Identification of Novel Target Genes of the PTH-activated αNAC Transcriptional Coregulator

Hadla Hariri, Martin Pellicelli, Julie A. Miller, and René St-Arnaud

Dept. of Human Genetics, McGill University and Shriners Hospitals for Children – Canada, Montreal (Quebec) Canada H3G 1A6

Nascent-polypeptide-associated complex and coregulator alpha (αNAC) was shown to be a downstream transcriptional effector of the PTH – cAMP – PKA signaling cascade in bone cells. Upon PTH treatment of osteoblasts, cAMP accumulates to activate PKA, which in turn phosphorylates αNAC on residue Ser99. This leads to nuclear translocation of the αNAC protein. Little is known about potential transcriptional targets of the PTH-activated, PKA-phosphorylated αNAC protein in osteoblasts. In order to identify such targets, we performed ChIP-seq and RNA-seq experiments in PTH-treated MC3T3-E1 osteoblast-like cells. Genes showing differential responses in both data sets were further validated using conventional ChIP and qRT-PCR. These experiments identified the potential αNAC target genes USP53 and NFIL3. Conventional ChIP assays in PTH-treated MC3T3-E1 cells confirmed a 4- and 5-fold enrichment of αNAC binding at the USP53 and NFIL3 promoter regions, respectively. PTH treatment also increased the mRNA expression of NFIL3 and USP53 by 6- and 3-fold, respectively. We then established shRNA-mediated αNAC knockdown MC3T3-E1 cells. Decreased expression of αNAC mRNA and protein levels were confirmed by qRT-PCR amplification and Western blotting. Interestingly, PTH-driven transcriptional induction of USP53 and NFIL3 was decreased by 50% following αNAC knockdown. These experiments have identified two novel αNAC target genes downstream of the PTH signal transduction pathway. We plan to uncover the biological impact of those targets on osteoblast biology to further our understanding of how the PTH-PKA-αNAC cascade contributes to optimal bone development.