Rabbit Anti-Human FHL1 Polyclonal antibody

Histone three lysine 27 (H3K27) methyltransferase enhancer of zeste homolog 2 (EZH2) is a critical epigenetic modifier, which regulates gene transcription through the trimethylation of the H3K27 residue leading to chromatin compaction and gene repression. EZH2 has previously been identified to regulate human bone marrow-derived mesenchymal stem cells (MSC) lineage specification. MSC lineage specification is regulated by the presence of EZH2 and its H3K27me3 modification or the removal of the H3K27 modification by lysine demethylases 6A and 6B (KDM6A and KDM6B). This study used a bioinformatics approach to identify novel genes regulated by EZH2 during MSC osteogenic differentiation. In this study, we identified the EZH2 targets, ZBTB16, MX1, and FHL1, which were expressed at low levels in MSC. EZH2 and H3K27me3 were found to be present along the transcription start site of their respective promoters. During osteogenesis, these genes become actively expressed coinciding with the disappearance of EZH2 and H3K27me3 on the transcription start site of these genes and the enrichment of the active H3K4me3 modification. Overexpression of EZH2 down-regulated the transcript levels of ZBTB16, MX1, and FHL1 during osteogenesis. Small interfering RNA targeting of MX1 and FHL1 was associated with a downregulation of the key osteogenic transcription factor, RUNX2, and its downstream targets osteopontin and osteocalcin. These findings highlight that EZH2 not only acts through the direct regulation of signaling modules and lineage-specific transcription factors but also targets many novel genes important for mediating MSC osteogenic differentiation.

The ability to accurately phenotype cells differentiated from human induced pluripotent stem cells (hiPSCs) is essential for their application in modeling developmental and disease processes, yet also poses a particular challenge without the context of anatomical location. Our specific objective was to determine if single-cell gene expression was sufficient to predict the electrophysiology of iPSC-derived cardiac lineages, to evaluate the concordance between molecular and functional surrogate markers. To this end, we used the genetically encoded voltage indicator ArcLight to profile hundreds of hiPSC-derived cardiomyocytes (hiPSC-CMs), thus identifying patterns of electrophysiological maturation and increased prevalence of cells with atrial-like action potentials (APs) between days 11 and 42 of differentiation. To profile expression patterns of cardiomyocyte subtype-associated genes, single-cell RNA-seq was performed at days 12 and 40 after the populations were fully characterized with the high-throughput ArcLight platform. Although we could detect global gene expression changes supporting progressive differentiation, individual cellular expression patterns alone were not able to delineate the individual cardiomyocytes into atrial, ventricular, or nodal subtypes as functionally documented by electrophysiology measurements. Furthermore, our efforts to understand the distinct electrophysiological properties associated with day 12 versus day 40 hiPSC-CMs revealed that ion channel regulators SLMAP, FGF12, and FHL1 were the most significantly increased genes at day 40, categorized by electrophysiology-related gene functions. Notably, FHL1 knockdown during differentiation was sufficient to significantly modulate APs toward ventricular-like electrophysiology. Thus, our results establish the inability of subtype-associated gene expression patterns to specifically categorize hiPSC-derived cells according to their functional electrophysiology, and yet, altered FHL1 expression is able to redirect electrophysiological maturation of these developing cells. Therefore, noncanonical gene expression patterns of cardiac maturation may be sufficient to direct functional maturation of cardiomyocytes, with canonical gene expression patterns being insufficient to temporally define cardiac subtypes of in vitro differentiation.