Name: P_5xNFAT->IgK->Nluc->bGH_polyA
Base Pairs: 1018bp
Origin: Homo sapiens
Properties: The transformation of calcium ion concentration is reported by fluorescence

Usage and Biology

At present, some people have successfully designed a series of repetitive pseudo-palindromic NFAT reaction element guided by nano-luciferase reporter gene system[1].

We developed a series of Ca2+induced NanoLuc reporting systems based on Ca2+ dependent activation of dimer NFAT to monitor the effects of increased Ca2+ concentration in downstream pathways following melatonin receptor response on cells. (Figure. 1)

The system is divided into two parts: the signal response element (encoded by Part:BBa K5267007) and the detection report element (encoded by Part:BBa_K2728003).

HEK293 cells were co-transfected with a newly designed NanoLuc reporter expression plasmid to construct a NFAT response element (RE) -directed Nanoluc reporting system. With the NanoLuc reporter gene, we can detect the activation of the NFAT signaling pathway.[2]

Figure 1. Construction of a pseudo-palindromic NFAT-response element (RE)-directed Nanoluc reporter system.

Special design

In order to evaluate the biological implications of heightened intracellular calcium ion (Ca2+) levels, we have engineered a suite of Ca2+-responsive NanoLuc-reporting constructs predicated on the Ca2+-dependent engagement of nuclear factors of activated T cells (NFAT) dimers (Figure 1).

Given the subdued transcriptional impact of an isolated response element, it is a common practice to introduce multiple tandem iterations of said element into the genomic locus proximal to the reporter gene, thereby amplifying the efficacy of the signaling cascade initiation.[1]

The aforementioned reporter constructs incorporate a variable number of tandem repeats (1x, 5x, 6x, and 7x) derived from the NFAT response element (NFAT-RE) within the interleukin-4 (IL4) promoter sequence (5'-TACATTGGAAATTTTTAT-3'). This particular sequence is anticipated to facilitate the transcriptional activation of the NanoLuc reporter genes (Figure 2).

Figure 2. Schematic diagram of MT1 receptor activating the downstream NFAT pathway.

Consequently, this system offers a valuable tool for elucidating the dynamic changes within the signaling network following the activation of melatonin receptors.

Function test

To substantiate the functionality of the aforementioned constructs, human embryonic kidney 293 cells (HEK293) were co-transfected with expression vectors harboring the newly engineered NanoLuc-reporter genes.

Thapsigargin (TG) is a known ER stress inducer that increases intracellular calcium (Ca2+) concentration by inhibiting the calcium atpase (SERCA pump) in the ER. This increased calcium concentration can activate a variety of cell signaling pathways, including the NFAT (nuclear factor of activated T cells) pathway, thereby analyzing the sensitivity and activation threshold of the NFAT pathway.

At the cellular level, melatonin can affect the activity of calcium channels through its receptors, leading to changes in intracellular calcium concentration. The reporting system is designed to be responsive to oscillations in intracellular Ca2+ concentrations.

The optimal configuration of the reporting pathway was ascertained by evaluating and comparing the relative luminescence unit (RLU) expression profiles of the NanoLuc reporter genes, thereby discerning the most efficacious design among the various constructs.

Method

Initially, we co-transfected HEK293T cells with an expression vector encoding the NanoLuc reporter gene, followed by the induction of an intracellular calcium ion (Ca2+) response using thapsigargin. Each experimental condition was performed in triplicate, alongside a non-transfected control group lacking NFAT (Part:BBa K5267049).

Upon a 48-hour exposure to thapsigargin, the luminescence intensity of the NanoLuc reporter, expressed in relative light units (RLU), was measured across all experimental groups to evaluate the transcriptional activity evoked by thapsigargin stimulation.

Subsequently, the same reporter gene was co-transfected into HEK293T cells, and the intracellular Ca2+ response was provoked by melatonin. The experiments were conducted with three replicates each, and a control group was included, which was not subjected to melatonin stimulation.

After a 24-hour melatonin stimulation period, the luminescence intensity of the NanoLuc reporter element, quantified in RLU, was assessed in all experimental groups to determine the transcriptional activity induced by melatonin treatment.

Results

Figure 3. NFAT activation in response to calcium ion signaling. (Regulation by TG)

HEK-293T cells were transfected with plasmids containing different promoters with 1×/5×/6×/7×NFAT elements respectively. Data are mean±SD of NanoLuc expression levels measured at 48 h after thapsigargin stimulation (n = 3 independent experiments).Upon a 48-hour incubation period, stimulation of the 5xNFAT promoter with 10 nM thapsigargin resulted in a mean augmentation of the NanoLuc reporter gene expression to a magnitude that was 7.18-fold superior to that ascertained in the absence of thapsigargin induction.

Figure 4. NFAT activation in response to calcium ion signaling.(Regulation by MT)

HEK-293T cells were co-transfected with melatonin receptor plasmid pCJ008(PCMV-MTNR1A) and plasmids containing different promoters with various copy numbers of NFAT elements pNC008(PNFAT_1-IgK-Nluc), pNC004(PNFAT_5-IgK-Nluc), pNC012(PNFAT_6-IgK-Nluc) and pNC010(PNFAT_7-IgK-Nluc) melatonin stimulation. Data are mean±SD of NanoLuc expression levels measured at 24 h after melatonin stimulation (n = 3 independent experiments).

In the in the P_5xNFAT->IgK->Nluc->bGH_polyA system, the introduction of 1 nM melatonin did not elicit a statistically significant increase in the mean NanoLuc expression levels compared to the system devoid of melatonin treatment.

Reference

[1] W. Zhang, T. Takahara, T. Achiha, H. Shibata, and M. Maki, “Nanoluciferase Reporter Gene System Directed by Tandemly Repeated Pseudo-Palindromic NFAT-Response Elements Facilitates Analysis of Biological Endpoint Effects of Cellular Ca2+ Mobilization,” Int. J. Mol. Sci., vol. 19, no. 2, p. 605, Feb. 2018, doi: 10.3390/ijms19020605.
[2] K. A. Strait, P. K. Stricklett, R. M. Kohan, and D. E. Kohan, “Identification of Two Nuclear Factor of Activated T-cells (NFAT)-response Elements in the 5′-Upstream Regulatory Region of the ET-1 Promoter,” J. Biol. Chem., vol. 285, no. 37, pp. 28520–28528, Sep. 2010, doi: 10.1074/jbc.M110.153189.