Methylation of histone H3 at lysine 36 (H3K36me), a widely-distributed chromatin mark, largely results from association of the lysine methyltransferase (KMT) SET-2 with RNA polymerase II (RNAPII), but most eukaryotes also have additional H3K36me KMTs that act independently of RNAPII. These include the orthologs of ASH1, which are conserved in animals, plants, and fungi but whose function and control are poorly understood. We found that Neurospora crassa has just two H3K36 KMTs, ASH1 and SET-2, and were able to explore the function and distribution of each enzyme independently. While H3K36me deposited by SET-2 marks active genes, inactive genes are modified by ASH1 and its activity is critical for their repression. ASH1-marked chromatin can be further modified by methylation of H3K27, and ASH1 catalytic activity modulates the accumulation of H3K27me2/3 both positively and negatively. These findings provide new insight into ASH1 function, H3K27me2/3 establishment, and repression in facultative heterochromatin.

High-throughput chromosome conformation capture (Hi-C) analyses revealed that the 3D structure of the Neurospora crassa genome is dominated by intra- and interchromosomal links between regions of heterochromatin, especially constitutive heterochromatin. Elimination of trimethylation of lysine 9 on histone H3 (H3K9me3) or its binding partner Heterochromatin Protein 1 (HP1)—both prominent features of constitutive heterochromatin—have little effect on the Hi-C pattern. It remained possible that di- or trimethylation of lysine 27 on histone H3 (H3K27me2/3), which becomes localized in regions of constitutive heterochromatin when H3K9me3 or HP1 are lost, plays a critical role in the 3D structure of the genome. We found that H3K27me2/3, catalyzed by the Polycomb Repressive Complex 2 (PRC2) member SET-7 (SET domain protein-7), does indeed play a prominent role in the Hi-C pattern of WT, but that its presence in regions normally occupied by H3K9me3 is not responsible for maintenance of the genome architecture when H3K9me3 is lost. The Hi-C pattern of a mutant defective in the PRC2 member N. crassa p55 (NPF), which is predominantly required for subtelomeric H3K27me2/3, was equivalent to that of the set-7 deletion strain, suggesting that subtelomeric facultative heterochromatin is paramount for normal chromosome conformation. Both PRC2 mutants showed decreased heterochromatin–heterochromatin contacts and increased euchromatin–heterochromatin contacts. Cytological observations suggested elimination of H3K27me2/3 leads to partial displacement of telomere clusters from the nuclear periphery. Transcriptional profiling of Δdim-5, Δset-7, Δset-7; Δdim-5, and Δnpf strains detailed anticipated changes in gene expression but did not support the idea that global changes in genome architecture, per se, led to altered transcription.

IntroductionTREM-1 belongs to the immunoglobulin (Ig)-like superfamily of receptors. In humans, TREM-1 is expressed on neutrophils and CD14 high monocytes. 1 TREM-1 expression is further up-regulated by TLR ligands. Binding of TREM-1 to endogenous ligand(s) expressed on granulocytes and platelets and present in sera of septic patients, 2-4 as well as to exogenous ligands on Marburg and Ebola viruses, 5 has been described. The exact nature of TREM-1 ligand(s), however, remains elusive. Engagement of TREM-1 by agonistic monoclonal antibodies (mAbs) results in respiratory burst, degranulation, phagocytosis, secretion of pro-inflammatory cytokines such as TNF-␣, of the chemokines, IL-8 and monocyte chemotactic protein-1 (MCP-1), and in the up-regulation of cell surface expressed differentiation/activation markers. 1 In animal models of lipopolysacharide (LPS)-induced septic shock and microbial sepsis caused by live Escherichia coli, application of a soluble TREM-1-Ig fusion protein greatly increased survival of experimental animals indicating the importance of TREM-1 in the amplification of inflammation. 6 TREM-1 possesses a short intracellular part that lacks intrinsic signaling motifs. Instead, it is coupled to the immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor protein, DAP12. 1 TREM-1 engagement leads to Ca 2ϩ mobilization and phosphorylation of several proteins including DAP12, extracellular-signal regulated kinase (Erk1/2), phospholipase C␥ (PLC␥) 1 and the adaptor protein NTAL that further interacts with Grb2. 7 Recently, it was reported that the adaptor protein CARD9 is essential for TREM-1-induced secretion of TNF-␣, IL-2 and IL-12p40 by mouse bone marrow-derived dendritic cells (BMDCs). 8 Bruton tyrosine kinase (Btk), a member of the Tec family of protein tyrosine kinases (PTKs), is involved in signaling via a variety of receptors including the B-cell receptor (BCR), cytokine receptors and integrins. 9,10 Loss of Btk function causes X-linked agammaglobulinemia (XLA), a rare primary immunodeficiency disease. 11,12 Mutations causing XLA have been described in all Btk domains as well as in noncoding sequences of the gene. 13 XLA is manifested by severe defects in early B-cell development, resulting in an almost complete absence of peripheral B cells and Igs of all classes. Affected individuals suffer from recurrent bacterial and enteroviral infections. 9 In mice, the R28C point mutation in the pleckstrin homology (PH) domain of Btk leads to X-linked immunodeficiency (Xid). 14 In innate immune cells, the role of Btk is less clear. Btk has been implicated in several pathways in myeloid cells. 10 Btk associates with certain TLRs and their downstream signaling molecules. 15 Contradictory results were obtained from studies of TLR stimulation of monocytes of XLA patients. In one report, relative to healthy controls, monocytes of XLA patients secreted reduced amounts of TNF-␣ upon LPS stimulation. 16 On the other hand, Perez et al 17 described that monocytes of XLA patients showed similar TN...