Chromatin immunoprecipitation assays: Application of chip-on-chip for defining dynamic transcriptional mechanisms in bone cells

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.