Study of atrophy in Facioscapulohumeralmuscular dystrophy (FSHD)

Facioscapulohumeral muscular dystrophy (FSHD) is a progressive hereditary muscle disease related to chromatin opening in the 4q35 region which facilitates the expression of the DUX4 (Double Homeobox 4) gene encoding a transcription factor. It plays a major role in the development of FSHD and initiates a cascade of genes deregulation causing muscle atrophy, oxidative stress, defects in muscle differentiation and inflammation.
Several signaling pathways can lead to muscle atrophy including the NF-κB and the PI3K/AKT/FOXO pathway. The latter is involved in the balance between hypertrophy and atrophy in normal muscle. Our laboratory has shown that abnormal expression of DUX4 in FSHD muscle cells induced expression of the atrogenes MuRF1 and atrogin-1 (MAFbx) (skeletal muscle atrophy markers).
FOXO1 is a transcriptional activator of the atrogenes and we have identified two putative DUX4 binding sites in the FOXO1 promoter. Our aim is to investigate whether the atrogenes expression we observe in FSHD muscle cells is dependent on FOXO1 transactivation by DUX4.
We have built two reporter vectors containing the luciferase gene under the control of the FOXO1 promoter regions containing either one (pGL3-B) or two (pGL3-A+B) putative DUX4 binding sites. We transfected control or FSHD immortalized myoblasts with each vector and measured the luciferase activity. We found a 3-fold higher luciferase activity in FSHD myoblasts compared to control myoblasts. We also co-transfected C2C12 myoblasts with pGL3-A+B or pGL3-B and with a DUX4 expression vector to evaluate the effect of DUX4 on the FOXO1 promoter activity. As negative controls we used in parallel a DUX1 expression vector (a non-pathologic paralog protein) or an empty vector. The FOXO1 promoter (A+B or B sites) were activated by DUX4 in a dose-dependent manner. The inactivation by mutagenesis of the putative DUX4 binding B site in the FOXO1 promoter induced a decrease in the luciferase activity.
Our preliminary data showed that the FOXO1 promoter was activated in FSHD myoblasts. This activation is probably due to the DUX4 induction in these cells. Indeed our preliminary analyzes of the FOXO1 promoter activity underscores this hypothesis as our data following mutation of DUX4 putative binding sites.
Our results suggest that the atrogenes expression is probably due to a direct FOXO1 gene activation by DUX4. FOXO1 could therefore constitute a novel DUX4 target and would be a possible druggable target to interfere with FSHD muscle atrophy.