Bone Abstracts

Molecularly characterized epigenetic networks that control bone formation and are altered during aging are necessary to uncover new potential targets for osteoanabolic therapy. Our contribution to the iBONE consortium is to identify osteoanabolic epigenomic regulators by screening which are involved in osteoblast phenotype and differentiation. This study will be done by a 3 step approach including: siRNA screening for epigenomic regulators of osteoblastic differentiation and validation, identification of target genes by ChIP and functional analyses.

We are currently establishing the siRNA screening. To do this, we chose a colorimetric alkaline phosphatase assay and the determination of Runx2 transcriptional activity or activity of the canonical Wnt/β-catenin pathway using specific reporter plasmids to quantitate osteoanabolic activity. Reporter constructs contain the firefly luciferase cDNA under the control of either Runx2 (8OSE2Tk-luc) or the Wnt/β-catenin signaling pathway responsive elements (TOPflash).

The hFOB1.19 and the FhSO6 cell lines have been stably transfected with the reporter constructs using TALE nuclease or CRISPR/Cas9 technology. The FhSO6 cell line is more differentiated than hFOB, having higher alkaline phosphatase activity and reduced type 1 collagen expression. TALEN and CRISPR/Cas9 have been used to insert the two reporter constructs and their respective negative controls (Tk-luc and FOPflash) into the AAVS1 docking site by homologous recombination in both cell lines. In parallel we have developed an alkaline phosphatase assay for high-throughput screening in 96 well-plates.

We will use a custom siRNA library targeting 347 epigenetic regulators which contain siRNAs against histone and chromatin modifiers, DNA methylation partners and histone chaperones.

Currently, we have FhSO6 and hFOB stably transfected clones and their characterization is ongoing. Some results on the siRNA screening will be presented at the meeting. Through these efforts, we expect to identify some novel epigenomic regulators of osteoblast function.