title: PARP1 Differentially Interacts with Promoter region ofDUX4Gene in FSHD Myoblasts.
abstract: 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 theDUX4promoter 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 h had a suppression ofDUX4(2.6 fold, p<0.05) andZSCAN4, agene previously shown to be upregulated byDUX4,(1.6 fold, p<0.01) in FSHD myoblasts. Treatment with fisetin (0.5 mM), a polyphenol compound with PARP1 inhibitory property, for 24 h also suppressed the expression ofDUX4(44.8 fold, p<0.01) andZSCAN4(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 withDUX4promoter and may be involved in modulatingDUX4expression in FSHD.
authors.affiliation: Department of Molecular Medicine, George Washington University, Washington DC, USA.
authors.forename: Vishakha
authors.initials: V
authors.lastname: Sharma
authors.affiliation: Center for Genetic Medicine Research, Children's National Health System, Washington DC, USA.
authors.forename: Sachchida Nand
authors.initials: SN
authors.lastname: Pandey
authors.affiliation: Center for Genetic Medicine Research, Children's National Health System, Washington DC, USA.
authors.forename: Hunain
authors.initials: H
authors.lastname: Khawaja
authors.affiliation: Center for Genetic Medicine Research, Children's National Health System, Washington DC, USA.
authors.forename: Kristy J
authors.initials: KJ
authors.lastname: Brown
authors.affiliation: Center for Genetic Medicine Research, Children's National Health System, Washington DC, USA; Department of Integrative Systems Biology, George Washington University, Washington DC, USA.
authors.forename: Yetrib
authors.initials: Y
authors.lastname: Hathout
authors.affiliation: Center for Genetic Medicine Research, Children's National Health System, Washington DC, USA; Department of Integrative Systems Biology, George Washington University, Washington DC, USA.
authors.forename: Yi-Wen
authors.initials: YW
authors.lastname: Chen
keywords: DUX4 Gene; Facioscapulohumeral muscular dystrophy; Immunoblotting; Myoblast