Part:BBa_K5195002
DUX4-DBD (DNA Binding Domain)
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Description
Base pairs: 651
Origin: DUX4-FL, found in human chromosome 4q35
Properties: A shorter isoform of the full-length double homeodomain protein that lacks the C-terminus, including the transcription-activating domain
Usage and Biology
DUX4-DBD is utilized to outcompete the full length isoform in binding to DUX4 binding sites. Located on the human chromosome 4q35, the full length gene is found within the chromosome’s D4Z4 repeat array, which consists of an open reading frame that encodes the two homeodomains.[1] Its double homeodomains are responsible for sequence-specific binding, located on the N-terminus along with three other nuclear localization sequences.
In patients with FSHD, there is a reactivation of the DUX4 transcription factor, which is meant to be silenced following early embryonic development. DUX4 activates harmful downstream target genes through its transcription-activating domain (TAD).[2] Removing this TAD, we create DUX4-DBD.
By over-expressing DUX4-DBD, we aim to minimize the effect of DUX4-FL on downstream gene expression, thereby reducing disease symptoms. This can be done by inserting a plasmid containing the short form into muscle cells and expressing enough of it to “flush out” the DUX4-FL at competitive inhibition at the promoter binding sites.
Figure 1. Overview of general approach to cloning DUX4-DBD.
Design
The DUX4-DBD sequence is an isoform of DUX4-FL that contains 217 amino acids, slightly longer than the 157 amino acid sequence known as DUX4-S. [3] Instead of reducing our protein to the 157th amino acid - the length at which the homeodomains are located - our team wanted the new cloned construct to maintain most of its original form. In doing so, we wanted to prevent any cytoxic effects from happening due to DUX4 deletions.
Assembly
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. Therefore, the remaining backbone would be the same.
Figure 2. Gel electrophoresis imaging of DUX4-DBD backbone.
After amplifying and purifying the backbone, the backbone assembled on itself. Ultimately, the cloning process yielded the following plasmid, which was later modified to introduce a fluorescent tag.
Figure 3. Plasmid map of tet-inducible DUX4-DBD under control of the CMV-enhancer/promoter.
Characterization
Early data demonstrated how effective DBD was at suppressing FL activity, though it was too soon to suggest our data to be statistically significant. Our first round of cloning utilized an inducible DBD construct with a fluorescent tag for quantitation and tracking. At the time, our team relied on a Dual-Glo Luciferase Assay System, which utilized both firefly and luciferase, for qualitatively identifying the transcriptional activity in our cells. More information regarding this sequence and its results can be found at the page for composite part BBa_K5195010.
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
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