Genome-Wide Chromatin Landscape Transitions Identify Novel Pathways in Early Commitment to Osteoblast Differentiation

• Published in: PloS one 2016-02, Vol.11 (2), p.e0148619-e0148619
• Main Authors: Thompson, Bethtrice, Varticovski, Lyuba, Baek, Songjoon, Hager, Gordon L
• Format: Article
• Language: English
• Subjects: Adipocytes; Adipogenesis - genetics; Analysis; Binding Sites; Biocompatibility; Biology; Biology and Life Sciences; Bone remodeling; Cell Cycle - genetics; Cell Differentiation - genetics; Cell Line; Cellular communication; Chromatin; Chromatin - genetics; Chromatin - metabolism; Chromatin Immunoprecipitation; Cluster Analysis; Computational Biology - methods; Correlation analysis; Cues; Cytokines; Deoxyribonuclease; Deoxyribonucleic acid; Differentiation; DNA; Enhancers; Extracellular matrix; Gene expression; Gene Expression Profiling; Gene Expression Regulation; Gene sequencing; Genetic regulation; Genome-wide association studies; Genome-Wide Association Study - methods; Genomes; High-Throughput Nucleotide Sequencing; Hormones; Humans; Hypersensitivity; Laboratories; Medicine and Health Sciences; Mesenchyme; Molecular Sequence Annotation; Mutation; Nucleotide Motifs; Osteoblastogenesis; Osteoblasts; Osteoblasts - cytology; Osteoblasts - metabolism; Osteogenesis; Osteogenesis - physiology; Osteoporosis; Pathways; Promoters; Protein Binding; Regulatory Sequences, Ribonucleic Acid; Signal Transduction; Stem cells; Temperature; Transcription factors; Transcription Factors - metabolism; Transcriptome; Wnt protein
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0148619

Bone continuously undergoes remodeling by a tightly regulated process that involves osteoblast differentiation from Mesenchymal Stem Cells (MSC). However, commitment of MSC to osteoblastic lineage is a poorly understood process. Chromatin organization functions as a molecular gatekeeper of DNA functions. Detection of sites that are hypersensitive to Dnase I has been used for detailed examination of changes in response to hormones and differentiation cues. To investigate the early steps in commitment of MSC to osteoblasts, we used a model human temperature-sensitive cell line, hFOB. When shifted to non-permissive temperature, these cells undergo "spontaneous" differentiation that takes several weeks, a process that is greatly accelerated by osteogenic induction media. We performed Dnase I hypersensitivity assays combined with deep sequencing to identify genome-wide potential regulatory events in cells undergoing early steps of commitment to osteoblasts. Massive reorganization of chromatin occurred within hours of differentiation. Whereas ~30% of unique DHS sites were located in the promoters, the majority was outside of the promoters, designated as enhancers. Many of them were at novel genomic sites and need to be confirmed experimentally. We developed a novel method for identification of cellular networks based solely on DHS enhancers signature correlated to gene expression. The analysis of enhancers that were unique to differentiating cells led to identification of bone developmental program encompassing 147 genes that directly or indirectly participate in osteogenesis. Identification of these pathways provided an unprecedented view of genomic regulation during early steps of differentiation and changes related to WNT, AP-1 and other pathways may have therapeutic implications.