Posted by George Shaw on Feb 6, 2013
Mentor: Stephen J. Tapscott, MD, PhD
See related 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.
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.
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