Figure 6.

Mechanism of Pax8-activated gene expression. Human HEK293 cells carrying an integrated Pax reporter gene (A) driven by Pax Responsive Sequences (PRS) were used to characterize Pax8-mediated transcription activation. (B) Cells were treated with negative control short hairpin RNA(shN) or PTIP KO short hairpin RNA (shPTIP) and transfected with Pax8 or an shPTIP-resistant rescue plasmid (lane 4). Note that PTIP KOs fail to activate the GFP reporter gene in the presence of Pax8. (C–H) Chromatin from cells transfected as in (B) was immunoprecipitated with antibodies against the indicated proteins. Quantitative PCR was done with primers against the PRS sequence to determine protein occupancy. Note that Pax8 binds regardless of PTIP KOs. However, Pax8 recruits PTIP and components of the Mll3/4 complex (Ash2L and Rbbp5) to imprint high levels of H3K4me2 and H3K4me3. All ChIP assays were performed in triplicate, with error bars representing one SD from the mean.