Abstract
The DNA-binding protein PU.1 is a myeloid lineage-determining and pioneering transcription factor due to its ability to bind "closed" genomic sites and maintain "open" chromatin state for myeloid lineage-specific genes. The precise mechanism of PU.1 in cell type-specific programming is yet to be elucidated. The melanoma cell line B16BL6, although it is nonmyeloid lineage, expressed Toll-like receptors and activated the transcription factor NF-κB upon stimulation by the bacterial cell wall component lipopolysaccharide. However, it did not produce cytokines, such as IL-1β mRNA. Ectopic PU.1 expression induced remodeling of a novel distal enhancer (located ∼10 kbp upstream of the IL-1β transcription start site), marked by nucleosome depletion, enhancer-promoter looping, and histone H3 lysine 27 acetylation (H3K27ac). PU.1 induced enhancer-promoter looping and H3K27ac through two distinct PU.1 regions. These PU.1-dependent events were independently required for subsequent signal-dependent and co-dependent events: NF-κB recruitment and further H3K27ac, both of which were required for enhancer RNA (eRNA) transcription. In murine macrophage RAW264.7 cells, these PU.1-dependent events were constitutively established and readily expressed eRNA and subsequently IL-1β mRNA by lipopolysaccharide stimulation. In summary, this study showed a sequence of epigenetic events in programming IL-1β transcription by the distal enhancer priming and eRNA production mediated by PU.1 and the signal-dependent transcription factor NF-κB.
Highlights
The DNA-binding protein PU.1 is a myeloid lineage– determining and pioneering transcription factor due to its ability to bind “closed” genomic sites and maintain “open” chromatin state for myeloid lineage–specific genes
In B16 cells transfected with CCAAT/enhancer-binding protein (C/EBP)␣ and PU.1, robust transcription of IL-1 mRNA was detected in response to TLR2, -4, and -9 ligands (Fig. 1A) without affecting the levels of ERK, p38, and NF-B activation (Fig. 1B)
Because C/EBP␣, together with PU.1, was shown to be involved in establishing monocyte-specific enhancers [43], we further examined whether both C/EBP␣ and PU.1 were required for programming IL-1 mRNA transcription
Summary
The transcription factor PU. mediates enhancer-promoter looping that is required for IL-1 eRNA and mRNA transcription in mouse melanoma and macrophage cell lines. Enhancers and promoters of these genes are in poised status, harboring active histone marks and transcriptional machinery that is waiting to be activated immediately by extracellular signals [31, 32] Among those cytokines, interleukin (IL)-1 (derived from the proteolytic processing of pro-IL-1 gene product) is a potent inflammatory cytokine that is rapidly induced by activated Toll-like receptors (TLRs), such as TLR4, activated by the Gram-negative bacterial component lipopolysaccharide (LPS). PU. is crucial for establishing poised promoter architecture and, looping between the promoter and a remote enhancer located about Ϫ3,000 bp from the transcription start site (TSS) of IL-1 gene [36, 37] Both promoter and enhancer elements are expected to be similar in mice, the distal regulatory elements and mechanism of PU. in regulating IL-1 transcription in mice are less well-understood. The PU.1-dependent enhancer-promoter looping and H3K27ac were independently required for subsequent signal-dependent and co-dependent events: NF-B recruitment and further H3K27ac, both of which were required for the eRNA and subsequent IL-1 mRNA transcription
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