Transcriptional changes after YAP/TAZ siRNA-mediated knockdown, and BMP-2 stimulation and SMAD1 ChIP-Seq in BMP2-treated human fetal osteoblasts

Osteogenesis is influenced by a variety of factors including growth factors - especially those of the TGF-beta superfamily - and Hippo signaling. Consequently, we hypothesize that the YAP/TAZ pathway is intertwining with TGF-beta and BMP signaling resulting in osteogenic differentiation. Therefore, we performed a comprehensive RNA-Seq study including both YAP/TAZ depletion by siRNA and BMP stimulation conditions in human fetal osteoblasts cell line hFOB 1.10. In order to identify the direct SMAD1 target genes corresponding to our RNA-Seq data, we performed an SMAD1 chromatin immunoprecipitation followed by sequencing. To deplete YAP and TAZ, cells were transfected with human siRNA SMARTpool targeting YAP1 or WWTR1. 48h post-transfection cells were starved in 0% FCS medium for 3h and then stimulated with 5nM BMP-2 for 1h. Afterwards, cells were lysed and total RNA was isolated. For each experimental condition, three biological replicates were analyzed. For ChIP Seq BMP-2 treated cells (5nM, 90min) were fixed in 1% Formaldehyde and quenched by adding glycine 125 mM for 5 minutes at 4°C. After this, cells were harvested and lysed according to the protocol described by Lee et al (2006) and chromatin was sheared by sonication using the Bioruptor ultra sonicator (Diagenode®). Sheared chromatin was immunoprecipitated using 10 µg of total SMAD1 antibody (Cell Signaling®, #9743) or the normal rabbit IgG antibody (Cell signaling®, #2729) as a control, followed by incubation with Protein G magnetic beads (Invitrogen®). DNA was eluted in elution buffer and DNA purification was performed using DNA Purification Buffers and Spin Columns kit, according to the manufacturer’s instructions (Cell Signaling®).