Background: Osteogenesis is a highly regulated developmental process and continues during the turnover and repair of mature bone. Runx2, the master regulator of osteoblastogenesis, directs a transcriptional program essential for bone formation through genetic and epigenetic mechanisms. While individual Runx2 gene targets have been identified, further insights into the broad spectrum of Runx2 functions required for osteogenesis are needed. Results: By performing genome-wide characterization of Runx2 binding at the three major stages of osteoblast differentiation - proliferation, matrix deposition and mineralization - we identify Runx2-dependent regulatory networks driving bone formation. Using chromatin immunoprecipitation followed by high-throughput sequencing over the course of these stages, we identify approximately 80,000 significantly enriched regions of Runx2 binding throughout the mouse genome. These binding events exhibit distinct patterns during osteogenesis, and are associated with proximal promoters and also non-promoter regions: upstream, introns, exons, transcription termination site regions, and intergenic regions. These peaks were partitioned into clusters that are associated with genes in complex biological processes that support bone formation. Using Affymetrix expression profiling of differentiating osteoblasts depleted of Runx2, we identify novel Runx2 targets including Ezh2, a critical epigenetic regulator; Crabp2, a retinoic acid signaling component; Adamts4 and Tnfrsf19, two remodelers of the extracellular matrix. We demonstrate by luciferase assays that these novel biological targets are regulated by Runx2 occupancy at non-promoter regions. Conclusions: Our data establish that Runx2 interactions with chromatin across the genome reveal novel genes, pathways and transcriptional mechanisms that contribute to the regulation of osteoblastogenesis.

Breast cancer remains the most common malignant disease in women worldwide. Despite advances in detection and therapies, studies are still needed to understand the mechanisms underlying this cancer. Cancer stem cells (CSC) play an important role in tumor formation, growth, drug resistance, and recurrence. Here, it is demonstrated that the transcription factor RUNX1, well known as essential for hematopoietic differentiation, represses the breast cancer stem cell (BCSC) phenotype and suppresses tumor growth in vivo. The current studies show that BCSCs sorted from premalignant breast cancer cells exhibit decreased RUNX1 levels, whereas ectopic expression of RUNX1 suppresses tumorsphere formation and reduces the BCSC population. RUNX1 ectopic expression in breast cancer cells reduces migration, invasion, and in vivo tumor growth (57%) in mouse mammary fat pad. Mechanistically, RUNX1 functions to suppress breast cancer tumor growth through repression of CSC activity and direct inhibition of ZEB1 expression. Consistent with these cellular and biochemical results, clinical findings using patient specimens reveal that the highest RUNX1 levels occur in normal mammary epithelial cells and that low RUNX1 expression in tumors is associated with poor patient survival.Implications: The key finding that RUNX1 represses stemness in several breast cancer cell lines points to the importance of RUNX1 in other solid tumors where RUNX1 may regulate CSC properties.