Part:BBa_K5195010

mNG-P2A-DUX4-DBD

The 2024 Stanford iGEM team designed a fusion protein consisting of mNeonGreen ([1] BBa_K5195004), a P2A linker ([2] BBa_K1442039), and a truncated version of the DUX4 protein coding for only the first 217 amino acids (DUX4-DBD) ([3] BBa_K5195002). This design was created in order be able to quantify how much DUX4-DBD protein would be produced in cells upon transfection. Because the P2A linker causes ribosomal “skipping” during translation, this effectively separates the mNeonGreen and DUX4-DBD proteins. In designing the coding sequence in this way, we enabled quantification of DUX4-DBD without directly attaching another large protein to it.

This coding sequence was used in a plasmid containing a doxycycline-inducible Tet-On Tight TRE promoter and the coding sequence for the full length DUX4 protein. Primers to amplify the entire plasmid except for the end of the DUX4-fl coding sequence to exclude the transcriptional activation domain. Then, an mNeonGreen-P2A fragment, replicated by PCR from an existing plasmid pK381, provided to us by one of our mentors, Dr. Phillip Kyriakakis, from the Stanford Coleman lab, was gel-purified and then Gibson Assembled with the DUX4-DBD fragment to create our imNG-P2A-DUX4-DBD plasmid.

Usage and Biology

Located on the human chromosome 4q35, the DUX4 gene a transcription factor encoding the protein DUX4 and is found within the chromosome’s D4Z4 repeat array[1]. The DUX4 full length protein (DUX4-fl) is composed of two double homeodomains, which are responsible for sequence-specific binding to DNA, along with a transcriptional activation domain (TAD) that activates harmful downstream genes[2].

In patients with facioscapulohumeral musclular dystrophy (FSHD), there is a reactivation of the DUX4 transcription factor, which is normally silenced following early embryonic development.

By over-expressing DUX4-DBD, a shortened isoform of DUX4-fl that lacks the TAD, the 2024 Stanford iGEM team hopes to competitively inhibit DUX4-fl binding to the DUX4 binding sites, replacing DUX4-fl binding with DUX4-DBD. Because it lacks the TAD, DUX4-DBD should not activate the downstream genes in the way that DUX4-fl does.

Experimental Approach

For testing this device, we first conducted a series of transfections HEK293T cells, seeded at a density of 100,000 cells/well in 24-well plates. Three different plasmids were used - one containing the mNeonGreen-P2A-DUX4-DBD sequence, one containing DUX4-FL, and a DUX4 reporter containing DUX4 binding sites upstream of mScarlet. These plasmids were cloned with PCR and assembled via Gibson Assembly, then transformed into DH5a or DH10b bacteria, miniprepped, and sequenced to verify that the resulting DNA sequences were the same as the intended ones. Varying ratios of DUX4-DBD to DUX4 full length (DUX4-fl) were transfected alongside a DUX4 reporter that produces mScarlet (red) fluorescent protein upon the activation of the DUX4 binding sites located upstream of the fluorescent protein in the plasmid. 48 hours, the plates were imaged using a fluorescent microscope and a fluorescence plate reader assay was performed.

Proof of expression and function

This transfection tested the potential for DUX4-DBD to act as a competitive inhibitor, because with more DUX4-DBD added, we expect the mScarlet fluorescent signal produced by the reporter to decrease because DUX4-DBD binds to the same region of DNA as DUX4-fl, but lacks the transcriptional activation domain responsible for recruiting transcriptional machinery to activate downstream gene expression. Our transfection results are shown below:

Here, the red fluorescent protein, our DUX4 reporter readout, decreases with increasing ratios of DUX4-DBD to DUX4-fl. The increasing amount of DUX4-DBD is implied from the increasing mNeonGreen (GFP) signal.

The above graph displays the plate reader assay results from 4 replicates of this transfection. It supports the visual results from the fluorescence microscope, showing that with increasing DUX4-DBD to DUX4-fl ratios, the DUX4 reporter expression level is driven down very close to its baseline level.

To obtain more precise, single-cell level data from our transfection experiments, we used flow cytometry to view individual cells and their fluorescence, which gives us a continuum of signals. The flow data supports our idea that as DUX4-DBD:DUX4-fl ratio increases, the corresponding reporter activity in those cells also decreases.

In the above figure, C12, C6, C1, and B8 represent different ratios of DUX4-DBD to DUX4-fl from highest to lowest (C12 is 25:1 DBD:FL, C6 is 10:1, C1 is 1:1, and B8 is 0:1).

These experiments were completed in HEK293T cells, which, though are easy to work with, were not the most representative of the environment our construct is ultimately aimed to be used in. Therefore, we tried to repeat our experiments in C2C12 cells, which is a mouse myoblast cell line. However, we noticed that our transfection efficiency was decreased, and though this was improved after optimization of transfection conditions, it was more difficult to visually see the trend of the DUX4 reporter decreasing with increased DUX4-DBD. The C2C12 transfection visual results are shown below:

Our fluorescence plate reader assay for the C2C12 transfection demonstrated the same trend of increasing DUX4-DBD decreasing DUX4-fl binding:

These experiments validate both the expression and function of the DUX4-DBD construct. The DUX4 reporter used in these experiments provided a readout of DUX4-fl binding and its recruitment of RNA polymerase to initiate transcription of mScarlet, simulating the way that the transcriptional activating domain in DUX4-fl recruits transcriptional machinery to activate harmful downstream genes. Because the green fluorescence is correlated with the amount of DUX4-DBD present, and as green fluorescence increases, red fluorescence decreases, this mNG-P2A-DUX4-DBD coding sequence demonstrates promising steps towards overexpressing DUX4-DBD to competitively inhibit DUX4-fl as a novel therapeutic approach for treating FSHD.

References

[1] U.S. National Library of Medicine. (n.d.). Dux4 double homeobox 4 [homo sapiens (human)] - gene - NCBI. National Center for Biotechnology Information. https://www.ncbi.nlm.nih.gov/gene/100288687

[2] Bosnakovski, D., Gearhart, M. D., Toso, E. A., Ener, E. T., Choi, S. H., & Kyba, M. (2018). Low level DUX4 expression disrupts myogenesis through deregulation of myogenic gene expression. Scientific Reports, 8(1). https://doi.org/10.1038/s41598-018-35150-8

[3] Mitsuhashi, H., Ishimaru, S., Homma, S., Yu, B., Honma, Y., Beermann, M. L., & Miller, J. B. (2018). Functional domains of the FSHD-associated DUX4 protein. Biology Open. https://doi.org/10.1242/bio.033977