friends of FSH Research


Identifying New Therapies

Posted by George Shaw on February 6, 2013

See layman summary at bottom.

Barbara Page
Barbara Page

Mentor: Stephen J Tapscott MD PhD

A final report by Barbara D Page on the work done for the second of a 2-year project titled Using model organisms to identify new therapies and understand the pathogenesis of FSHD, FSH Scholar funded by Friends of FSH Research (see 2011 Grant).

Specific Aims

Aim 1 will test the hypothesis that a small molecule can be used to increase epigenetic silencing. We had previously obtained and characterized an S. pombe yeast strain with marker genes inserted flanking the mating type locus on chromosome II. This strain had the ura4 and ade6 genes located at the boundaries of the mating type repressive chromatin. We had also confirmed that mutations in the ribonucleotide reductase pathway (cdc22 and tds1) allowed for the spreading of repressive heterochromatin, as demonstrated by the 5-FOA resistance of this strain. After performing a number of optimization steps (cell number, media composition, growth conditions, ATP luciferase assay development), we successfully established a liquid media-based assay in a 96-well plate format. Using these testing conditions, we identified a compound that could serve as a positive in our screen (hydroxyurea). Given these promising preliminary results, we began a collaboration with Dr. Marc Ferrer, PhD at the NIH Chemical Genomics Center (NIHCGC) to initiate the beginning of a high throughput small molecule screen for compounds capable of increasing repressive chromatin structures. Since this assay at the NIHCGC required use of a 1536-well format, Dr. Ferrer and his colleagues began to optimize the miniaturization protocol for this platform. Initial attempts at this process demonstrated a high degree of well-to-well variability that would require further optimization. While this was occurring, the NIHCGC underwent a change with regard to requirements for funding collaborative screens. We had previously planned to write an internal NIH RO3 grant with his group to perform the high throughput screen, but unfortunately this funding mechanism was terminated. Given that the yeast screen still required significant optimization and the change in funding mechanism, this Aim was suspended to concentrate on Aim 2 for the remainder of the award period.

Comparison of double homeobox containing proteins in mouse, dog, human, and worm
Comparison of double homeobox containing proteins in mouse, dog, human, and worm

Aim 2 will test the hypothesis that a DUX4 ortholog from the model organism C. elegans can be used to understand the pathogenesis of FSHD. The C. elegans gene duxl-1 (DUX [vertebrate dual homeobox] like) encodes a dual homeobox protein with similarity to the human protein DUX4. We decided to take advantage of this similarity and the excellent genetic, molecular, and developmental biology of C. elegans to investigate the function of a dual homeobox protein. Because C. elegans has been extraordinarily useful in dissecting the molecular components and their functions in such biological processes as germ line stem cell regulation, programmed cell death, neuronal path finding and RNA interference, the C. elegans research community has organized collections of mutants and gene fusions that are available to all researchers. We obtained useful reagents such as a deletion of the duxl-1 gene and a gene fusion between the duxl-1 promoter and gfp. The strain containing duxl-1(tm956), a deletion allele of duxl-1, was studied under different conditions to determine when the DUXL-1 protein functions. The duxl-1 promoter fusion to gfp was examined to determine which tissues express DUXL-1. Using these tools, we have found that duxl-1 affects fertility and anatomy of the gonad. Our results suggest that duxl-1 plays a regulatory role in cellular remodeling, a process that requires the careful removal of part of a cell without damaging the rest of the cell. If the function of duxl-1 in C. elegans has similarity with the function of DUX4 in human cells, then the ectopic expression of DUX4 protein in skeletal muscle, as observed in patients with FSHD, might inappropriately initiate cellular remodeling of otherwise healthy, normal muscle. Immune cells, such as macrophages, could recognize this signal, and this response could cause slow loss of muscle tissue. It is hoped that this insight will lead to new therapies to prevent muscle destruction in patients with FSHD.

Layman Description

As a Friends of FSH Research Scholar, I have focused on using two different model organisms to identify new therapies for facioscapulohumeral muscular dystrophy (FSHD) and better understand how this disease occurs. Recent experiments suggest that FSHD may be caused in part by the inappropriate expression of the gene DUX4 due to a loss of gene silencing. Therefore, a possible line of therapy for patients with FSHD would be the use of drugs that could reestablish silencing of the DUX4 gene. To identify such drugs, my first project focused on developing an assay using the model organism S. pombe (a single-celled yeast) as a tool to screen for small molecules that can increase gene silencing. Our pilot studies suggest that this assay can be used for the large scale testing of compounds to identify potential therapeutics for patients with FSHD. My second project took advantage of the numerous genetic tools available in the model organism C. elegans (a very small worm) to investigate how genes like DUX4 function in normal development. We discovered a DUX4-like gene in C. elegans, which we named duxl-1. Our results suggest that duxl-1 functions in the remodeling of tissues by regulating an "eat me" signal important for organized cellular destruction. If DUX4 in human cells acts similarly, then the inappropriate expression of DUX4 in skeletal muscle, as observed in patients with FSHD, might mislabel healthy muscle cells with the "eat me" signal. Immune cells in the body would recognize this signal, leading to the slow loss of muscle tissue. It is hoped that this insight will lead to new therapies to prevent muscle destruction in patients with FSHD.

—Barbara D Page