ISSN: ISSN: 2157-7412
Vishakha Sharma, Sachchida Nand Pandey, Hunain Khawaja, Kristy J Brown, Yetrib Hathout and Yi-Wen Chen
Objective: The goal of the study is to identity proteins, which interact with the promoter region of double homeobox protein 4 (DUX4) gene known to be causative for the autosomal dominant disorder Facioscapulohumeral Muscular Dystrophy (FSHD). Methods: We performed a DNA pull down assay coupled with mass spectrometry analysis to identify proteins that interact with a DUX4 promoter probe in Rhabdomyosarcomca (RD) cells. We selected the top ranked protein poly (ADP-ribose) polymerase 1 (PARP1) from our mass spectrometry data for further ChIP-qPCR validation using patients’ myoblasts. We then treated FSHD myoblasts with PARP1 inhibitors to investigate the role of PARP1 in the FSHD myoblasts. Results: In our mass spectrometry analysis, PARP1 was found to be the top ranked protein interacting preferentially with the DUX4 promoter probe in RD cells. We further validated this interaction by immunoblotting in RD cells (2-fold enrichment compared to proteins pulled down by a control probe, p<0.05) and ChIP-qPCR in patients’ myoblasts (65-fold enrichment, p<0.01) Interestingly, the interaction was only observed in FSHD myoblasts but not in the control myoblasts. Upon further treatment of FSHD myoblasts with PARP1 inhibitors, we showed that treatment with a PARP1 inhibitor, 3-aminobenzamide (0.5 mM), for 24 hours had a suppression of DUX4 (2.6 fold, p<0.05) and ZSCAN4, a gene previously shown to be upregulated by DUX4, (1.6 fold, p<0.01) in FSHD myoblasts. Treatment with fisetin (0.5 mM), a polyphenol compound with PARP1 inhibitory property, for 24 hours also suppressed the expression of DUX4 (44.8 fold, p<0.01) and ZSCAN4 (2.2 fold, p<0.05) in the FSHD myoblasts. We further showed that DNA methyltransferase 1 (DNMT1), a gene regulated by PARP1 was also enriched at the DUX4 promoter in RD cells through immunoblotting (2-fold, p<0.01) and immortalized FSHD myoblasts (42-fold, p<0.01) but not control myoblasts through ChIP qPCR. Conclusions: Our results showed that PARP1 and DNMT1 interacted with DUX4 promoter and may be involved in modulating DUX4 expression in FSHD.