The til-1 locus was identified as a common retroviral integration site in virus-accelerated lymphomas of CD2-myc transgenic mice. We now show that viral insertions at til-1 lead to transcriptional activation of PEBP2␣A (CBFA1), a transcription factor related to the Drosophila segmentation gene product, Runt. Insertions are upstream and in the opposite orientation to the gene and appear to activate a variant promoter that is normally silent in T cells. Activity of this promoter was detected in rodent osteogenic sarcoma cells and primary osteoblasts, implicating bone as the normal site of promoter activity. The isoforms encoded by the activated gene all encompass the conserved runt DNAbinding domain and share a novel N terminus different from the previously reported PEBP2␣A products. Minor products include isoforms with internal deletions due to exon skipping and a novel C-terminal domain unrelated to known runt domain factors. The major isoform expressed from the activated til-1 locus (G1) was found to account for virtually all of the core binding factor activity in nuclear extracts from its corresponding lymphoma cell line. Another member of this gene family, AML1(CBFA2), is well known for its involvement in human hemopoietic tumors. These results provide evidence of a direct oncogenic role for PEBP2␣A and indicate that the Myc and Runt family genes can cooperate in oncogenesis.

We investigated alternate mechanisms employed by enhancers to position and remodel nucleosomes and activate tissue-specific genes in divergent cell types. We demonstrated that the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene enhancer is modular and recruits different sets of transcription factors in T cells and myeloid cells. The enhancer recruited distinct inducible tissue-specific enhanceosome-like complexes and directed nucleosomes to different positions in these cell types. In undifferentiated T cells, the enhancer was activated by inducible binding of two NFAT/AP-1 complexes which disrupted two specifically positioned nucleosomes (N1 and N2). In myeloid cells, the enhancer was remodeled by GATA factors which constitutively displaced an upstream nucleosome (N0) and cooperated with inducible AP-1 elements to activate transcription. In mast cells, which express both GATA-2 and NFAT, these two pathways combined to activate the enhancer and generate high-level gene expression. At least 5 kb of the GM-CSF locus was organized as an array of nucleosomes with fixed positions, but the enhancer adopted different nucleosome positions in T cells and mast cells. Furthermore, nucleosomes located between the enhancer and promoter were mobilized upon activation in an enhancer-dependent manner. These studies reveal that distinct tissue-specific mechanisms can be used either alternately or in combination to activate the same enhancer.

This is a repository copy of Standardized protocols for differentiation of THP-1 cells to macrophages with distinct M(IFNγ+LPS), M(IL-4) and M(IL-10) phenotypes.

Tumour invasion and metastasis is the most common cause of death from cancer. For epithelial cells to invade surrounding tissues and metastasise, an epithelial-mesenchymal transition (EMT) is required. We have demonstrated that FGFR1 expression is increased in bladder cancer and that activation of FGFR1 induces an EMT in urothelial carcinoma (UC) cell lines. Here, we created an in vitro FGFR1-inducible model of EMT, and used this model to identify regulators of urothelial EMT. FGFR1 activation promoted EMT over a period of 72 hours. Initially a rapid increase in actin stress fibres occurred, followed by an increase in cell size, altered morphology and increased migration and invasion. By using site-directed mutagenesis and small molecule inhibitors we demonstrated that combined activation of the mitogen activated protein kinase (MAPK) and phospholipase C gamma (PLCγ) pathways regulated this EMT. Actin stress fibre formation was regulated by PLCγ activation, and was also important for the increase in cell size, migration and altered morphology. MAPK activation regulated migration and E-cadherin expression, indicating that combined activation of PLCγand MAPK is required for a full EMT. We used expression microarrays to assess changes in gene expression downstream of these signalling cascades. COX-2 was transcriptionally upregulated by FGFR1 and caused increased intracellular prostaglandin E2 levels, which promoted migration. In conclusion, we have demonstrated that FGFR1 activation in UC cells lines promotes EMT via coordinated activation of multiple signalling pathways and by promoting activation of prostaglandin synthesis.

The mammalian Runx gene family (Runx1-3) are transcription factors that play essential, lineage-specific roles in development. A growing body of evidence implicates these genes as mutational targets in cancer where, in different contexts, individual family members have been reported to act as tumour suppressors, dominant oncogenes or mediators of metastasis. We are exploring these paradoxical observations by ectopic expression of RUNX genes in primary murine embryonic fibroblasts where, in common with a number of other dominant oncogenes, RUNX1 induces senescence-like growth arrest in the presence of an intact p19 ARF -p53 pathway. We now report that, in MEFs lacking functional p53, RUNX1 has apparently prooncogenic effects on cell growth that include cytoskeletal reorganization, reduced contact inhibition at confluence and accelerated tumour expansion in vivo. On the other hand, RUNX1 conferred no obvious growth advantage at low cell density and actually delayed entry of primary MEFs into S phase. We also found that ectopic RUNX1 interferes with the morphological and growth responses of p53-null MEFs to TGFb indicating that these effects are mediated by overlapping pathways. These observations help to elucidate the context-dependent consequences of loss and gain of Runx activity.

The closely linked IL-3 and GM-CSF genes are located within a cluster of cytokine genes co-expressed in activated T cells. Their activation in response to TCR signaling pathways is controlled by specific, inducible upstream enhancers. To study the developmental regulation of this locus in T lineage cells, we created a transgenic mouse model encompassing the human IL-3 and GM-CSF genes plus the known enhancers. We demonstrated that the IL-3/GM-CSF locus undergoes progressive stages of activation, with stepwise increases in active modifications and the proportion of cytokine-expressing cells, throughout the course of T cell differentiation. Looking first at immature cells, we found that the IL-3/GM-CSF locus was epigenetically silent in CD4/CD8 double positive thymocytes, thereby minimizing the potential for inappropriate activation during the course of TCR selection. Furthermore, we demonstrated that the locus did not reach its maximal transcriptional potential until after T cells had undergone blast cell transformation to become fully activated proliferating T cells. Inducible locus activation in mature T cells was accompanied by noncoding transcription initiating within the enhancer elements. Significantly, we also found that memory CD4 positive T cells, but not naive T cells, maintain a remodeled chromatin structure resembling that seen in T blast cells.

Protein-protein interactions are essential for the control of cellular functions and are critical for regulation of the immune system. One example is the binding of Fc regions of IgG to the Fc gamma receptors (FcγRs). High sequence identity (98%) between the genes encoding FcγRIIIa (expressed on macrophages and natural killer cells) and FcγRIIIb (expressed on neutrophils) has prevented the development of monospecific agents against these therapeutic targets. We now report the identification of FcγRIIIa-specific artificial binding proteins called "Affimer" that block IgG binding and abrogate FcγRIIIa-mediated downstream effector functions in macrophages, namely TNF release and phagocytosis. Cocrystal structures and molecular dynamics simulations have revealed the structural basis of this specificity for two Affimer proteins: One binds directly to the Fc binding site, whereas the other acts allosterically.

Multicellular 3-dimensional (3D) in vitro models of normal human breast tissue to study cancer initiation are required. We present a model incorporating three of the major functional cell types of breast, detail the phenotype and document our breast cancer initiation studies. Myoepithelial cells and fibroblasts were isolated and immortalised from breast reduction mammoplasty samples. Tri-cultures containing non-tumorigenic luminal epithelial cells HB2, or HB2 overexpressing different HER proteins, together with myoepithelial cells and fibroblasts were established in collagen I. Phenotype was assessed morphologically and immunohistochemically and compared to normal breast tissue. When all three cell types were present, polarised epithelial structures with lumens and basement membrane production were observed, akin to normal human breast tissue. Overexpression of HER2 or HER2/3 caused a significant increase in size, while HER2 overexpression resulted in development of a DCIS-like phenotype. In summary, we have developed a 3D tri-cellular model of normal human breast, amenable to comparative analysis after genetic manipulation and with potential to dissect the mechanisms behind the early stages of breast cancer initiation.