Figure 7.

Immunoprecipitation of H3K27-acetylated (H3K27ac) chromatin in Pit-1/0 cells expressing Nr4a2 and/or Pou1f1. Pit-1/0 cells, which express neither Nr4a2 nor significant levels of Pou1f1, were transfected with Nr4a2-IRES-GFP, Pou1f1-IRES-GFP, or a 1:1 mixture of both expression vectors, and control cells were transfected with an empty IRES-GFP vector. Cells transfected with the 1:1 mixture of the Pou1f1 and Nr4a2 expression vectors express lower levels of each protein due to the concentration of each expression vector being halved during transfection to keep the total amount of DNA constant in each transfection. GFP⁺ cells were isolated by FACS and chromatin was isolated. ChIP was performed using an antibody that recognizes H3K27ac. Following ChIP, qRT-PCR was performed to assay the levels of H327 acetylation at the Gh promoter, the Prl promoter, and the MyoD promoter as a negative control. IgG controls (data not shown) were also included in this assay for all samples. In all cases, ChIP performed with IgG yielded <1% of input. *P < 0.05.