Facioscapulohumeral dystrophy (FSHD) is the third most prevalent muscular dystrophy, with no available treatment. FSHD develops as a consequence of the expression of Double Homeobox 4 (DUX4) in skeletal muscle. As such, DUX4 expression provides a measurable context to better understand mechanisms that regulate FSHD biology and may eventually guide the development of targeted therapeutics for this disease.
DUX4 is a transcription factor encoded by a retrogene, a DNA copied back from RNA by reverse transcription, and is embedded in each unit of the D4Z4 macrosatellite repeat, a large tandem DNA repeat known to impact gene regulation and human health. Such repetitive DNA sequences constitute an abundant yet enigmatic part of the human genome. Despite increasing evidence on the functionality of these repeats, their biologic role is still elusive and controversial. In healthy individuals, DUX4 is only expressed in a small number of tissues, including the early pre-implantation embryo and the testis. However, in FSHD patients, DUX4 is found to be expressed in skeletal muscle caused by decreased epigenetic repression of the D4Z4 macrosatellite array.
In most somatic tissues, the D4Z4 arrays appear to be silenced via multiple mechanisms, including DNA methylation, histone modification, and repressive chromatin proteins, which are disrupted in FSHD. Nevertheless, the mechanisms that establish and maintain DUX4 repression in somatic tissues are still unknown, thus highlighting a need for a broader understanding of DUX4 gene regulation. To do this, Dr. Amy Campbell together with colleagues in the Tapscott laboratory in the Human Biology Division used CRISPR-based locus-specific proteomics to identify proteins that bind to these repetitive sequences containing DUX4. Specifically, the authors carried out CRISPR/Cas9 engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) followed by mass spectrometry (MS) to identify regulators of the D4Z4 macrosatellite repeat. Their study, published in the journal eLIFE, identified a total of 261 proteins, including components of the Nucleosome Remodeling Deacetylase (NuRD) complex, which was further confirmed by chromatin immunoprecipitation experiments showing that the complex bound to the D4Z4 macrosatellite repeat in human muscle cells.
This approach, combined with a methodical series of gene depletion experiments, revealed that the NuRD complex, together with the Chromatin Assembly Factor 1 (CAF-1) complex, function to epigenetically repress DUX4 expression in human skeletal muscle and induced pluripotent stem cells. These studies also uncovered a role for DUX4-induced expression of the MBD3L family of methyl-CpG-binding proteins (MBD3L2-5) in relieving NuRD complex silencing activity and amplifying DUX4 levels in FSHD muscle cells.
Dr. Campbell explained how the interaction of these complexes may play out during development: “DUX4 is expressed for a short time during preimplantation development but is rapidly silenced and remains repressed in most somatic tissues. We expect that NuRD and CAF-1, together with other modes of transcriptional repression (such as DNA methylation), play a role in repressing DUX4 throughout development, with potentially different relative requirements for each pathway depending on the tissue and/or developmental stage. For example, there are reports of DUX4 expression in thymus and germ cells but the regulation of DUX4 in these cellular contexts may differ from that found in muscle and stem cells.” Dr. Stephen Tapscott pointed out that “NuRD and CAF-1 are only two of the many complexes implicated in the regulation of DUX4, raising the question of whether this complexity reflects simple redundancy or different specific regulatory roles for each complex in development.”
The Tapscott lab seeks to understand DUX4 regulation and function in normal development and disease; this study has definitely shed light on the NuRD and CAF-1 complexes being important players. It has also provided several novel approaches to designing new therapies for FSHD. However, the work has only just begun. Dr. Tapscott pointed out that: “For this study, we used control myoblasts that do not express DUX4 to isolate the D4Z4-associated proteins. One of our next goals will be to do similar studies from cells that do express DUX4 to see whether expression is associated with new factors or simply the loss of repressors.”
Although the authors only focused on a small number of proteins that were identified to bind to the D4Z4 repeat, Dr. Campbell revealed that there is much more in the pipeline. “We are now exploring the function of several other of the D4Z4-associated proteins we identified with special interest in those that play a positive transcriptional role at the locus.” As for the enChIP-MS approach, Dr. Campbell said: “We are initiating collaborations to extend the CRISPR-based proteomics approach used in this study to other cellular contexts relevant to DUX4 and/or muscle biology.”
Campbell AE, Shadle SC, Jagannathan S, Lim J, Resnick R, Tawil R, van der Maarel SM, Tapscott SJ. 2018. NuRD and CAF-1-mediated silencing of the D4Z4 array is modulated by DUX4-induced MBD3L proteins. eLIFE. 7:e31023
Funding was provided by the National Institutes of Health, the FSH Society and Friends of FSH Research.