Figure 6

The IRF1 TSS is disrupted in MEN1-depleted cells. (a) 5' RACE-PCR of RNA collected from shRNA-MEN1 and shRNA-NS cells, with or without treatment with IFN-γ. PCR products were generated using the 5' RACE primer along with a reverse primer designed to the third exon of IRF1. Arrows indicate the base position on the reverse strand of chromosome 5 to which cDNA ends mapped and the percentages reflect the frequency that a site was observed. Asterisks mark the position of major CTSSs determined in [39] with the large asterisk indicating the RefSeq TSS. Red indicates other minor CTSSs. 2fTGH cells, +/- IFN-γ data were the same as the shRNA-NS data. (b) 3' RACE-PCR products were generated using a polydT primer along with a forward primer designed to the third exon or the tenth exon of IRF1. Inverted triangles indicate observed cleavage sites on the IRF1 mRNA. Poly(A) signal sequences are in red. (c) 5' RACE-PCR of 2-PCPA treated and untreated cells, with or without IFN-γ induction. Primers are depicted in Figure 1c. 2-PCPA: trans-2-phenylcyclopropylamine; CTSS: cap analysis of gene expression tag starting site; IFN: interferon; MEN1: multiple endocrine neoplasia type 1; NS: nonsilencing; PCR: polymerase chain reaction; RACE: rapid amplification of cDNA ends; TSS: transcription start site.