Figure 3

ChIP analysis of histone modification at the promoter region of the AR gene. Allele-specific ChIP analysis for H3 acetylation and H3 K4 methylation was performed using the highly polymorphic CAG repeat region in the 5' coding region of the androgen receptor in Xq12. A. "Normal" isoX fibroblasts with highly skewed inactivation (GM00088) and cloned fibroblasts from normal (1871) and ICF (PT4) cultures were subjected to ChIP and the immunoprecipitated material was amplified for AR analysis in two stages; nested FAM-labeled primers were used in the second stage for size analysis on an automated sequencer. The ethidium bromide-stained agarose gel of the secondary PCR products show strong signals in the input (Inp), anti-H3 dimethyl K4 antibody (mK4), and anti-H3 acetyl K9, K14 (acH3), but little or no signal in the "no antibody" controls (-). The weak bands with slower migration correspond to heteroduplexes with the primary amplimer, but these do not interfere with the allele-specific analysis of the denatured product. B. and C. Allele-specific analyses of the products shown in A, using an automated sequencer, revealed that the active X alleles (Xa) in normal cells (1871 and GM00088) are hypermethylated at H3 K4 (H3meK4) and hyperacetylated at H3 K9, K14 (AcetylH3), whereas the corresponding inactive X (Xi) alleles are hypomethylated and hypoacetylated. ICF cells (PT4) showed the same active X versus inactive X modifications as normal cells. "Shadow bands" (s) probably correspond to PCR errors. Allele assignment was ascertained by a similar method using allele-specific RT-PCR with DNased RNA.