To test our hypothesis that shortened DUX4-DBD is capable of competitively inhibiting natural DUX4 binding to DNA, the wet lab team made heavy use of the DBTL design cycle throughout our experimental efforts.

DBTL cycles (wet lab)

Cycle 1: Creating Initial Constructs (iDUX4-DBD, cDUX4-fl, and mScarlet DUX4 reporter)

Design
Prior to any wet lab experimentation, the first step of our project was to design the constructs we would use to carry out our project. In our first round of cloning, we designed and created 3 plasmids: an inducible mNG-P2A-DUX4-DBD plasmid, a constitutive DUX4-FL plasmid, and an mScarlet DUX4 reporter.

imNG-P2A-DUX4-DBD:
Upon obtaining a Tet-On doxycycline-inducible DUX4-fl plasmid (Addgene Plasmid #99281), we constructed a DUX4-DBD plasmid by cloning out part of the DUX4-fl coding sequence and leaving only the first 217 amino acids of DUX4. These 217 amino acids constitute the homeodomains and some of the C-terminal tail of the original DUX4-fl protein, but lack the transcriptional activation domain. In order to be able to quantify the DUX4-DBD protein in cells, we also inserted an mNeonGreen (mNG) sequence followed by a Peptide 2A (P2A) linker upstream of the DUX4-DBD. The P2A linker causes ribosomal “skipping” during translation, and this effectively separates the mNG and DUX4-DBD. In designing the coding sequence in this way, we enabled quantification of DUX4-DBD without directly attaching another large protein to it.

mTagBFP2-P2A DUX4-fl:
We wanted to harness the inducible Tet-On system to more easily modulate the amount of DUX4-DBD produced, but because we did not want to modulate the amount of DUX4-fl in a co-transfection, we also created a constitutive version of the DUX4-fl from the original Tet-On inducible DUX4-fl plasmid we obtained from Addgene. We replaced the original tight TRE promoter with an SV40 promoter and inserted an mTagBFP2 coding sequence followed by a P2A linker downstream of the DUX4-fl coding sequence.

mScarlet DUX4 reporter:
We obtained a luciferase-based DUX4 reporter plasmid containing 6xDUX4 binding sites (pGL4-6X-DUX4-ffy) from the Kyba Lab of the University of Minnesota Twin Cities. This construct produces firefly luciferase as its readout, which requires an endpoint assay to quantify. In order to overcome this, we designed a reporter with mScarlet fluorescent readout instead of luciferase, as this would allow us to skip the assay step, enable us to take images of fluorescent readout at multiple time points throughout an experiment, and set up live cell fluorescent imaging experiments.

Build

We first designed primers to cut out the fragments of interest for assembling our plasmids. Using PCR, we amplified the fragments of the original DUX4-fl Addgene plasmid and the luciferase DUX4 reporter plasmid that we obtained. After amplification, we ensured the purity and accuracy of the PCR fragments with gel purification, quantification by Nanodrop, transformed into DH10B and DH5a cells, plating with antibiotic selection, and finally, mini-prepping and sequencing (Plasmidsaurus).

For the imNG-P2A-DUX4-DBD plasmid, we first designed primers to amplify the entire plasmid except for the end of the DUX4-fl coding sequence to exclude the transcriptional activation domain. Then, we amplified an mNG-P2A fragment from an existing plasmid pK 381, provided to us by one of our mentors, Dr. Phillip Kyriakakis, from the Stanford Coleman lab. The mNG-P2A fragment was then Gibson Assembled with the DUX4-DBD fragment to create our imNG-P2A-DUX4-DBD plasmid.

To create the constitutive DUX4-fl plasmid, we amplified the original Addgene plasmid, but without the Tight TRE promoter, and then Gibson assembled that fragment with a Geneblock containing an mTagBFP2-P2A sequence.

To create our DUX4 reporter construct, we amplified a backbone of the Kyba Lab reporter that excluded the firefly luciferase fusion protein (fLuc Fusion) sequence. In parallel, we amplified an mScarlet sequence from the plasmid pPK 381, which was provided to us by one of our mentors, Phil Kyriakakis, from the Stanford Coleman Lab. After amplifying both fragments, we inserted the mScarlet sequence into the vector, pGL4-Amp(R)-6X-DUX4. The resulting plasmid pGL4-DUX4-6X-UAS-mScarlet was subsequently utilized for nearly all other transfections as our fluorophore reporter alternative.

The following schematic (from the 2023 Stanford iGEM team) describes our cloning methodology:

The below diagrams summarize our cloning efforts for our first round of cloning:

The key components of the final 3 plasmids we utilized for subsequent transfections are sketched below:

Test

We first needed to validate the functionality of our reporter constructs as it is critical to almost every experiment we do from here. After that, we also used these initial constructs to test our hypothesis of competitive inhibition.

To validate our reporter constructs, we transfected doxycycline-inducible DUX4-fl with DUX4 luciferase reporter at different doxycycline concentrations. This was done in HEK293T cells, as they are an easily transfectable and maintainable cell line.

We transfected dox-inducible DUX4-fl with mScarlet DUX4 reporter to see if our new reporter construct reported DUX4-fl activity accurately, and we also transfected imNG-P2A-DUX4-DBD by itself to check if it produces green fluorescent protein. After observing that both our fluorescent reporter and DBD functioned as expected, we conducted 4 repeat trials of transfection testing competition between DUX4 and DBD at different ratios.

In these experiments, RFP served as the reporter readout, and GFP was the DBD fluorescence readout.

These experiments were all completed in HEK293T cells, which, though easy to work with, were not the most representative of the environment our construct is targeting. Therefore, we repeated our experiments in C2C12 cells, which is a mouse muscle cell line. However, we noticed that our transfection efficiency became significantly lower, leading us to optimize the Lipofectamine 3000 transfection protocol. After that, we were able to conduct competitive inhibition experiments with C2C12 cells, yielding similar results to our HEK293T experiments.

To obtain higher quality data from our experiments, we used flow cytometry to view individual cells and their fluorescence, which gave us a continuum of signals. The flow data supported our idea that as the DBD:FL ratio increased, the corresponding reporter activity in those cells decreased.

Learn

The reporter proved a critical part in verifying the competition between DUX4-FL and DUX4-DBD, offering a more quantitative outlook. Both our luciferase reporter and mScarlet reporter functioned well, setting us up for future experiments and validating our cloning and transfection techniques.

Cycle 2: Creating DBD-KRAB

Design

After achieving successful DUX4 reporter knockdown with our DUX4-DBD construct, we explored fusing a repression domain to the end of the DBD in order to further prevent the activation of any genes near the DUX4 binding sites.

Build

Our KRAB repressor domain sequence was derived from AddGene plasmid pHAGE EF1α dCas9-KRAB, which was first referenced as being an effective transcriptional repressor. We ordered the KRAB domain sequence as a gene block, which we later Gibson Assembled with the backbone of the mNG-DBD 217 construct.

Test

Similar transfections to Cycle 1 have been done to test the competitive inhibition capabilities of our DBD-KRAB construct. mNG-DBD-KRAB is transfected into HEK cells alongside full length and reporter fluorescence was measured. DBD-KRAB consistently knocks down DUX4 fluorescent reporter activity at greater levels than DBD alone (note the logarithmic scale).

DBD-KRAB appears to cause an exponential reporter knockdown, while DBD knockdown is linear. However, more repeats and data points are needed to prove this hypothesis. (two repeat trials)

However, when the construct is tested in C2C12 cells, the inhibition trends are not as strong or apparent.

Learn

From these transfections, we can conclude that KRAB can increase the inhibitive properties of DBD in HEK cells. Though this still needs to be tested by more repeat trials and more intermediate data points. However, when it comes to C2C12, KRAB seems to disrupt normal inhibition function. Since this was one trial, it could be human error that led to this result. All together, more testing is needed to confirm KRAB functionality in muscle adjacent systems, but the preliminary results in HEK cells are promising.

Cycle 3: Creating NES Constructs (DBD-mScarlet-NES (designed but never built), NLS-DBD-mScarlet-NoLS-NES and for testing, DBD-GFP)

Design

In attempts to create a more “spreadable” DUX4-DBD that would enhance propagation of myoblasts and myofibers, we planned to add an additional pair of NLS/NES to the plasmid. As a control, utilized as a way to visualize migration differences between constructs, we made a constitutive DUX4-DBD fused directly to GFP. Finally, to further assess the potency of a standalone NES in spreading DBD to distinct myonuclei, we planned to make a DBD-mScarlet-NES construct; though we synthesized and ordered its parts, it was never cloned.

Build

We amplified the backbone of inducible mNG-DUX4-DBD, which excluded the fluorophore and DBD coding sequences. For each backbone, we utilized different primers that would add overhangs and bind to their respective gene fragments. The gene fragments, synthesized as gBlocks through IDT, consisted of NLS-DBD-mScarlet-NES, DBD-GFP, and DBD-mScarlet-NES. During assembly, we inserted the fragments at a 3:1 insert to vector ratio, due to initial difficulty growing isolated bacterial colonies with the fragments integrated into their sequence. Furthermore, transformation via electroporation proved ineffective due to lack of bacterial growth on plates. Subsequently, we transitioned to high-efficiency heat shock, eventually yielding our desired constructs.

Test

We designed and carried out an initial live-cell imaging experiment in which we co-transfected C2C12 cells with DBD-GFP and NLS-DBD-mScarlet-NES, then lifted them and added them to a denser well plate of C2C12s and differentiated them. We live-imaged the cells for 48 hours.

Learn

The live-cell imaging experiment was inconclusive because the fluorescent protein signal did not appear to move much beyond individual cells. This is likely due to the imaging time frame being too short - imaging for 5-7 days would provide better visualization of C2C12 muscle cell fusion. We hope to carry out this experiment in the future and observe results in more fully-formed myotubes.

Cycle 4: reporter cell line and inducible full length cell line

Design

In order to achieve more accurate and precise results from our C2C12 transfections, we created a C2C12 cell line stably expressing our mScarlet DUX4 reporter. With this cell line, all of the cells in a culture would contain the reporter and therefore be a better experimental model than transfecting transiently, since not all cells are transfected in that method. We also hoped to eventually use this cell line for live imaging of cell fusion, as it would enable us to visualize reporter expression and DUX4 repression on the nuclear level.

Build

We transfected the DUX4 reporter into C2C12 cells, then added hygromycin as the DUX4 reporter plasmid contained a hygromycin resistance gene, so this allowed us to select for the cells that got transfected and where the DUX4 reporter DNA was integrated into the cells’ genome. We continuously selected and cultured cells for 14 days, until the control well with no DNA added died off while small colonies of the reporter-containing cells remained.

Test

Next, we grew up the small remaining colonies and transfected them with DUX4-fl to test if the reporter could successfully be activated. The below figure shows that the reporter was successfully activated, but its expression level is not optimal. This may also be because of low Dux4-fl transfection efficiency.

Learn

Transfecting the DUX4 reporter cell line with FL showed activation of DUX4 reporter, but we hope to further increase its level of activation. We also created a DUX4 reporter + inducible DUX4-fl cell line, but have yet to test its efficacy. In the future, we hope to generate stable cell lines through the use of lentiviral vectors.

Cloning Overview

Cloning Subsection
Stanford iGEM 2024
To develop our cloning constructs, we followed a deliberate process of creating our plasmid vectors, connecting fragments, and supplying exogenous DNA for bacterial cells.

For our entire cloning efforts, they consisted of resuspending primers and DNA fragments, then utilizing them to PCR template backbones or add overhangs to linearized fragments. From there, we ran gel electrophoresis to validate PCR products, imaged, and purified through gel extraction. The subsequent steps included Gibson Assembly, electroporation, liquid culture, and mini-prep, yielding transfectable stocks of cloned DNA constructs.

Through our initial tet-inducible DUX4-FL plasmid, AddGene pCW57.1-DUX4-CA, we yielded a multitude of constructs. We made a constitutive BFP-P2A-DUX4-FL, in which an additional fluorophore mTAG-BFP2 fused to a P2A linker was inserted into the backbone, preceding the DUX4-FL coding sequence. The fluorophore was attached in attempts to view the binding of FL to the reporter.

From the aforementioned tet-inducible FL, we produced a doxycycline inducible DUX4-DBD, essentially removing the C-terminal, including the transcription-activating domain. Building off of the inducible DUX4-DBD, we also added an mNeonGreen protein attached to a P2A linker for fluorescent readout. The use of a P2A linker allowed us to quantify the presence of our DUX4-DBD in a way that didn’t require the fluorescent protein to be directly fused to it. In this manner, we were also able to visualize the competitive inhibition between both DUX4 isoforms.

We also designed a construct in which we only used the two homeodomains of the DBD, 157 amino acids. Initially, our design included 217 amino acids to maintain most of the original DUX4-FL sequence, though we shortened its length to test the effect of the extra C-terminal region.

Using the same DUX4-DBD backbone, we made three additional constructs, the first of those being Myospreader DBD. In this case, we had attached an additional SV40 NLS on the N-terminus, an Alyref NES and HIV rev NoLS on the C-terminus, and fused with a fluorescent mScarlet protein. We intended for the addition of an NLS/NES pair to facilitate the propagation of DBD into more distant and scattered myonuclei.

As a control, we also made a constitutive DBD-GFP, where DBD was directly bound to GFP, in order to co-transfect these and visualize migration differences. We also made a version of DBD with an attached repressor domain, Krüppler-associated box (KRAB), to yield our DUX4-DBD-KRAB construct. We intended for the additional KRAB domain to efficiently repress both DUX4-FL and its target genes in FSHD myocytes, targeting the promoter or exon1 of DUX4.

We initially utilized a previously developed DUX4 reporter which featured 6 DUX4 binding sites of known DUX4 binding motifs upstream of a luciferase luminescent protein. The process proved somewhat inefficient, and we decided to rely on fluorescent readouts instead. To obtain this construct, we replaced the luciferase sequence with fluorescent mScarlet, yielding the plasmid 6x-DUX4-UAS-mScarlet.

And ultimately, utilizing the plasmid pAAV EF1A-eGFP-WPRE-hGHpA as a backbone, we cloned the plasmid pAAV-EF1a-DBD-eGFP, which contains our desired cargo and is ready for transduction to create our AAV therapeutic.

References:
[1] Poukalov, K. K., Valero, M. C., Muscato, D. R., Adams, L. M., Chun, H., Lee, Y. il, Andrade, N. S., Zeier, Z., Sweeney, H. L., & Wang, E. T. (2023). Myospreader Improves Gene Editing in Skeletal Muscle by Myonuclear Propagation. https://doi.org/10.1101/2023.11.06.565807
[2] Himeda, C. L., Jones, T. I., Virbasius, C.-M., Zhu, L. J., Green, M. R., & Jones, P. L. (2018). Identification of epigenetic regulators of DUX4-FL for targeted therapy of facioscapulohumeral muscular dystrophy. Molecular Therapy, 26(7), 1797–1807. https://doi.org/10.1016/j.ymthe.2018.04.019