Genome-wide occupancy profiling reveals critical roles of FOXO1 in regulating extracellular matrix and circadian rhythm genes in human chondrocytes.

Objective Osteoarthritis (OA) is the most common age-related joint disease. With aging and inOA, the expression of FoxO transcription factors is reduced, diminishing their chondroprotective actions. In order to elucidate the molecular mechanisms by which FoxO1 protects chondrocytes, we sought to identify the genome-wide occupancy profile of FoxO1. Methods We performed FoxO1 chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) on human primary chondrocytes. ChIP-Seq data were integrated withRNAsequencing (RNA-Seq) data sets. Bioinformatics results were confirmed in primary chondrocytes that were treated with a FoxO1 inhibitor. Results Analysis of FoxO1 ChIP-Seq on human primary chondrocytes showed that pathways implicated inOApathogenesis are mainly regulated by FoxO1 binding to tissue-specific enhancers with suboptimal binding sites (20% of the peaks), while more ubiquitous FoxO1 pathways are regulated at the promoter level through interaction with its canonical binding motif (7% of the peaks). Integrating FoxO1 occupancy data withRNA-Seq data comparingOAand healthy human cartilage revealed 428 putative FoxO1 target genes that are dysregulated inOA. Pathway analysis showed enrichment for genes belonging to the senescence pathway (logP= -6.73), extracellular matrix (ECM) pathway (logP= -12.97), and circadian clock pathway (logP= -6.30), which suggests that FoxO1 dysregulation plays an important role in their abnormal expression inOA. Using an inhibitor of FoxO1, we confirmed that FoxO1 regulates these pathways in cultured human chondrocytes. Conclusion FoxO1 regulates ubiquitous and cartilage-specific genes in chondrocytes by using different mechanisms. The FoxO1 transcriptional network is a key player in regulating homeostasis,ECM, and circadian clock genes and plays an important role in the abnormal expression of these pathways observed inOApathogenesis.