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Functional domains of the FSHD-associated DUX4 protein

Hiroaki Mitsuhashi¹, Satoshi Ishimaru², Sachiko Homma³

¹Department of Applied Biochemistry, School of Engineering, Tokai University, Kanagawa 259-1207, Japan hmitsuhashi@tsc.u-tokai.ac.jp millerjb@bu.edu.

|April 6, 2018

Abstract:

Aberrant expression of the full-length isoform of DUX4 (DUX4-FL) appears to underlie pathogenesis in facioscapulohumeral muscular dystrophy (FSHD). DUX4-FL is a transcription factor and ectopic expression of DUX4-FL is toxic to most cells. Previous studies showed that DUX4-FL-induced pathology requires intact homeodomains and that transcriptional activation required the C-terminal region. In this study, we further examined the functional domains of DUX4 by generating mutant, deletion, and fusion variants of DUX4. We compared each construct to DUX4-FL for (i) activation of a DUX4 promoter reporter, (ii) expression of the DUX4-FL target gene ZSCAN4, (iii) effect on cell viability, (iv) activation of endogenous caspases, and (v) level of protein ubiquitination. Each construct produced a similarly sized effect (or lack of effect) in each assay. Thus, the ability to activate transcription determined the extent of change in multiple molecular and cellular properties that may be relevant to FSHD pathology. Transcriptional activity was mediated by the C-terminal 80 amino acids of DUX4-FL, with most activity located in the C-terminal 20 amino acids. We also found that non-toxic constructs with both homeodomains intact could act as inhibitors of DUX4-FL transcriptional activation, likely due to competition for promoter sites.This article has an associated First Person interview with the first author of the paper.

Insights

The C-terminal region of DUX4-FL, particularly the last 20 amino acids, drives its toxicity in facioscapulohumeral muscular dystrophy (FSHD). Non-toxic DUX4 variants can inhibit DUX4-FL activity by competing for promoter sites.

Area of Science:

Molecular Biology
Genetics
Cell Biology

Background:

Facioscapulohumeral muscular dystrophy (FSHD) pathogenesis is linked to aberrant expression of the full-length DUX4 (DUX4-FL) isoform.
Ectopic DUX4-FL expression is toxic to most cells, with previous research indicating a requirement for intact homeodomains and the C-terminal region for transcriptional activation and pathology.
Understanding the specific functional domains of DUX4 is crucial for developing targeted therapies for FSHD.

Purpose of the Study:

To further delineate the functional domains of DUX4 responsible for its pathogenic effects in FSHD.
To investigate the relationship between DUX4 transcriptional activity and its impact on cellular properties.
To explore the potential of DUX4 variants as inhibitors of DUX4-FL activity.

Main Methods:

Generation of mutant, deletion, and fusion variants of DUX4.
Comparison of DUX4 variants with DUX4-FL across multiple assays: DUX4 promoter reporter activation, ZSCAN4 expression, cell viability, caspase activation, and protein ubiquitination.
Assessment of the role of specific DUX4 domains, particularly the C-terminal region, in mediating transcriptional activity and cellular toxicity.

Main Results:

The transcriptional activity of DUX4-FL, measured by reporter gene activation and ZSCAN4 expression, correlated directly with its effects on cell viability, caspase activation, and protein ubiquitination.
Transcriptional activity was primarily mediated by the C-terminal 80 amino acids of DUX4-FL, with the most significant contribution from the C-terminal 20 amino acids.
Non-toxic DUX4 variants retaining intact homeodomains demonstrated inhibitory effects on DUX4-FL transcriptional activation, suggesting a competitive mechanism for promoter binding.

Conclusions:

The C-terminal region of DUX4-FL is critical for its transcriptional activity and subsequent cellular toxicity relevant to FSHD.
DUX4 transcriptional activity is a key determinant of its impact on multiple molecular and cellular properties.
Intact DUX4 homeodomain-containing variants can act as dominant-negative inhibitors of DUX4-FL, offering a potential therapeutic strategy for FSHD.

Read full analysis

Keywords:

DUX4 Facioscapulohumeral dystrophy Homeodomains Muscular dystrophy Skeletal muscle Transactivation domain

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