Latest revision as of 13:35, 2 October 2024

Ca-Dependent Synthetic NF-AT

Is a synthetic NFAT transcription factor, after an increase in calcium, will enter the nucleus and bind to a specific minimal promoter. It uses the TALE system for binding to DNA and VP16 as the activation domain. Under homeostatic conditions, it is anchored in the plasma membrane via the KRφ peptide. It has both Myc and FLAG tags for immunohistochemical analysis and Western blotting

Sequence and Features

Usage and Biology

This chimeric protein is composed of three main components:

NFAT Transcription Factor cn Domain:

In its natural state, NFAT contains two Nuclear Localization Signals (NLS) that enable nuclear translocation. However, in this engineered version, one NLS has been removed, leaving only a single NLS to reduce background transcription. The NFAT domain is activated through phosphorylation by calcineurin in response to calcium signaling. This domain is critical for calcium-dependent activation in cellular processes, as the increase in cytosolic calcium activates calmodulin, which, in turn, activates calcineurin.

TALE Binding Domain and Calcineurin Fusion:

The TALE domain, which recognizes specific DNA sequences, is fused to the calcineurin domain via a GS10 flexible linker. The TALE sequence has high affinity for 10 TALE binding sites, which can be placed at various positions within the DNA, such as upstream of the TATA box to activate transcription of target genes.

VP16 Activation Domain and KRφ Peptide:

Transcription activation is achieved through the VP16 activation domain, which recruits the necessary cellular machinery, such as RNA polymerase II, to the promoter region. To prevent background transcription, a KRφ peptide is fused to the C-terminus of the protein, which anchors the protein to the inner plasma membrane. Upon elevated calcium levels, the protein is released from the membrane and translocates to the nucleus via the remaining NLS, initiating transcription. After calcium levels normalize, the protein is dephosphorylated and returns to the cytosol, where it re-anchors to the membrane, resetting for further activation.

The chimeric protein is also tagged at the N-terminus with Myc and FLAG tags to facilitate detection and purification using techniques like Western blotting, immunohistochemistry, and affinity purification. Overexpression of this chimeric protein is recommended to achieve robust promoter activation and significant transcriptional output

Tale "Highly specific DNA sequences, known as TALE-binding sites, are engineered to have strong affinity for TALE proteins, which are designed to recognize specific nucleotide sequences. These binding sites can be inserted at precise locations within the genome to guide synthetic proteins to specific loci. For example, in the case of minimal promoters, TALE-binding sites can be positioned upstream of the TATA box to enhance the targeted binding of transcription factors containing the corresponding TALE domains.

TALEs (Transcription Activator-Like Effectors) are a fascinating system, first characterized in plant pathogens, and in many ways, they share similarities with the CRISPR-Cas9 system, serving as its predecessor. TALEs are modular and can be customized to target any DNA sequence by modifying specific amino acids. By fusing TALEs with various proteins, such as nucleases, they can be used as powerful genetic engineering tools to cut target DNA, or as activators or inhibitors to regulate specific regions of the genome.

The core of the TALE system consists of 33 to 35 amino acid repeats, with the specificity for DNA binding determined by the 12th and 13th residues of each repeat. These two residues form the Repeat-Variable Diresidue (RVD), which dictates which nucleotide the repeat will bind to:

NI → binds to Adenine (A)

HD → binds to Cytosine (C)

NG → binds to Thymine (T)

NN → binds to Guanine (G) (and sometimes Adenine)

By assembling these repeats, each of which targets a specific nucleotide, TALEs can be designed to bind almost any sequence of choice. Furthermore, one TALE sequence can target multiple TALE binding sites, provided the proximity between the sites is sufficiently low. These binding sites can be positioned anywhere, whether in central or peripheral DNA regions. By assembling these repeats, each of which targets a specific nucleotide, TALEs can be designed to bind almost any sequence of choice. Furthermore, one TALE sequence can target multiple TALE binding sites, provided the proximity between the sites is sufficiently low. These binding sites can be positioned anywhere, whether in central or peripheral DNA regions.

In this particular case, a TALE sequence is fused with the NFAT transcription factor to bind to 10 TALE binding sites upstream of a minimal promoter. Upon receiving a stimulus, this setup activates transcription with high specificity

Zhang S, Chen H, Wang J. Generate TALE/TALEN as Easily and Rapidly as Generating CRISPR. Mol Ther Methods Clin Dev. 2019 Feb 19;13:310-320. doi: 10.1016/j.omtm.2019.02.004. PMID: 30923728; PMCID: PMC6423989.

Meško M, Lebar T, Dekleva P, Jerala R, Benčina M. Engineering and Rewiring of a Calcium-Dependent Signaling Pathway. ACS Synth Biol. 2020 Aug 21;9(8):2055-2065. doi: 10.1021/acssynbio.0c00133. Epub 2020 Jul 20. PMID: 32643923; PMCID: PMC7467823.

Structural Design and Experiments

Introduction to the Project

When performing immunocytochemistry on the original plasmid pCMV-MycNFAT provided by Professor Meško and her team, significant background noise was observed. In contrast, the production of NFAT was successfully detected via Western blot analysis. To address the background issue, we decided to add a FLAG tag to the N-terminus of the Myc tag through subcloning. For this purpose, we utilized the pCMV-FLAG-TRIM32 construct (BBa_K5490034), which was readily available in the lab. To facilitate the subcloning process, it was necessary to remove the TRIM32 sequence downstream of the FLAG tag while extracting the NFAT insert from the original construct. We needed to identify suitable enzymes to maintain directionality and preserve the open reading frame (ORF). We developed two different strategies to achieve this outcome.

Strategy 1: Blunt-End Cloning

Insert Preparation:

We identified a HindIII site upstream of the NFAT insert, which will be used for blunt-end cloning, and an XbaI site downstream to ensure directionality.

Vector Preparation:

For the vector, we located an XhoI site upstream of the TRIM32 gene for blunt-end cloning and an XbaI site downstream to maintain directionality.

Strategy 2: Partial Digestion Cloning

Insert Preparation:

We first identified a BglII site at the borders of the NFAT insert; however, an additional BglII site was present within the NFAT sequence, necessitating a partial digestion.

Vector Preparation:

For the vector, we identified a BamHI site at the borders of the TRIM32 gene. BamHI is isoschizomeric to BglII, but it cuts within the TRIM32 gene, which is acceptable as we only require the backbone from this construct.

Building a new composite part

Strategy 1: Blunt-End Cloning

Preparation of the Insert:

First, we linearized the construct using HindIII, followed by treatment with the Klenow fragment to convert the sticky ends into blunt ends. We then digested the linearized product with XbaI and isolated the insert through gel extraction.

Preparation of the Vector:

We linearized the vector with XhoI, converted the sticky ends to blunt ends using the Klenow fragment, performed another digestion with XbaI, and isolated the backbone via gel extraction.

Strategy 2: Partial Digestion Cloning

Preparation of the Insert:

Initially, we linearized the pCMV-MycNFAT construct with XbaI, then performed partial digestion with BglII at four different time points: 10, 20, 30, and 45 minutes. We identified the correct band through gel electrophoresis and isolated it via gel extraction.

Preparation of the Vector:

Complete digestion of the vector was carried out using BamHI, followed by isolation of the vector and subsequent digestion with XbaI. The backbone fragment was then removed through gel extraction.

Ligation Step

For the ligation we mixed the insert and vector in a 3:1 molar ratio and used T4 ligase. Notably, for the partial digestion strategy, we employed a buffer specialized for blunt-end ligation. The constructs were then amplified in DH5α cell lines.

Testing of another tagged version of the synthetic NFAT

Unfortunately, in the first scenario using the Klenow fragment, only a single colony was produced. Upon screening via restriction digest analysis, this colony did not yield the desired cutting pattern when viewed on a gel. In contrast, in the second scenario, multiple colonies were obtained, and suitable colonies were identified through restriction digest analysis. A midi prep was subsequently conducted to extract sufficient plasmid quantities, with measurements performed using software that analyzed light intensity from the gel, alongside NanoDrop readings.

Learning from a successful cloning

One important lesson learned during this project is that not all antibody tags are suitable for performing immunocytochemistry, as their effectiveness can vary depending on the specific protein being studied. Additionally, we gained valuable experience in subcloning techniques and realized that multiple strategies can achieve the same goal, which is beneficial since one method may not always be successful. This adaptability is crucial in molecular cloning, as it increases the likelihood of obtaining the desired constructs.

• 1.pCMV-FlagNFAT(BglII/BamHIxXbaI)-22-XhoI
• 2.blank well
• 3.pCMV-FlagNFAT(BglII/BamHIxXbaI)-22-PciI
• 4.pCMV-FlagNFAT(BglII/BamHIxXbaI)-22-HindIII
• 5.pCMV-FlagNFAT(BglII/BamHIxXbaI)-22-supercoiled
• 6.1kb ladder plus by NEB

Functional Experiments

For this experiment, we aim to investigate the correlation between ionophore concentration, which increases calcium levels in the cytosol, and promoter activity, as measured by a luciferase reporter.

ICC

In this experiment, two cell lines, Neuro-2a (mouse neuroblastoma) and SHSY-5Y (human neuroblastoma), were transfected with a FLAG-tagged NFAT construct at two different ratios: 20% and 80% of the total DNA. The remaining DNA was supplemented differently for each condition.

For the 20% FLAG-NFAT condition, 60% of the total DNA was pcDNA 3.1, an empty vector used to balance the total DNA content, while 20% of the DNA was AcGFP-N1, a plasmid encoding green fluorescent protein (GFP) to facilitate visualization.

In the 80% FLAG-NFAT condition, 20% of the total DNA was AcGFP-N1, with the remaining DNA supplied by the FLAG-NFAT construct.

Following transfection, the Neuro-2a cells were treated with ionomycin at four concentrations: 0 μM, 0.5 μM, 2 μM, and 5 μM, and were observed at two time points, 4 hours and 24 hours, post-treatment. The aim was to explore the effects of increasing ionomycin concentrations over time.

Meanwhile, the SHSY-5Y cells were treated with ionomycin at three concentrations: 0 μM, 2 μM, and 5 μM. The cells were observed after 4 hours of incubation for all three concentrations, and at the 24-hour mark for the 2 μM concentration only.

Immunocytochemistry (ICC) was frequently performed to detect the presence and localization of FLAG-tagged NFAT using a specific anti-FLAG antibody. The goal of the experiment was to assess NFAT expression and translocation under varying ionomycin treatments. By investigating how ionomycin, a calcium ionophore, influences NFAT, the experiment aimed to understand the dynamics of NFAT activation, particularly its calcium-dependent translocation from the cytoplasm to the nucleus.

ICC results and discussion:

For the Neuro-2a cells, we observed strong anchoring of the synthetic NFAT protein to the plasma membrane, with translocation to the nucleus occurring primarily after prolonged exposure to high concentrations of ionomycin. Notably, nuclear localization was most prominent in the 24-hour treatment group at 2 μM and higher ionomycin concentrations.

For the SHSY-5Y cells, the 20% FLAG-NFAT transfection did not yield detectable results, as the NFAT concentration at this transfection rate was too low for reliable antibody detection. However, similar to the Neuro-2a cells, NFAT exhibited strong anchoring to the plasma membrane, with translocation to the nucleus only observed under specific conditions. Nuclear translocation occurred primarily in the 24-hour treatment group at 2 μM ionomycin, and in the 4-hour group at 5 μM, though the latter concentration appeared to be toxic to the cells.

Overall, these results suggest that the synthetic NFAT protein translocates to the nucleus only after prolonged exposure to high concentrations of calcium in the cytoplasm. This pattern is consistent with conditions associated with cellular stress or pathogenesis, such as infection by West Nile Virus (WMV) in neural cells.

After the application of ionomycin, we occasionally observe cellular snapshots where it is strongly nuclear, and this is encouraging because NFAT has very rapid oscillation in its movement to the nucleus and a short residence time there. Additionally, its mobility is also affected by the presence of other isoforms in various cells.

https://doi.org/10.3390/ijms22052725

Zhong, QH., Zha, SW., Lau, A.T.Y. et al. Recent knowledge of NFATc4 in oncogenesis and cancer prognosis. Cancer Cell Int 22, 212 (2022). https://doi.org/10.1186/s12935-022-02619-6

https://doi.org/10.3389/fimmu.2018.00032

Second round of ICC experiments:

We repeated the experiment with new time points and ionomycin incubation concentrations, using a new plasmid as a control: NFAT fused with EGFP (pGFP-NFAT). For the Neuro2a cells, we incubated at 0 µM and 3 µM ionomycin for 18 hours, and for the SHSY-5Y cells, at 0 µM and 1 µM ionomycin for 6 hours. Both constructs, FLAG-NFAT and pGFP-NFAT, were used at 80%, with the remaining 20% consisting of dsRED.

Second round of ICC results and discussion:

It is evident that in the Neuro2A cell line FLAG-NFAT shows a more cytoplasmic distribution compared to GFP-NFAT, where NFAT is found in the nucleus even without ionomycin treatment. Although we did not capture the time point when NFAT translocates to the nucleus, the luciferase assay indicates this translocation.

The same results observed in Neuro2a were also seen in the SHSY-5Y cell line. FLAG-NFAT exhibited a more cytoplasmic distribution, while GFP-NFAT showed NFAT localized in the nucleus, even without ionomycin treatment.

Luciferase Assay

After the ICC assay we performed a luciferase assay to monitor promoter activity. The results , firstly ,show proof that indeed the NFAT translocates to the nucleus , since we have luciferase activity and secondly helped establish optimal ionophore concentrations for future experiments, providing a benchmark for calcium-induced promoter activation.

Experiments in Neuro2a Cell Line

Initially, Neuro2a cells were transfected with the dsRED plasmid and observed under a fluorescence microscope to measure transfection efficiency.

We performed transfections on the cell lines, using the following plasmids as controls to predict luminescence, which could then be compared with our experimental groups. The well containing 10% dsRED and 90% CMV-EGFP-C1 plasmids will not produce any luminescence, as it lacks the necessary luciferase component. This serves as our negative control.

The other wells contain positive controls, arranged in incremental order based on expected luminescence production:

10% dsRED and 90% Basic-LUC vector

10% dsRED and 90% SV40 Enhancer-LUC vector

10% dsRED and 90% SV40 Promoter-LUC vector

10% dsRED and 90% SV40-LUC control vector

10% dsRED and 90% CMV-LUC vector

Note: These types of controls were utilized for both the viral reporter assay and the pRE-LUC assays and in Neuro2a and SHSY-5Y cell lines.

For the main control group, we aimed to assess whether background transcription was occurring under various conditions. The first three control groups consisted of:

10% dsRED + 70% pcDNA3.1 + 20% pMIN-LUC

10% dsRED + 30% pcDNA3.1 + 60% pMIN-LUC

10% dsRED + 30% pcDNA3.1 + 60% pMIN-LUC + 3 µM of ionomycin incubated for 18 hours

This setup was designed to evaluate whether the minimal promoter was active in the absence of NFAT under high and low cytosolic calcium concentrations.

The second set of three control groups included:

10% dsRED + 70% pcDNA3.1 + 20% pMIN-LUC

10% dsRED + 30% pcDNA3.1 + 60% pMIN-LUC

10% dsRED + 30% pcDNA3.1 + 60% pMIN-LUC + incubation in 3 µM of ionomycin for 18 hours

This arrangement aimed to assess whether the cells were producing luminescence in the absence of luciferase.

For this set of experiments, we aim to assess the luminescent intensity under three different ratios of pMIN-LUC to pFLAG-NFAT, namely 1:3, 1:1, and 3:1, with and without ionomycin incubation at 3 µM for 18 hours.

Six cultures were transfected with the following combinations:

10% dsRED + 10% pcDNA3.1 + 20% pMIN-LUC + 60% pFLAG-NFAT 10% dsRED + 45% pMIN-LUC + 45% pFLAG-NFAT 10% dsRED + 10% pcDNA3.1 + 60% pMIN-LUC + 20% pFLAG-NFAT 10% dsRED + 10% pcDNA3.1 + 20% pMIN-LUC + 60% pFLAG-NFAT + (ionomycin incubation at 3 µM for 18 hours) 10% dsRED + 45% pMIN-LUC + 45% pFLAG-NFAT + (ionomycin incubation at 3 µM for 18 hours) 10% dsRED + 10% pcDNA3.1 + 60% pMIN-LUC + 20% pFLAG-NFAT + (ionomycin incubation at 3 µM for 18 hours)

Results and discussion:

As shown in both the diagram and the table, we observe the expected results across all control groups. There is a clear increase in luminescence from the plasmids anticipated to produce higher amounts of luciferase protein. Normalization was performed using the total negative control 10%DsRED+90% CMV-EGFP-C1.

We observed a spike in luminescence in groups 9 and 13, indicating that NFAT effectively transcribes the luciferase gene under high calcium concentrations at these specific ratios. Additionally, in groups 10 and 11, we noted that when a 1:1 molar ratio is used, the system appears to be ionomycin-independent.

Experiments in SHSY-5Y Cell Line

Initially, SHSY-5Ycells were transfected with the dsRED plasmid and observed under a fluorescence microscope to measure transfection efficiency. Note: Pictures are not provided due to the low intensity observed under the microscope.

We performed transfections on the cell lines, using the following plasmids as controls to predict luminescence, which could then be compared with our experimental groups. The well containing 10% dsRED and 90% CMV-EGFP-C1 plasmids will not produce any luminescence, as it lacks the necessary luciferase component. This serves as our negative control.

The other wells contain positive controls, arranged in incremental order based on expected luminescence production:

10% dsRED and 90% Basic-LUC vector

10% dsRED and 90% SV40 Enhancer-LUC vector

10% dsRED and 90% SV40 Promoter-LUC vector

10% dsRED and 90% SV40-LUC control vector

10% dsRED and 90% CMV-LUC vector

Note: These types of controls were utilized for both the viral reporter assay and the pRE-LUC assays and in Neuro2a and SHSY-5Y cell lines.

In this setup, cells were transfected under five different conditions as control groups to evaluate potential background transcription and cell luminescence:

10% dsRED + 70% pcDNA3.1 + 20% pRE-LUC 10% dsRED + 30% pcDNA3.1 + 60% pRE-LUC 10% dsRED + 30% pcDNA3.1 + 60% pRE-LUC + incubation at 1 µM for 6 hours 10% dsRED + 10% pcDNA3.1 + 20% pRE-LUC + 60% pFLAG-NFAT 10% dsRED + 10% pcDNA3.1 + 60% pRE-LUC + 20% pFLAG-NFAT

For the experimental group, cells were incubated in 1 µM ionomycin for 6 hours and transfected with two different ratios of pRE-LUC to pFLAG-NFAT, namely 1:3 and 3:1. The remaining DNA was supplemented with 10% dsRED and 10% pcDNA3.1.

Results and discussion:

As shown in both the diagram and the table, we observe the expected results across all control groups. There is a clear increase in luminescence from the plasmids anticipated to produce higher amounts of luciferase protein. Normalization was performed using the total negative control 10%DsRED+90% CMV-EGFP-C1.

The ionomycin induction did not work for the SHSY-5Y cell line. The duration of the treatment may have been too short, preventing the cells from having sufficient time to produce the enzyme. Repeating the experiment with a higher concentration of ionomycin or extending the treatment duration could provide a clearer result for the human cell line.

SHSY-5Y cells produce at least ten times lower levels of luciferase compared to Neuro2a cells, resulting in lower RLU/µg values of total protein.

We observed significant differences between the neural cell lines. In the case of Neuro2a, the expected results were obtained. However, in the human cell line SHSY-5Y, there were no statistically significant differences between the experimental and control groups.

IMPORTANT NOTICE

The NFAT construct was kindly provided by Benčina M, with the goal of studying its translocation to the nucleus after ionophore stimulation. For detection, we used an immunohistochemical approach targeting the Myc-tag epitope, which was fused to the N-terminus of the NFAT construct. However, the antibody against the Myc-tag epitope generated significant background noise. While it had some affinity for its target, it also bound nonspecifically to other molecules, leading to an inconclusive image under the microscope. Despite this issue, we confirmed the expression of the synthetic protein via Western blot, which motivated us to explore alternatives. Specifically, we decided to add a Flag tag in front of the Myc-tag epitope, leading us to try two different cloning strategies to achieve this.

First, we attempted to fuse the Flag tag using subcloning. In this approach, we extracted the insert and blunt-ended one side of both the vector and the insert while keeping the other side sticky to maintain directionality during the ligation step. Unfortunately, this strategy was unsuccessful. The second strategy, which worked, involved performing a partial digestion, as one of the restriction enzyme sites was located within the NFAT insert itself. We then performed directional cloning into a new vector containing the Flag tag .

Nomination: Best New Composite Part

We nominate ourselves for the award of the Best New Composite Part for:

Adding a new epitope to a synthetic NFAT, driven by the CMV promoter and enhancer, thus created a system that offers both high expression levels and easy detection through the FLAG epitope.

IMPORTANT TIP

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