Abstract
Normal cell growth and differentiation of bone cells requires the sequential expression of cell type specific genes to permit lineage specification and development of cellular phenotypes. Transcriptional activation and repression of distinct sets of genes support the anabolic functions of osteoblasts and the catabolic properties of osteoclasts. Furthermore, metastasis of tumors to the bone environment is controlled by transcriptional mechanisms. Insights into the transcriptional regulation of genes in bone cells may provide a conceptual basis for improved therapeutic approaches to treat bone fractures, genetic osteopathologies, and/or cancer metastases to bone. Chromatin immunoprecipitation (ChIP) is a powerful technique to establish in vivo binding of transcription factors to the promoters of genes that are either activated or repressed in bone cells. Combining ChIP with genomic microarray analysis, colloquially referred to as “ChIP-on-chip,” has become a valuable method for analysis of endogenous protein/DNA interactions. This technique permits assessment of chromosomal binding sites for transcription factors or the location of histone modifications at a genomic scale. This chapter discusses protocols for performing chromatin immunoprecipitation experiments, with a focus on ChIP-on-chip analysis. The information presented is based on the authors' experience with defining interactions of Runt-related (RUNX) transcription factors with bone-related genes within the context of the native nucleosomal organization of intact osteoblastic cells.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
1. Ren, B., Robert, F., Wyrick, J. J., et al. (2000). Genome-wide location and function of DNA binding proteins. Science 290, 2306–2309.
2. Pratap, J., Javed, A., Languino, L. R., et al. (2005). The Runx2 osteogenic transcription factor regulates matrix metalloproteinase 9 in bone metastatic cancer cells and controls cell invasion. Mol Cell Biol 25, 8581–8591.
3. Javed, A., Barnes, G. L., Pratap, J., et al. (2005). Impaired intranuclear trafficking of Runx2 (AML3/CBFA1) transcription factors in breast cancer cells inhibits osteolysis in vivo. Proc Natl Acad Sci USA 102, 1454–1459.
4. Blyth, K., Cameron, E. R., Neil, J. C. (2005). The runx genes: gain or loss of function in cancer. Nat Rev Cancer 5, 376–387.
5. Young, D. W., Hassan, M. Q., Yang, X.-Q., et al. (2007). Mitotic retention of gene expression patterns by the cell fate determining transcription factor Runx2. Proc Natl Acad Sci USA 104, 3189–3194.
6. Young, D. W., Hassan, M. Q., Pratap, J., et al. (2007). Mitotic occupancy and lineage-specific transcriptional control of rRNA genes by Runx2. Nature 445, 442–446.
7. Galindo, M., Pratap, J., Young, D. W., et al. (2005). The bone-specific expression of RUNX2 oscillates during the cell cycle to support a G1 related anti-proliferative function in osteo blasts. J Biol Chem 280, 20274–20285.
8. Shen, J., Hovhannisyan, H., Lian, J. B., et al. (2003). Transcriptional induction of the osteo calcin gene during osteoblast differentiation involves acetylation of histones H3 and H4. Mol Endocrinol 17, 743–756.
9. Shen, J., Montecino, M. A., Lian, J. B., et al. (2002). Histone acetylation in vivo at the osteo calcin locus is functionally linked to vitamin D dependent, bone tissue-specific transcription. J Biol Chem 277, 20284–20292.
10. Hassan, M. Q., Javed, A., Morasso, M. I., et al. (2004). Dlx3 transcriptional regulation of osteoblast differentiation: temporal recruitment of Msx2, Dlx3, and Dlx5 homeodomain pro teins to chromatin of the osteocalcin gene. Mol Cell Biol 24, 9248–9261.
11. O'Geen, H., Nicolet, C. M., Blahnik, K., et al. (2006). Comparison of sample preparation methods for ChIP-chip assays. Biotechniques 41, 577–580.
12. Scacheri, P. C., Crawford, G. E., Davis, S. (2006). Statistics for ChIP-chip and DNase hyper sensitivity experiments on NimbleGen arrays. Methods Enzymol 411, 270–282.
13. Qi, Y., Rolfe, A., MacIsaac, K. D., et al. (2006). High-resolution computational models of genome binding events. Nat Biotechnol 24, 963–970.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
van der Deen, M. et al. (2008). Chromatin Immunoprecipitation Assays: Application of ChIP-on-Chip for Defining Dynamic Transcriptional Mechanisms in Bone Cells. In: Westendorf, J.J. (eds) Osteoporosis. Methods In Molecular Biology™, vol 455. Humana Press. https://doi.org/10.1007/978-1-59745-104-8_13
Download citation
DOI: https://doi.org/10.1007/978-1-59745-104-8_13
Publisher Name: Humana Press
Print ISBN: 978-1-58829-828-7
Online ISBN: 978-1-59745-104-8
eBook Packages: Springer Protocols