Transgenic Drosophila for Investigating DUX4 and FRG1, Two Genes Associated with Facioscapulohumeral Muscular Dystrophy (FSHD)

Facioscapulohumeral muscular dystrophy (FSHD) is typically an adult onset dominant myopathy. Epigenetic changes in the chromosome 4q35 region linked to both forms of FSHD lead to a relaxation of repression and increased somatic expression of DUX4-fl (DUX4-full length), the pathogenic alternative splicing isoform of the DUX4 gene. DUX4-fl encodes a transcription factor expressed in healthy testis and pluripotent stem cells; however, in FSHD, increased levels of DUX4-fl in myogenic cells lead to aberrant regulation of target genes. DUX4-fl has proven difficult to study in vivo; thus, little is known about its normal and pathogenic roles. The endogenous expression of DUX4-fl in FSHD-derived human muscle and myogenic cells is extremely low, exogenous expression of DUX4-fl in somatic cells rapidly induces cytotoxicity, and, due in part to the lack of conservation beyond primate lineages, viable animal models based on DUX4-fl have been difficult to generate. By contrast, the FRG1 (FSHD region gene 1), which is linked to FSHD, is evolutionarily conserved from invertebrates to humans, and has been studied in several model organisms. FRG1 expression is critical for the development of musculature and vasculature, and overexpression of FRG1 produces a myopathic phenotype, yet the normal and pathological functions of FRG1 are not well understood. Interestingly, DUX4 and FRG1 were recently linked when the latter was identified as a direct transcriptional target of DUX4-FL. To better understand the pathways affected in FSHD by DUX4-fl and FRG1, we generated transgenic lines of Drosophila expressing either gene under control of the UAS/GAL4 binary system. Utilizing these lines, we generated screenable phenotypes recapitulating certain known consequences of DUX4-fl or FRG1 overexpression. These transgenic Drosophila lines provide resources to dissect the pathways affected by DUX4-fl or FRG1 in a genetically tractable organism and may provide insight into both muscle development and pathogenic mechanisms in FSHD

The DUX4 model of FSHD.

<p>(A) A model of the human system, which represents a summary of published work relevant to understanding FRG1 and DUX4 in relation to FSHD supplied to aid the reader with context, showing the FSHD-associated human chromosome 4q35 D4Z4 macrosatellite in healthy (upper) and FSHD (lower) subjects. In healthy subjects, the D4Z4 array consists of between 11 and ~120 D4Z4 repeat units (RU) and is epigenetically repressed (black lollipops). FRG1 is ubiquitously expressed, but there is no (or very little) polyadenylated DUX4-fl mRNA expression. In FSHD1 subjects, the D4Z4 array consists of between 1 and 10 D4Z4 RU, is epigenetically derepressed (yellow lollipops), and a significantly higher fraction of cells than in healthy subjects express polyadenlylated DUX4-fl mRNA. The DUX4-FL protein, a transcription factor that regulates many genes, can bind to an enhancer located in intron 2 of <i>FRG1</i>, and expression levels of FRG1 are moderately increased in these DUX4-FL expressing cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150938#pone.0150938.ref034" target="_blank">34</a>]. (B) The polyadenylated DUX4-fl mRNA is derived from the distal repeat of the D4Z4 array. There are two potential translation start sites for DUX4-fl, termed MKG and MAL. Transgenic <i>Drosophila UAS-DUX4-fl</i>^<i>MKG</i> and <i>UAS-DUX4-fl</i>^<i>MAL</i> contain the coding sequence, codon optimized for <i>Drosophila</i>, from the indicated start codon through the stop codon (red *) in exon 1. Please see the following reviews for relevant references relating to FSHD depicted in this figure [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150938#pone.0150938.ref017" target="_blank">17</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150938#pone.0150938.ref021" target="_blank">21</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150938#pone.0150938.ref024" target="_blank">24</a>].</p

FRG1 is highly conserved between human and <i>Drosophila</i>.

<p>(A) Alignment of the predicted amino acid sequence for the human (Hs) and <i>D</i>. <i>melanogaster</i> (Dm) FRG1 orthologs. Conserved amino acids are highlighted in yellow with identical amino acids indicated by the letter and similar amino acids indicated by a +. The functional domains, including the nuclear localization signals (NLS) and RNA binding domain (RBD), are indicated. The peptides used as antigens for generating the DM1 and DM2 antibodies are boxed in red. (B) DmFRG1 expression was assayed by qRT-PCR at day 2 (D2) and day 10 (D10) in the thorax or head and abdomen combined, as indicated. Expression was normalized to <i>rp49</i> RNA levels and presented as fold expression compared to D2 or D10 thorax. Induction of DmFRG1 mRNA was significant (* p < 0.01 by Student’s t-test) for both lines when compared with <i>w</i> and <i>DJ667GAL4</i> alone. (C) Left panel: Western blots of protein extracts from 0–17h embryos (lane 1 and 4) or head/thorax of adult flies (lanes 2, 3, 5, and 6) of <i>yw</i> strain (lanes 1, 2, 4, and 5) or <i>tubP-GAL4</i>,<i>UAS-DmFRG1</i> flies (lanes 3 and 6), probed with the DM1 or DM2 antibody as indicated. Beta-tubulin was used as a loading control. (D) Thoracic muscle tissue immunostained for DmFRG1 (green) and counterstained with DAPI (blue) and phalloidin (red). Endogenous DmFRG1 is not detectable in the nuclei of <i>yw</i> flies (white arrows) and accumulates in nuclei when overexpressed (yellow arrows). Zoomed regions displayed in the lower panels are outlined in the upper panels by dotted white boxes.</p

Nuclear localization of endogenous and overexpressed DmFRG1 is conserved.

<p>Salivary gland tissue (A-G) and polytene chromosome spreads (H-M) from <i>w</i> (A, B) and <i>tubP-GAL4</i>, <i>UAS-DmFRG1</i> were immunostained for total DmFRG1 using the DM2 antibody (red; A-E, G, I, K, L) or specifically overexpressed DmFRG1 using the HA antibody (green; F, G, J, K, M), and stained with DAPI (blue) to show the nuclei (A, C) or chromosomes (H, L, M). Endogenous DmFRG1 is predominantly cytoplasmic (A, B), while overexpressed DmFRG1 accumulates in the nucleus (E, F, G). In the nucleus, the endogenous and overexpressed DmFRG1 localizations mostly overlap (yellow; G, K).</p

Ectopic <i>DUX4-fl</i> expression by <i>lGMR-GAL4</i> produces a readily screenable eye phenotype.

<p>A-C) Compound eyes of <i>lGMR-GAL4</i> consist of precisely organized and pigmented ommatidia and bristles. D-F) Eyes of <i>lGMR-GAL4/+</i>, <i>UASp-DUX4/+</i> are the usual size but lack organized ommatidia. Corneal lenses and pigment cells are completely missing; bristles are formed, but irregularly dispersed. Bar = 100 μm in panel D; Bar = 50 μm in panels E and F.</p

The Effects of Disease Models of Nuclear Actin Polymerization on the Nucleus

Actin plays a crucial role in regulating multiple processes within the nucleus, including transcription and chromatin organization. However, the polymerization state of nuclear actin remains controversial, and there is no evidence for persistent actin filaments in a normal interphase nucleus. Further, several disease pathologies are characterized by polymerization of nuclear actin into stable filaments or rods. These include filaments that stain with phalloidin, resulting from point mutations in skeletal α-actin, detected in the human skeletal disease intranuclear rod myopathy, and cofilin/actin rods that form in response to cellular stressors like heatshock. To further elucidate the effects of these pathological actin structures, we examined the nucleus in both cell culture models as well as isolated human tissues. We find these actin structures alter the distribution of both RNA polymerase II and chromatin. Our data suggest that nuclear actin filaments result in disruption of nuclear organization, which may contribute to the disease pathology

Abnormal musculature in DM-FRG1 overexpressing flies.

<p>H&E stained paraffin sections of thoraces from <i>DJ667GAL4</i> (A and C) and of <i>DJ667GAL4; DmFRG1</i> (B and D) adult flies. (B) Overexpression of DmFRG1 leads to disorganized dorsal longitudinal muscles (DLMs, indirect wing depressors) exhibiting variable numbers and shapes of muscles suggestive of muscle degeneration (arrowheads) compared with the highly organized musculature observed in the controls (A), which exhibit the characteristic 6 distinct DLMs on each side. (C and D) H&E stained paraffin sections of the dorso-ventral tergosternal muscle bundles (indirect wing levators) show histological features characteristic of muscle degeneration (arrowheads). * denotes the same muscle in these flies for a reference. Bar = 500 μm.</p

DUX4 expression phenotypes.

<p>DUX4 expression phenotypes.</p