Figure 4.
Histone modifications at the Il9 locus. (a) VISTA plot of conserved non-coding sequences adjacent to the Il9 locus. (b,c) Naïve CD4+ T cells were isolated and analyzed directly or differentiated under TH1, TH2, TH9, TH17 or iTreg culture conditions for five days. Cells were analyzed for histone modifications using chromatin immunoprecipitation for the indicated histone modifications and primers for Il9 CNS. (d,e) Wild-type and Sfpi1lck−/− naïve CD4+ T cells were cultured under TH9 conditions and used for ChIP analysis as described in b performed for AcH3 at CNS1 and CNS2 (d), or for AcH4 and me3H3K27 at CNS1 (e). (f) Top, consensus PU.1 binding sites in the Il9 CNS1. Bottom, DAPA analysis of PU.1 binding from TH2 or TH9 extracts to site 2. For competition, extracts were incubated with a 5-fold excess of unlabelled PU.1 consensus double-stranded oligonucleotide. Expression of PU.1 in each cell extract is shown as input. Results are representative of three experiments. (g) Naïve CD4+ T cells were differentiated under TH1, TH2, or TH9 culture conditions for five days. Cells were analyzed for PU.1 binding by ChIP with primers for Il9CNS1. Results of ChIP experiments are shown as average ± SD of percent input with control IgG subtracted (a-e) or with anti-PU.1 and IgG control ChIP values shown separately (g) and are representative of 2-4 experiments.