Revision as of 20:38, 29 September 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

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.

IMPORTANT NOTICE<h1> 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 .