PRMT4 is a novel coactivator of c-Myb-dependent transcription in haematopoietic cell lines.

Protein arginine methyltransferase 4 (PRMT4)-dependent methylation of arginine residues in histones and other chromatin-associated proteins plays an important role in the regulation of gene expression. However, the exact mechanism of how PRMT4 activates transcription remains elusive. Here, we identify the chromatin remodeller Mi2α as a novel interaction partner of PRMT4. PRMT4 binds Mi2α and its close relative Mi2ß, but not the other components of the repressive Mi2-containing NuRD complex. In the search for the biological role of this interaction, we find that PRMT4 and Mi2α/ß interact with the transcription factor c-Myb and cooperatively coactivate c-Myb target gene expression in haematopoietic cell lines. This coactivation requires the methyltransferase and ATPase activity of PRMT4 and Mi2, respectively. Chromatin immunoprecipitation analysis shows that c-Myb target genes are direct transcriptional targets of PRMT4 and Mi2. Knockdown of PRMT4 or Mi2α/ß in haematopoietic cells of the erythroid lineage results in diminished transcriptional induction of c-Myb target genes, attenuated cell growth and survival, and deregulated differentiation resembling the effects caused by c-Myb depletion. These findings reveal an important and so far unknown connection between PRMT4 and the chromatin remodeller Mi2 in c-Myb signalling.

Homo-oligomerization of the activating natural killer cell receptor NKp30 ectodomain increases its binding affinity for cellular ligands.

The natural cytotoxicity receptors, comprised of three type I membrane proteins NKp30, NKp44, and NKp46, are a unique set of activating proteins expressed mainly on the surface of natural killer (NK) cells. Among these, NKp30 is a major receptor targeting virus-infected cells, malignantly transformed cells, and immature dendritic cells. To date, only few cellular ligands of NKp30 have been discovered, and the molecular details of ligand recognition by NKp30 are poorly understood. Within the current study, we found that the ectodomain of NKp30 forms functional homo-oligomers that mediate high affinity binding to its corresponding cellular ligand B7-H6. Notably, this homo-oligomerization is strongly promoted by the stalk domain of NKp30. Based on these data, we suggest that homo-oligomerization of NKp30 in the plasma membrane of NK cells, which might be favored by IL-2-dependent up-regulation of NKp30 expression, provides a way to improve recognition and lysis of target cells by NK cells.

MYB induces the expression of the oncogenic corepressor SKI in acute myeloid leukemia.

Acute myeloid leukemia (AML) arises through clonal expansion of transformed myeloid progenitor cells. The SKI proto-oncogene is highly upregulated in different solid tumors and leukemic cells, but little is known about its transcriptional regulation during leukemogenesis. MYB is an important hematopoietic transcription factor involved in proliferation as well as differentiation and upregulated in most human acute leukemias. Here, we find that MYB protein binds within the regulatory region of the SKI gene in AML cells. Reporter gene assays using MYB binding sites present in the SKI gene locus show MYB-dependent transcriptional activation. SiRNA-mediated depletion of MYB in leukemic cell lines reveals that MYB is crucial for SKI gene expression. Consistently, we observed a positive correlation of MYB and SKI expression in leukemic cell lines and in samples of AML patients. Moreover, MYB and SKI both were downregulated by treatment with histone deacetylase inhibitors. Strikingly, differentiation of AML cells induced by depletion of MYB is attenuated by overexpression of SKI. Our findings identify SKI as a novel MYB target gene, relevant for the MYB-induced differentiation block in leukemic cells.

Identification and in silico structural analysis of Gallus gallus protein arginine methyltransferase 4 (PRMT4).

Protein arginine methyltransferase 4 (PRMT4) is an essential epigenetic regulator of fundamental and conserved processes during vertebrate development, such as pluripotency and differentiation. Surprisingly, PRMT4 homologs have been identified in nearly all vertebrate classes except the avian genome. This raises the possibility that in birds PRMT4 functions are taken over by other PRMT family members. Here, we reveal the existence of a bona fide PRMT4 homolog in the chicken, Gallus gallus. Using a biochemical approach, we initially purified a putative chicken PRMT4 protein and thus provided the first evidence for the presence of an endogenous PRMT4-specific enzymatic activity toward histone H3 arginine 17 (H3R17) in avian cells. We then isolated a G. gallus PRMT4 (ggPRMT4) transcript encompassing the complete open reading frame. Recombinant ggPRMT4 possesses intrinsic methyltransferase activity toward H3R17. CRISPR/Cas9-mediated deletion of ggPRMT4 demonstrated that the transcript identified here encodes avian PRMT4. Combining protein-protein docking and homology modeling based on published crystal structures of murine PRMT4, we found a strong structural similarity of the catalytic core domain between chicken and mammalian PRMT4. Strikingly, in silico structural comparison of the N-terminal Pleckstrin homology (PH) domain of avian and murine PRMT4 identified strictly conserved amino acids that are involved in an interaction interface toward the catalytic core domain, facilitating for the first time a prediction of the relative spatial arrangement of these two domains. Our novel findings are particularly exciting in light of the essential function of the PH domain in substrate recognition and methylation by PRMT4.