The Kat in the HAT: The Histone Acetyl Transferase Kat6b (MYST4) Is Downregulated in Murine Macrophages in Response to LPS.

Epigenetic modulators, including histone methylases, demethylases, and deacetylases, have been implicated previously in the regulation of classical and alternative macrophage activation pathways. In this study, we show that the histone acetyl transferase (HAT) Kat6B (MYST4) is strongly suppressed (>80%) in macrophages by lipopolysaccharide (LPS) (M1 activation), while Kat6A, its partner in the MOZ/MORF complex, is reciprocally upregulated. This pattern of expression is not altered by LPS together with the adenosine receptor agonist NECA (M2d activation). This is despite the observation that miR-487b, a putative regulator of Kat6B expression, is mildly stimulated by LPS, but strongly suppressed by LPS/NECA. Other members of the MYST family of HATs (Kat5, Kat7, and Kat8) are unaffected by LPS treatment. Using the pLightswitch 3'UTR reporter plasmid, the miR-487b binding site in the Kat6b 3'UTR was found to play a role in the LPS-mediated suppression of Kat6B expression, but other as-yet unidentified factors are also involved. As Kat6B is a HAT that has the potential to modulate gene expression by its effects on chromatin accessibility, we are continuing our studies into the potential roles of this epigenetic modulator in macrophage activation pathways.

3'UTR AU-Rich Elements (AREs) and the RNA-Binding Protein Tristetraprolin (TTP) Are Not Required for the LPS-Mediated Destabilization of Phospholipase-Cß-2 mRNA in Murine Macrophages.

We have shown previously that bacterial lipopolysaccharide (LPS)-mediated suppression of phospholipase-Cß-2 (PLCß-2) expression is involved in M1 (inflammatory) to M2-like (wound healing) phenotypic switching of macrophages triggered by adenosine. This suppression is mediated post-transcriptionally by destabilization of PLCß-2 mRNA (messenger ribonucleic acid). To investigate the mechanism of this LPS-mediated destabilization, we examined the roles of RNA-binding agents including microRNAs and RNA-binding proteins that are involved in regulating stability of mRNAs encoding growth factors, inflammatory mediators, and proto-oncogenes. Adenylate and uridylate (AU)-rich elements (AREs) in 3'UTRs are specific recognition sites for RNA-binding proteins including tristetraprolin (TTP), HuR, and AUF1 and for microRNAs that are involved in regulating mRNA stability. In this study, we investigated the role of TTP and AREs in regulating PLCß-2 mRNA stability. The 3'UTR of the PLCß-2 gene was inserted into the pLightswitch luciferase reporter plasmid and transfected into RAW264.7 cells. LPS suppressed luciferase expression from this reporter. Luciferase expression from mutant 3'UTR constructs lacking AREs was similarly downregulated, suggesting that these regions are not required for LPS-mediated suppression of PLCß-2. TTP was rapidly upregulated in both primary murine macrophages and RAW264.7 cells in response to LPS. Suppression of PLCß-2 by LPS was examined using macrophages from mice lacking TTP (TTP-/-). LPS suppressed PLCß-2 expression to the same extent in wild type (WT) and TTP-/- macrophages. Also, the rate of decay of PLCß-2 mRNA in LPS-treated macrophages following transcriptional blockade was similar in WT and TTP-/- macrophages, clearly indicating that TTP is not involved in LPS-mediated destabilization of PLCß-2 mRNA in macrophages.