Gene expression during normal and FSHD myogenesis

- expression normal and FSHD myogenesis Tsumagari et al. Open Access Gene expression during normal and FSHD myogenesis Koji Tsumagari1, Shao-Chi Chang1, Michelle Lacey2,3, Carl Baribault2,3, Sridar V Chittur4, Janet Sowden5, Rabi Tawil5, Gregory E Crawford6 and Melanie Ehrlich1,3* Background: Facioscapulohumeral muscular dystrophy (FSHD) is a dominant disease linked to contraction of an array of tandem 3.3-kb repeats (D4Z4) at 4q35. Within each repeat unit is a gene, DUX4, that can encode a protein containing two homeodomains. A DUX4 transcript derived from the last repeat unit in a contracted array is associated with pathogenesis but it is unclear how. Methods: Using exon-based microarrays, the expression profiles of myogenic precursor cells were determined. Both undifferentiated myoblasts and myoblasts differentiated to myotubes derived from FSHD patients and controls were studied after immunocytochemical verification of the quality of the cultures. To further our understanding of FSHD and normal myogenesis, the expression profiles obtained were compared to those of 19 non-muscle cell types analyzed by identical methods. Results: Many of the ~17,000 examined genes were differentially expressed (> 2-fold, p < 0.01) in control myoblasts or myotubes vs. non-muscle cells (2185 and 3006, respectively) or in FSHD vs. control myoblasts or myotubes (295 and 797, respectively). Surprisingly, despite the morphologically normal differentiation of FSHD myoblasts to myotubes, most of the disease-related dysregulation was seen as dampening of normal myogenesisspecific expression changes, including in genes for muscle structure, mitochondrial function, stress responses, and signal transduction. Other classes of genes, including those encoding extracellular matrix or pro-inflammatory proteins, were upregulated in FSHD myogenic cells independent of an inverse myogenesis association. Importantly, the disease-linked DUX4 RNA isoform was detected by RT-PCR in FSHD myoblast and myotube preparations only at extremely low levels. Unique insights into myogenesis-specific gene expression were also obtained. For example, all four Argonaute genes involved in RNA-silencing were significantly upregulated during normal (but not FSHD) myogenesis relative to non-muscle cell types. Conclusions: DUX4s pathogenic effect in FSHD may occur transiently at or before the stage of myoblast formation to establish a cascade of gene dysregulation. This contrasts with the current emphasis on toxic effects of experimentally upregulated DUX4 expression at the myoblast or myotube stages. Our model could explain why DUX4s inappropriate expression was barely detectable in myoblasts and myotubes but nonetheless linked to FSHD. Background Differentiation of myoblasts to myotubes is one of the best cell culture models for vertebrate differentiation. However, there has been only limited expression profiling of well characterized myoblast cell strains and of myoblasts differentiated in vitro to myotubes [1-3]. In this study, we profiled expression of control myoblasts * Correspondence: 1Human Genetics Program, Tulane Medical School, New Orleans, LA, USA Full list of author information is available at the end of the article and myotubes as well as analogous cells from patients with facioscapulohumeral muscular dystrophy (FSHD). Importantly, we were able to compare control and FSHD myoblasts and myotubes with 19 different nonmuscle cell types subjected to identical expression profiling. The data are directly comparable because the same experimental and computational techniques were used for all the cell types. This allowed us to identify myogenesis-specific as well as disease-associated differences in expression. We are particularly interested in regenerative myogenesis [4], as opposed to embryonic myogenesis [5], because of its role in limiting atrophy due to muscle damage, aging, and disease. FSHD is a dominant disease whose pathogenesis is still perplexing despite new insights into its genetic linkage [6-8]. It is progressively debilitating and painful and mainly affects skeletal muscle. FSHD is linked to contraction at 4q35 of a tandem array of 3.3-kb repeats, D4Z4, from about 11-100 to 1-10 copies [9]. It is usually diagnosed in the second decade, and the patients lifespan is generally not affected. Initially, the pathology is limited to a small set of skeletal muscles, often asymmetrically. There is apparently no involvement of smooth muscle. No efficacious treatment is available. Although other expression profiling studies of FSHD vs. normal- or disease-control muscle biopsies have been done [6,10-13], no clear consensus has emerged as to the genes that lead to the muscle pathology. Usually, only modest up- or downregulation of gene expression was observed. FSHD is likely to involve defects in muscle cell precursors [10]; therefore, studies of FSHD myoblasts and myotubes should also elucidate normal myogenesis. In analyses of muscle tissue, myogenesis-specific, disease-related changes in expression are obscured by the very low percentages of (activated) satellite cells. Upon expression profiling of FSHD and control myoblasts (but not myotubes) in 2003, Winokur et al. found ~20 genes were FSHD-dysregulated; among them were genes involved in the response to oxidative stress [14]. Accordingly, they demonstrated and Barro et al. confirmed [14,15] that FSHD myoblasts are significantly more sensitive to the lethal effects of drug-induced oxidative stress than normal-control and disease-control myoblasts. Nonetheless, Barro et al. demonstrated that this hypersensitivity did not affect growth rates or the ability of myoblasts to differentiate to myotubes. A recent expression profiling study of FSHD and control myoblasts and myotubes by Cheli et al. [16] provided no characterization of the purity of the myoblast or myotube samples and paradoxically reported no musclerelated terms among 177 functional terms for genes with differential expression in normal-control myoblast vs. normal-control myotube preparations, which is very different from what we have found, as described below. A number of 4q35 genes have been considered as candidates for the initially dysregulated gene during FSHD pathogenesis, namely, FRG1, DUX4, DUX4C, ANT1 (SLC25A4), FRG2, TUBB4Q, and FAT1 [6,17-24]. Recently implicated in FSHD pathogenesis from genetic mapping is DUX4, a 1.6-kb gene that resides within each 3.3-kb repeat unit of D4Z4. DUX4 encodes a protein containing two homeodomains [25,26]. The protein is strongly pro-apoptotic when highly overexpressed in experimental models [21,27-29]. DUX4 transcripts are normally difficult to detect probably because of heterochromatinization of normal long D4Z4 arrays inhibiting their transcription [30,31] and the lack of a polyadenylation signal within DUX4 [32], which generally leads to DUX4 mRNA being unstable. However, in patients, a polyadenylation signal is provided for the most distal DU (...truncated)