The novel methyltransferase SETD4 regulates TLR agonist-induced expression of cytokines through methylation of lysine 4 at histone 3 in macrophages

The production of inflammatory cytokines is closely related to pathogen-associated molecular pattern (PAMP)-triggered activation of the Toll-like receptor (TLR), intracellular signal transduction pathways such as MAPK and NF-kappa B, and histone modifications. Histone methylation, a type of histone modifications, is mainly accomplished by a class of SET family proteins containing highly conserved SET domains. In the present study, we found that SET domain-containing protein 4 (SETD4) regulated inflammatory cytokines in response to TLR agonists. LPS stimulation led to the enhanced SETD4 expression, while the increased IL-6 and TNF-alpha release from LPS-stimulated RAW264.7 cells was attenuated by depletion of SETD4 using RNA interference. The results were further confirmed in BMDMs and pM phi isolated from SETD4-deficient mice where SETD4(-/-) macrophages treated with LPS, BLP or Poly(I:C) showed down-regulated IL-6 and TNF-alpha mRNA and protein levels when compared with SETD4(+/+) macrophages. Moreover, the mRNA levels of all NF-kappa B-dependent genes including IL-1 beta, IL-10, NFKBA, DUSP1, CCL2, CCLS, and CXCL10 in SETD4(-/-) macrophages were substantially reduced. To further clarify the regulatory mechanism(s) by which SETD4 modulates inflammatory cytokines, we examined the effect of SETD4 on the activation of MAPK and NF-kappa B signalling pathways, and found that knockout of SETD4 had no effect on phosphorylation of p38, ERK, JNK, p65, and I kappa B alpha. Notably, SETD4 translocated quickly from the cytosol to the nucleus upon LPS stimulation, suggesting that SETD4 may exert its regulatory function downstream of the MAPK and NF-kappa B pathways. To characterize this, we performed an in vitro HMTase assay to measure histone methyltransferase (HMTase) activity of SETD4. H3K4mel and H3K4me2 levels were enhanced dramatically with the supplementation of SETD4, whereas both H3K4mel and H3K4me2 were strongly attenuated in SETD4(-/-) BMDMs. Moreover, the LPS-stimulated recruitment of H3K4mel and H3K4me2 at both TNF-alpha and IL-6 promoters was severely impaired in SETD4(-/-) BMDMs. Collectively, these results demonstrate that SETD4 positively regulates IL-6 and TNF-alpha expression in TLR agonist-stimulated macrophages by directly activating H3K4 methylation.