IRF8 and PU.1 binding and the H3K4me1 enhancer signature at the Klf4 gene locus. (A) UCSC genome browser image of tag density plots for IRF8, PU.1, and H3K4me1 ChIP-seq and input DNA at the Klf4 gene locus in empty MSCV-transduced Tot2 cells or IRF8-transduced Tot2 cells. H3K4me1 ChIP-seq peaks identified by SICER and PU.1 and IRF8 ChIP-seq peaks by HOMER are indicated as bars above the density plots. The genomic sequences of each IRF8 ChIP-seq peak are shown (peaks A-F; see also supplemental Figure 2). (B) Direct activation of the Klf4 gene by IRF8. IRF8-estrogen receptor (ER) –transduced Tot2 cells were treated with β-estradiol (Est) (1 µM) and cycloheximide (CHX) (1 µg/mL). CHX was added 10 minutes before the addition of Est. Transcript levels of Klf4 and Myc were quantified in biological triplicates by qRT-PCR. Data were normalized by Actb levels and shown as values relative to those in ER alone–transduced Tot2 cells treated under the same conditions (mean ± standard error). (*) P < .05 (Student t test). NS, not significant.