Mark P. Kamps

Specificity in Toll-like receptor signalling through distinct effector functions of TRAF3 and TRAF6

Toll-like receptors (TLRs) are activated by pathogen-associated molecular patterns to induce innate immune responses and production of pro-inflammatory cytokines, interferons and anti-inflammatory cytokines. TLRs activate downstream effectors through adaptors that contain Toll/interleukin-1 receptor (TIR) domains, but the mechanisms accounting for diversification of TLR effector functions are unclear. To dissect biochemically TLR signalling, we established a system for isolating signalling complexes assembled by dimerized adaptors. Using MyD88 as a prototypical adaptor, we identified TNF receptor-associated factor 3 (TRAF3) as a new component of TIR signalling complexes that is recruited along with TRAF6. Using myeloid cells from TRAF3- and TRAF6-deficient mice, we show that TRAF3 is essential for the induction of type I interferons (IFN) and the anti-inflammatory cytokine interleukin-10 (IL-10), but is dispensable for expression of pro-inflammatory cytokines. In fact, TRAF3-deficient cells overproduce pro-inflammatory cytokines owing to defective IL-10 production. Despite their structural similarity, the functions of TRAF3 and TRAF6 are largely distinct. TRAF3 is also recruited to the adaptor TRIF (Toll/IL-1 receptor domain-containing adaptor-inducing IFN-β) and is required for marshalling the protein kinase TBK1 (also called NAK) into TIR signalling complexes, thereby explaining its unique role in activation of the IFN response.

fos-associated cellular p39 is related to nuclear transcription factor AP-1

Proto-oncogene fos encodes a nuclear protein that appears to be involved in the transcriptional regulation of some genes. fos protein (p55fos) is associated with other nuclear proteins in complexes that bind to regulatory regions containing TPA-responsive promoter elements (TREs). The enhancer-binding activator protein 1 (AP-1), which is the specific binding factor of a TRE, is the product of the nuclear proto-oncogene jun. We show that one of the fos-associated proteins, p39, is immunologically and structurally related to nuclear factor AP-1. The sequence TGACTCA, which is the consensus minimal binding site of AP-1, is also recognized by gel-purified p39. In HeLa cell extracts p55fos and p39jun are associated in a nucleoprotein complex that interacts with a TRE. Cooperation between nuclear oncoproteins fos and jun is required for full activation of transcription from a TPA-responsive promoter element in transfected mammalian cells.

Acid and base hydrolysis of phosphoproteins bound to immobilon facilitates analysis of phosphoamino acids in gel-fractionated proteins

Immobilon, a membrane of polyvinylidene difluoride to which gel-fractionated proteins can be transferred electrophoretically, was found to be an excellent matrix for the analysis of the phosphoamino acid content of phosphoproteins. Hydrolysis of 32P-labeled proteins bound to Immobilon with 5.7 N HCl resulted in the release of 90% of the 32P in the form of Pi, phosphoamino acids, and phosphopeptides. Two-dimensional electrophoretic analysis of the released phosphoamino acids yielded undistorted patterns. Because direct hydrolysis of proteins transferred to Immobilon eliminated the need for both preparative extraction of proteins from a gel and recovery by precipitation, analysis was rapid and yields of phosphoamino acids were extremely consistent. The yield of phosphoamino acids from proteins bound to Immobilon, unlike that from proteins eluted from gels, was independent of the size of the protein. The detection of 32P-labeled, phosphotyrosine-containing proteins in sodium dodecyl sulfate-polyacrylamide gels has been shown to be substantially improved by incubation of the gel in 1.0 N KOH for 2 h at 55 degrees C. Base hydrolysis of proteins bound to Immobilon proved to be faster and more sensitive than hydrolysis of proteins in gels. Less than 10% of bound protein was lost from Immobilon during the 2-h incubation at 55 degrees C in 1.0 N KOH. The autoradiographic image after alkaline hydrolysis of proteins on Immobilon was sharper than that obtained after hydrolysis of proteins in the gel. In addition, unlike base-treated gels, the dimensions of the Immobilon filter were unaffected by treatment with base.(ABSTRACT TRUNCATED AT 250 WORDS)

NUP98-NSD1 links H3K36 methylation to Hox-A gene activation and leukaemogenesis.

Nuclear receptor-binding SET domain protein 1 (NSD1) prototype is a family of mammalian histone methyltransferases (NSD1, NSD2/MMSET/WHSC1, NSD3/WHSC1L1) that are essential in development and are mutated in human acute myeloid leukemia (AML), overgrowth syndromes, multiple myeloma and lung cancers. In AML, the recurring t(5;11)(q35;p15.5) translocation fuses NSD1 to nucleoporin-98 (NUP98). Here, we present the first characterization of the transforming properties and molecular mechanisms of NUP98–NSD1. We demonstrate that NUP98–NSD1 induces AML in vivo, sustains self-renewal of myeloid stem cells in vitro, and enforces expression of the HoxA7, HoxA9, HoxA10 and Meis1 proto-oncogenes. Mechanistically, NUP98–NSD1 binds genomic elements adjacent to HoxA7 and HoxA9, maintains histone H3 Lys 36 (H3K36) methylation and histone acetylation, and prevents EZH2-mediated transcriptional repression of the Hox-A locus during differentiation. Deletion of the NUP98 FG-repeat domain, or mutations in NSD1 that inactivate the H3K36 methyltransferase activity or that prevent binding of NUP98–NSD1 to the Hox-A locus precluded both Hox-A gene activation and myeloid progenitor immortalization. We propose that NUP98–NSD1 prevents EZH2-mediated repression of Hox-A locus genes by colocalizing H3K36 methylation and histone acetylation at regulatory DNA elements. This report is the first to link deregulated H3K36 methylation to tumorigenesis and to link NSD1 to transcriptional regulation of the Hox-A locus.

Monoclonal antibodies to phosphotyrosine

Phosphotyrosine coupled to KLH, BSA, and OVA was used for the production and screening of antibodies to phosphotyrosine. 800 hybridomas secreting antibodies that bound to phosphotyrosine were detected by ELISA. The most reactive 100 of these 800 were tested subsequently for their ability to bind phosphotyrosine-containing proteins on Western blots. Eight stable hybridoma cell lines were selected for further study, cloned by limiting dilution, and grown as ascites. These antibodies were purified by three different methods, and it was found that affinity chromatography on phosphotyrosine-affigel provided the most rapid and effective method. Many phosphotyrosine-containing proteins were detected by using these antibodies in Western blotting and immunoaffinity purification procedures. Binding of anti-phosphotyrosine antibody could be competed by phosphotyrosine or phenylphosphate but not by phosphoserine, phosphothreonine, or free phosphate. These antibodies should be useful for the identification and purification of proteins phosphorylated on tyrosine residues in transformed and growth factor-treated cells.

The pentapeptide motif of Hox proteins is required for cooperative DNA binding with Pbx1, physically contacts Pbx1, and enhances DNA binding by Pbx1

The vertebrate Hox genes, which represent a subset of all homeobox genes, encode proteins that regulate anterior-posterior positional identity during embryogenesis and are cognates of the Drosophila homeodomain proteins encoded by genes composing the homeotic complex (HOM-C). Recently, we demonstrated that multiple Hox proteins bind DNA cooperatively with both Pbx1 and its oncogenic derivative, E2A-Pbx1. Here, we show that the highly conserved pentapeptide motif F/Y-P-W-M-R/K, which occurs in numerous Hox proteins and is positioned 8 to 50 amino acids N terminal to the homeodomain, is essential for cooperative DNA binding with Pbx1 and E2A-Pbx1. Point mutational analysis demonstrated that the tryptophan and methionine residues within the core of this motif were critical for cooperative DNA binding. A peptide containing the wild-type pentapeptide sequence, but not one in which phenylalanine was substituted for tryptophan, blocked the ability of Hox proteins to bind cooperatively with Pbx1 or E2A-Pbx1, suggesting that the pentapeptide itself provides at least one surface through which Hox proteins bind Pbx1. Furthermore, the same peptide, but not the mutant peptide, stimulated DNA binding by Pbx1, suggesting that interaction of Hox proteins with Pbx1 through the pentapeptide motif raises the DNA-binding ability of Pbx1.

The pancreatic islet factor STF-1 binds cooperatively with Pbx to a regulatory element in the somatostatin promoter: importance of the FPWMK motif and of the homeodomain.

A number of homeodomain proteins have been shown to regulate cellular development by stimulating the transcription of specific target genes. In contrast to their distinct activities in vivo, however, most homeodomain proteins bind indiscriminately to potential target sites in vitro, suggesting the involvement of cofactors which specify target site selection. One such cofactor, termed extradenticle, has been shown to influence segmental morphogenesis in Drosophila melanogaster by binding cooperatively with certain homeodomain proteins to target regulatory elements. Here we demonstrate that STF-1, an orphan homeodomain protein required for pancreatic development in mammals, binds cooperatively to DNA with Pbx, the mammalian homolog of extradenticle. Cooperative binding with Pbx requires a pentapeptide motif (FPWMK) which is well conserved among a large subset of homeodomain proteins. The FPMWK motif is not sufficient to confer Pbx cooperativity on other homeodomain proteins, however; the N-terminal arm of the STF-1 homeodomain is also essential. As cooperative binding with Pbx occurs on only a subset of potential STF-1 target sites, our results suggest that Pbx may specify target gene selection in the developing pancreas by forming heterodimeric complexes with STF-1.

Rous sarcoma virus transforming protein lacking myristic acid phosphorylates known polypeptide substrates without inducing transformation.

Mutagenesis of glycine 2 of p60src, the transforming protein of Rous sarcoma virus (RSV), yields a protein that is neither myristylated nor bound to cellular membranes. Although these mutant viruses retain full tyrosine protein kinase activity, they are transformation-defective. We examined in detail tyrosine phosphorylation of cellular polypeptides and the phenotype induced by infection with two such viruses. Infection failed to cause growth in agar, cytoskeletal reorganization, or changes in fibronectin synthesis and protease secretion. Strikingly, tyrosine phosphorylation of the known substrates of p60src was extensive, and differed from that found in wild-type transformed cells only quantitatively. There was no apparent correlation between the extent to which any of eight known protein substrates of p60src were phosphorylated and the phenotype of infected cells. We suggest that the phosphorylation of as yet unidentified proteins, which are probably found in cellular membranes, is essential for transformation by RSV.

Quantitative production of macrophages or neutrophils ex vivo using conditional Hoxb8

Differentiation mechanisms and inflammatory functions of neutrophils and macrophages are usually studied by genetic and biochemical approaches that require costly breeding and time-consuming purification to obtain phagocytes for functional analysis. Because Hox oncoproteins enforce self-renewal of factor-dependent myeloid progenitors, we queried whether estrogen-regulated Hoxb8 (ER-Hoxb8) could immortalize macrophage or neutrophil progenitors that would execute normal differentiation and normal innate immune function upon ER-Hoxb8 inactivation. Here we describe methods to derive unlimited quantities of mouse macrophages or neutrophils by immortalizing their respective progenitors with ER-Hoxb8 using different cytokines to target expansion of different committed progenitors. ER-Hoxb8 neutrophils and macrophages are functionally superior to those produced by many other ex vivo differentiation models, have strong inflammatory responses and can be derived easily from embryonic day 13 (e13) fetal liver of mice exhibiting embryonic-lethal phenotypes. Using knockout or small interfering RNA (siRNA) technologies, this ER-Hoxb8 phagocyte maturation system represents a rapid analytical tool for studying macrophage and neutrophil biology.

Transcriptional competence and the active marking of tissue-specific enhancers by defined transcription factors in embryonic and induced pluripotent stem cells

We reported previously that well-characterized enhancers but not promoters for typical tissue-specific genes, including the classic Alb1 gene, contain unmethylated CpG dinucleotides and evidence of pioneer factor interactions in embryonic stem (ES) cells. These properties, which are distinct from the bivalent histone modification domains that characterize the promoters of genes involved in developmental decisions, raise the possibility that genes expressed only in differentiated cells may need to be marked at the pluripotent stage. Here, we demonstrate that the forkhead family member FoxD3 is essential for the unmethylated mark observed at the Alb1 enhancer in ES cells, with FoxA1 replacing FoxD3 following differentiation into endoderm. Up-regulation of FoxD3 and loss of CpG methylation at the Alb1 enhancer accompanied the reprogramming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem (iPS) cells. Studies of two genes expressed in specific hematopoietic lineages revealed that the establishment of enhancer marks in ES cells and iPS cells can be regulated both positively and negatively. Furthermore, the absence of a pre-established mark consistently resulted in resistance to transcriptional activation in the repressive chromatin environment that characterizes differentiated cells. These results support the hypothesis that pluripotency and successful reprogramming may be critically dependent on the marking of enhancers for many or all tissue-specific genes.