Browsing by Author "Romero, Brigette"
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Item DNA Methylation Analysis Reveals Distinct Patterns in Satellite Cell–Derived Myogenic Progenitor Cells of Subjects with Spastic Cerebral Palsy(Journal of Personalized Medicine, 2022-11-30) Robinson, Karyn G.; Marsh, Adam G.; Lee, Stephanie K.; Hicks, Jonathan; Romero, Brigette; Batish, Mona; Crowgey, Erin L.; Shrader, M. Wade; Akins, Robert E.Spastic type cerebral palsy (CP) is a complex neuromuscular disorder that involves altered skeletal muscle microanatomy and growth, but little is known about the mechanisms contributing to muscle pathophysiology and dysfunction. Traditional genomic approaches have provided limited insight regarding disease onset and severity, but recent epigenomic studies indicate that DNA methylation patterns can be altered in CP. Here, we examined whether a diagnosis of spastic CP is associated with intrinsic DNA methylation differences in myoblasts and myotubes derived from muscle resident stem cell populations (satellite cells; SCs). Twelve subjects were enrolled (6 CP; 6 control) with informed consent/assent. Skeletal muscle biopsies were obtained during orthopedic surgeries, and SCs were isolated and cultured to establish patient–specific myoblast cell lines capable of proliferation and differentiation in culture. DNA methylation analyses indicated significant differences at 525 individual CpG sites in proliferating SC–derived myoblasts (MB) and 1774 CpG sites in differentiating SC–derived myotubes (MT). Of these, 79 CpG sites were common in both culture types. The distribution of differentially methylated 1 Mbp chromosomal segments indicated distinct regional hypo– and hyper–methylation patterns, and significant enrichment of differentially methylated sites on chromosomes 12, 13, 14, 15, 18, and 20. Average methylation load across 2000 bp regions flanking transcriptional start sites was significantly different in 3 genes in MBs, and 10 genes in MTs. SC derived MBs isolated from study participants with spastic CP exhibited fundamental differences in DNA methylation compared to controls at multiple levels of organization that may reveal new targets for studies of mechanisms contributing to muscle dysregulation in spastic CP.Item Enhanced myogenesis through lncFAM-mediated recruitment of HNRNPL to the MYBPC2 promoter(Nucleic Acids Research, 2022-12-19) Chang, Ming-Wen Chang; Yang, Jen-Hao; Tsitsipatis, Dimitrios; Yang, Xiaoling; Martindale, Jennifer L.; Munk, Rachel; Pandey, Poonam R.; Banskota, Nirad; Romero, Brigette; Batish, Mona; Piao, Yulan; Mazan-Mamczarz, Krystyna; De, Supriyo; Abdelmohsen, Kotb; Wilson, Gerald M.; Gorospe, MyriamThe mammalian transcriptome comprises a vast family of long noncoding (lnc)RNAs implicated in physiologic processes such as myogenesis, through which muscle forms during embryonic development and regenerates in the adult. However, the specific molecular mechanisms by which lncRNAs regulate human myogenesis are poorly understood. Here, we identified a novel muscle-specific lncRNA, lncFAM71E1-2:2 (lncFAM), which increased robustly during early human myogenesis. Overexpression of lncFAM promoted differentiation of human myoblasts into myotubes, while silencing lncFAM suppressed this process. As lncFAM resides in the nucleus, chromatin isolation by RNA purification followed by mass spectrometry (ChIRP-MS) analysis was employed to identify the molecular mechanisms whereby it might promote myogenesis. Analysis of lncFAM-interacting proteins revealed that lncFAM recruited the RNA-binding protein HNRNPL to the promoter of MYBPC2, in turn increasing MYBPC2 mRNA transcription and enhancing production of the myogenic protein MYBPC2. These results highlight a mechanism whereby a novel ribonucleoprotein complex, lncFAM-HNRNPL, elevates MYBPC2 expression transcriptionally to promote myogenesis.