Loss of PRDM11 promotes MYC-driven lymphomagenesis.

The PR-domain (PRDM) family of genes encodes transcriptional regulators, several of which are deregulated in cancer. By using a functional screening approach, we sought to identify novel tumor suppressors among the PRDMs. Here we demonstrate oncogenic collaboration between depletion of the previously uncharacterized PR-domain family member Prdm11 and overexpression of MYC. Overexpression of PRDM11 inhibits proliferation and induces apoptosis. Prdm11 knockout mice are viable, and loss of Prdm11 accelerates MYC-driven lymphomagenesis in the Eµ-Myc mouse model. Moreover, we show that patients with PRDM11-deficient diffuse large B-cell lymphomas (DLBCLs) have poorer overall survival and belong to the nongerminal center B-cell-like subtype. Mechanistically, genome-wide mapping of PRDM11 binding sites coupled with transcriptome sequencing in human DLBCL cells evidenced that PRDM11 associates with transcriptional start sites of target genes and regulates important oncogenes such as FOS and JUN. Hence, we characterize PRDM11 as a putative novel tumor suppressor that controls the expression of key oncogenes, and we add new mechanistic insight into B-cell lymphomagenesis.

Cell-free expression and functionality analysis of the tobacco lectin.

The Nicotiana tabacum lectin, called Nictaba, is a nucleocytoplasmic plant lectin expressed in tobacco leaves after exogenous application of specific jasmonates and upon insect herbivory. Since the lectin concentrations are rather low, huge amounts of plant material are needed to purify milligram quantities of the protein. In addition, the purified lectin fractions are always contaminated with low molecular weight compounds such as phenols. In an attempt to improve and facilitate the purification of the tobacco lectin in reasonable amounts, an in vitro-coupled transcription/translation system based on an Escherichia coli lysate was used to express the lectin gene. Recombinant expression levels could be enhanced by an adapted codon usage. Recombinant lectin was purified, biochemically characterized and found to be biologically active. The biological activity of the recombinant lectin towards insect epithelial midgut cells was clearly demonstrated in a functional bio-assay and the internal cellular localization was analyzed using immunocytochemical techniques.

The sorLA cytoplasmic domain interacts with GGA1 and -2 and defines minimum requirements for GGA binding.

We report that the Vps10p domain receptor sorLA binds the adaptor proteins GGA1 and -2, which take part in Golgi-endosome sorting. The GGAs bind with differential requirements via three critical residues in the C-terminal segment of the sorLA cytoplasmic tail. Unlike in sortilin and the mannose 6-phosphate receptors, the GGA-binding segment in sorLA contains neither an acidic cluster nor a dileucine. Our results support the concept of sorLA as a potential sorting receptor and suggest that key residues in sorLA and sortilin conform to a new type of motif (psi-psi-X-X-phi) defining minimum requirements for GGA binding to cytoplasmic receptor domains.

Tomosyn interacts with the SUMO E3 ligase PIASγ.

Protein modification by Small Ubiquitin-like MOdifier (SUMO) entities is involved in a number of neuronal functions, including synaptogenesis and synaptic plasticity. Tomosyn-1 (syntaxin-binding protein 5; STXPB5) binds to t-SNARE (Soluble NSF Attachment Protein Receptor) proteins to regulate neurotransmission and is one of the few neuronal SUMO substrate proteins identified. Here we used yeast two-hybrid screening to show that tomosyn-1 interacts with the SUMO E3 ligase PIASγ (Protein Inhibitor of Activated STAT; PIAS4 or ZMIZ6). This novel interaction involved the C-terminus of tomosyn-1 and the N-terminus of PIASγ. It was confirmed by two-way immunoprecipitation experiments using the full-length proteins expressed in HEK293T cells. Tomosyn-1 was preferentially modified by the SUMO-2/3 isoform. PIASγ-dependent modification of tomosyn-1 with SUMO-2/3 presents a novel mechanism to adapt secretory strength to the dynamic synaptic environment.

Transcriptional effects of transfection: the potential for misinterpretation of gene expression data generated from transiently transfected cells.

Transfection is used to introduce a gene of interest into a cell. To interpret the downstream results, understanding which effects are the true biological responses to the gene and which, if any, are off-target effects can be difficult. In order to discriminate true biological effects from off-target effects, we transfected a breast cancer cell line, MCF7, with a vector encoding either a reporter gene or the identical vector without the reporter gene insert. Both resulted in similar numbers of differentially expressed transcripts, suggesting that very few of the responses were directly due to the introduction of the reporter gene. We postulate that many differentially expressed transcripts are the result of the introduction of foreign DNA, as the biological processes associated with these genes are primarily associated with an immune response to a viral infection. Interestingly, different transfection reagents resulted in > 10-fold difference in the number of differentially expressed transcripts. This suggests the importance of testing multiple reagents and selecting the best transfection reagent along with the appropriate vector within the context of the experimental model system to ensure that the majority of the observed responses are biological effects of the gene of interest and not based on a particular transfection process used.

Two distinct genes drive expression of seven tomosyn isoforms in the mammalian brain, sharing a conserved structure with a unique variable domain.

Tomosyn was previously identified as a syntaxin-binding protein that inhibits soluble NSF (n-ethylmaleimide-sensitive fusion protein) attachment protein receptor (SNARE)-mediated secretion. We set out to investigate the distribution of tomosyn mRNA in the mammalian brain and found evidence for the presence of two paralogous genes designated tomosyn-1 and -2. In a collection of tomosyn-2 cDNA clones, we observed four splice variants (named xb-, b-, m- and s-tomosyn-2) derived from the skipping of exons 19 and 21. This feature is conserved with tomosyn-1 that encodes three splice variants. To compare the expression pattern of tomosyn-1 and -2, we performed in situ hybridization experiments with gene-specific probes. Both genes were expressed in the nervous system, clearly following distinct spatial and developmental expression patterns. Real-time quantitative PCR experiments indicated that tomosyn-1 expression was up-regulated less than threefold between developmental stages E10 and P12, whereas tomosyn-2 expression increased 31-fold. Not only the transcription level, but also the splice composition of tomosyn-2 mRNA shifted during development. We conclude that two distinct genes drive expression of seven tomosyn isoforms. Their expression patterns support a role in regulating neuronal secretion. All isoforms share conserved WD40 and SNARE domains separated by a hypervariable module, the function of which remains to be clarified.

FuGENE 6 Transfection Reagent: the gentle power.

FuGENE 6 Transfection Reagent has been commercially available since 1997. Since that time, its popularity has increased due to its ease of use, minimal to no cytotoxicity, and the high level of transfection in many different cell lines. FuGENE 6 Transfection Reagent is gentle on the cells. Adherent cells can be trypsinized and transfected by the DNA:FuGENE 6 reagent complex prior to plating, making it a strong candidate for high throughput applications. Additionally, low cell numbers can be transfected in 96-well plates. As with most reagents, the complex formation step is critical and special handling is required because the reagent is supplied in 80% ethanol. For example, contact with plastic must be avoided as inhibitors of transfection can leach from some plastics. We investigated parameters that have been reported to affect the transfection efficiency including the use of common antibiotics, passage level of the cells, and length of time for complex formation. These parameters are often cell line dependent and can be optimized to increase transfection efficiency for a specific cell line.