Critical Role for Transcription Factor C/EBP-β in Regulating the Expression of Death-Associated Protein Kinase 1

The Med1 Subunit of Transcriptional Mediator Plays a Central Role in Regulating CCAAT/Enhancer-binding Protein-β-driven Transcription in Response to Interferon-γ

“…We have shown earlier that IFNinduced expression of irf9 mRNA is regulated by C/EBP-␤ (14). We have recently found that the death-associated protein kinase 1 gene (dapk1) is also regulated by IFN-␥ through C/EBP-␤ (29). Therefore, we examined if IFN-␥-induced expression of these two genes was influenced by the loss of Med1 by stimulating isogenic med1 ϩ/ϩ and med1 Ϫ/Ϫ cells with IFN-␥ and monitored the expression levels of irf9 and dapk1 mRNA by real time PCR.…”

“…Mut T3 A contained an alanine in place of threonine, and Mut T3 D contained an aspartate in place of threonine. We have recently shown that this threonine residue is phosphorylated by ERK1/2 in response to IFN-␥ treatment (29). Both mutants were transfected into cebpb Ϫ/Ϫ cells, and their IFN-induced binding to Med1 was compared with that of wild type C/EBP-␤ (Fig.…”

“…We used primers that could detect C/EBP-␤ binding to the critical IFN-induced regulatory element, CRE, of the dapk1 promoter (29). Since dapk1 was induced in a delayed manner by IFN-␥, med1 ϩ/ϩ and med1 Ϫ/Ϫ MEFs were stimulated with IFN-␥ for 8 h, and soluble chromatin was subject to the ChIP assay.…”

“…The DNA recovered from ChIP products was used for quantitative PCR with specific primer pairs (supplemental Table 3). The dapk1 primer pair detects the IFN-induced recruitment of C/EBP-␤ to a recently identified CRE/ATF site in dapk1 promoter (29). DNA extracted from soluble chromatin was used as input control.…”

“…irf9-luc was described elsewhere (28). dapk1-luc contains a 1.2-kb fragment from the mouse dapk1 promoter upstream of the luciferase gene (29). Luciferase activity was normalized to that of ␤-galactosidase.…”