Runx2 regulates chromatin accessibility to direct the osteoblast program at neonatal stages

Copyright and License

© 2022 The Authors

Acknowledgement

We thank Drs. Henry M. Kronenberg, Clifford J. Tabin, Kevin A. Peterson, Yuichi Nishi, Lick Pui Lai, Sean M. Hasso, and Lori L. O′Brien for helpful input; David Butler and Peter Maye for sharing Col2-ECFP and Col10a1-mCherry mice; and Jill McMahon, Charles Nicolet, Selene Tyndale, Helen Truong, Ryoko Kudo, Motoki Goto, Daisuke Tanaka, Asuka Uchida, Chie Kataoka, and Nozomi Nagumo for providing technical assistance. The supercomputing resources were provided by the Human Genome Center at the University of Tokyo. This work utilized the core research facility of the Center for Disease Biology and Integrative Medicine at the Graduate School of Medicine and the One-stop Sharing Facility Center for Future Drug Discoveries at the Graduate School of Pharmaceutical Sciences, The University of Tokyo. 

Funding

This study was funded by Japan Society for the Promotion of Science (JSPS) postdoctoral fellowships for research abroad (to H.H.); grants-in-aid for science research from JSPS (16H06279 [PAGS], 17H05106, 18K19636, and 20H03885 to H.H.; 21H04952 to U.C.; and 17H04403 and 21H03142 to S. Ohba); Rising Star Awards from the American Society for Bone and Mineral Research (to H.H. and S. Ohba); Mochida Memorial Foundation research grants (to H.H. and S.O.^∗); the Uehara Memorial Foundation Research Grant (to H.H. and S.O.^∗); the Takeda Science Foundation Research Grant (to H.H. and S.O.^∗); the Japan Agency for Medical Research and Development (AMED; JP21bm0704071 and JP21zf0127002 to H.H.); the Japan Science and Technology Agency through the Center of Innovation Program (JPMJCE1304 to U.C.); and the National Institutes of Health (DK056246 to A.P.M.).

Contributions

Conceptualization, H.H., S. Ohba, and A.P.M.; methodology, T.S., S.T., Q.G., M.K., K.N., A.A., and S. Ohba; investigation, H.H., H.O., X.H., S. Onodera, T.A., M.S., and Y.S; writing – original draft, H.H., U.C., A.P.M., and S. Ohba; funding acquisition, H.H., U.C., A.P.M., and S. Ohba; supervision, H.H., A.P.M., and S. Ohba.

Data Availability

ATAC-seq, ChIP-seq, and RNA-seq data have been deposited at Gene Expression Omnibus (GEO: GSE178293). scRNA-seq data have been deposited at the DDBJ BioProject database (DDBJ BioProject database: PRJDB8387).

Supplemental Material

• Document S1. Figures S1–S7.
• Table S1. All coordinates of identified cell-type specific chromatin-accessible regions and associated genes in the top 10 GREAT GO terms related to the biological process, related to Figure 1.
• Table S2. Gene expression profiles in C2-Cho, C10-Cho, and Sp7-Ob cells, determined by RNA-seq, related to Figure 1.
• Table S3. All coordinates of Runx2 ChIP-seq peaks in osteoblasts and chondrocytes and associated genes, related to Figure 2.
• Table S4. All coordinates of osteoblast- and chondrocyte-distinct Runx2 ChIP-seq peaks in osteoblasts and chondrocytes, Runx2 shared peaks in both cell types, and associated genes, related to Figure 3.
• Table S5. All coordinates of significantly changed ATAC-seq signals upon Runx2 deficiency in osteoblasts and associated genes, related to Figure 4.
• Table S6. All coordinates of the Runx2-responsive regions in NIH 3T3 cells and associated genes, related to Figure 5.
• Table S7. All oligo DNA information used in this study, related to Figure 6. These include the gRNA sequence, oligo DNA sequence, and targeted putative enhancer coordinates for CROP-seq; the DNA sequences of the probes for EMSA and primer sequences for qPCR are listed.
• Document S2. Article plus supplemental information.