Schreier, S. | bioRxiv Science

bioRxiv2019

A simple and sensitive SYBR Gold-based assay to quantify DNA-protein interactions

A simple, accessible, and inexpensive assay to quantify the strength of DNA-protein interactions was developed. The assay relies on capturing DNA-protein complexes using an affinity resin that binds tagged, recombinant proteins. Sequential washes with filtration spin cups and centrifugation remove non-specific interactions in a gentle, uniform manner and a final elution isolates specific DNA-protein complexes. SYBR Gold nucleic acid stain is added to the eluted product and the fluorescence intensity accurately quantifies the amount of captured DNA, ultimately illustrating the relative strength of the DNA-protein interaction. The major utility of the assay resides in the versatility and quantitative nature of the SYBR Gold:nucleic acid interaction, eliminating the need for customized or labeled oligos and permitting relatively inexpensive quantification of binding capacity. The assay also employs DNA-protein complex capture by the very common purification tag, 6xHis, but other tags could likely be utilized. Further, SYBR Gold fluorescence is compatible with a wide variety of instruments, including UV transilluminators, a staple to any molecular biology laboratory. This assay was used to compare the binding capacities of different Auxin Response Factor (ARF) transcription factors to various dsDNA targets, including the classical AuxRE motif and several divergent sequences. Results from dose-response assays suggest that different ARF proteins might show distinct comparative affinities for AuxRE variants, emphasizing that specific ARF-AuxRE binding strengths likely contribute to the complex and fine-tuned cellular auxin response.

plant biology↗

bioRxiv2018

Transgenic mice expressing tunable levels of DUX4 develop characteristic facioscapulohumeral muscular dystrophy-like pathophysiology ranging in severity

BackgroundAll types of facioscapulohumeral muscular dystrophy (FSHD) are caused by the aberrant myogenic activation of the somatically silent DUX4 gene, which initiates a cascade of cellular events ultimately leading to FSHD pathophysiology. Therefore, FSHD is a dominant gain-of-function disease that is amenable to modeling by DUX4 overexpression. However, there is large variability in the patient population. Typically, progressive skeletal muscle weakness becomes noticeable in the second or third decade of life, yet there are many genetically FSHD individuals who develop symptoms much later in life or remain relatively asymptomatic throughout their lives. Conversely, in rare cases, FSHD may present clinically prior to 5-10 yrs of age, ultimately manifesting as a very severe early onset form of the disease. Thus, there is a need to control the timing and severity of pathology in FSHD-like models.\n\nMethodsWe have recently described a line of conditional DUX4 transgenic mice, FLExDUX4, that develop a myopathy upon induction of human DUX4-fl expression in skeletal muscle. Here, we use the FLExDUX4 mouse crossed with the skeletal muscle-specific and tamoxifen inducible line ACTAl-MerCreMer to generate a highly versatile bi-transgenic mouse model with chronic, low-level DUX4-fl expression and mild pathology, that can be induced to develop more severe FSHD-like pathology in a dose-dependent response to tamoxifen. We identified conditions to reproducibly generate models exhibiting mild, moderate, or severe DUX4-dependent pathophysiology, and characterized their progression.\n\nResultsWe assayed DUX4-fl mRNA and protein levels, fitness, strength, global gene expression, histopathology, and immune response, all of which are consistent with an FSHD-like myopathic phenotype. Importantly, we identified sex-specific and muscle-specific differences that should be considered when using these models for preclinical studies.\n\nConclusionsThe ACTA1-MCM;FLExDUX4 bi-transgenic mouse model expresses a chronic low level of DUX4-fl and has mild pathology and detectable muscle weakness. The onset and progression of moderate to severe pathology can be controlled via tamoxifen injection to provide consistent and readily screenable phenotypes for assessing therapies targeting DUX4-fl mRNA and protein. Thus, these FSHD-like mouse models can be used to study a range of DUX4-fl expression and pathology dependent upon investigator need, through controlled mosaic expression of DUX4.

cell biology↗