Phospho-sensitive chromatin immunoprecipitation of RNA Polymerase II (Prot 48)
RNA polymerase II (RNAPII) is responsible for the transcription of protein-coding genes, in addition to a large number of non-coding RNAs. The C-terminal domain (CTD) of its largest subunit consists of multiple heptad repeats (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7; 52 copies in mammals) that are targeted for a wide range of post-translational modifications, providing a platform for interaction with chromatin modifiers and RNA processing machinery (Brookes and Pombo, 2009). Ser5 residues become phosphorylated during transcription initiation and Ser2 residues during productive transcription elongation.
Chromatin immunoprecipitation (ChIP) analysis has made it possible to determine the genomic location of many proteins involved in gene regulation, including RNAPII. However, understanding the roles of RNAPII modification in gene regulation requires knowledge of its phosphorylation status, which dictates the cohort of factors that associate with RNAPII at different steps of the transcription cycle, in active and primed states of gene expression. This requires a reliable ChIP protocol that preserves RNAPII phosphorylation and that works well with phospho-specific antibodies, some of which are IgM antibodies.
We have optimised a sensitive and robust ChIP protocol for the detection of different phosphorylated forms of RNAPII, compatible with a wide range of RNAPII antibodies including phospho-specific antibodies against phosphorylated Ser2 (Ser2P), Ser5 (Ser5P) and Ser7(Ser7P). In addition, we have used this protocol to efficiently detect a range of other transcriptional regulators (e.g. CDK9, NELF, TBP, CBX7, Ring1B) and histone modifications (e.g. H2Aub1, H3K27me3, H3K9ac, H3K4me2, H3K4me3, H3K36me3) in a number of different mouse and human cell lines and primary cells. The protocol was initially adapted from Soutoglou and Talianidis (2002), and its first version published in Stock et al. (2007). Here, we report the most recent modifications.
1 Genome Function Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK.
2Present address: Cancer Research Technology Discovery Laboratory, Wolfson Institute for Biomedical Research, The Cruciform Building, Gower Street, London, WC1E 6BT, UK.
Corresponding author: Ana Pombo, MRC Clinical Sciences Centre, Imperial College School of Medicine,Hammersmith Hospital Campus, Du Cane Road, London W12 0NN,UK.