ETS-1 and ETS-2 are upregulated in a transgenic mouse model of pigmented ocular neoplasm

Purpose: Choroidal melanoma is the most common primary malignant ocular tumor in human adults. Relevant mouse models of human uveal melanoma still remain to be developed. We have studied the transgenic mouse strain, Tyrp-1-TAg, to try to gain insight into possible molecular mechanisms common to pigmented ocular neoplasms occurring spontaneously in the eyes of these mice and human choroidal melanoma. The role of two members of the ETS (E26 avian leukemia oncogene) family of transcription factors, ETS-1 and ETS-2, has been investigated in many cancers but has not yet been studied in ocular tumors. Methods: This is the first study describing the production and distribution of ETS-1 and ETS-2 mRNAs and proteins using in situ hybridization and immunohistochemistry in murine ocular tissue sections of normal control eyes and tumoral eyes from mice of the same age. Using semi-quantitative reverse-transcription polymerase chain reaction (RT–PCR) and western blots experiments, we compared changes in ETS-1 and ETS-2 expression, their protein levels, and the regulation of some of their target gene expressions at different stages of the ocular tumoral progression in the transgenic mouse model, Tyrp-1-TAg, with those in normal eyes from control mice of the same age. Results: In normal control adult mouse eyes, ETS-1 was mostly present in the nuclei of all neuroretinal layers whereas ETS-2 was mostly localized in the cytosol of the cell bodies of these layers with a smaller amount present in the nuclei. Both were found in the retinal pigmentary epithelium (RPE). ETS-1 and ETS-2 mRNA and protein levels were much higher in the ocular tissues of Tyrp-1-TAg mice than in control ocular tissues from wild-type mice. This upregulation was correlated with tumor progression. We also demonstrated upregulation of ETS-1 and ETS-2 target expressions in Tyrp-1-TAg mice when comparing with the same target expressions in control mice. Conclusions: Our findings suggest that ETS-1 and ETS-2 are upregulated in ocular tumors derived from the retinal epithelium and may be involved in one or several signaling pathways that activate the expression of a set of genes involved in ocular tumor progression such as those encoding ICAM-1 (intercellular adhesion molecule-1), PAI-1 (Plasminogen activator inhibitor-1), MCP-1 (monocyte chemoattractant protein-1) and p16 (Cyclin dependent kinase inhibitor 2A).

A conserved transcriptional enhancer that specifies Tyrp1 expression to melanocytes

Friedrich Beermann

Pigment cells of mammals originate from two different lineages: melanocytes arise from the neural crest, whereas cells of the retinal pigment epithelium (RPE) originate from the optic cup of the developing forebrain. Previous studies have suggested that pigmentation genes are controlled by different regulatory networks in melanocytes and RPE. The promoter of the tyrosinase-related family gene Tyrp1 has been shown to drive detectable transgene expression only to the RPE, even though the gene is also expressed in melanocytes as evident from Tyrp1-mutant mice. This indicates that the regulatory elements responsible for Tyrp1 gene expression in the RPE are not sufficient for expression in melanocytes. We thus searched for a putative melanocyte-specific regulatory sequence and demonstrate that a bacterial artificial chromosome (BAC) containing the Tyrp1 gene and surrounding sequences is able to target transgenic expression to melanocytes and to rescue the Tyrp1b (brown) phenotype. This BAC contains several highly conserved non-coding sequences that might represent novel regulatory elements. We further focused on a sequence located at -15 kb, which we identified as a melanocyte-specific enhancer as shown by cell culture and transgenic mice experiments. In addition, we show that the transcription factor Sox10 can activate this conserved enhancer. The presence of a distal Tyrp1 regulatory element, which specifies melanocyte-specific expression, supports the idea that separate regulatory sequences can mediate differential gene expression in melanocytes and RPE.

2006

Mammalian TOR as a central modulator of the transcriptional response of keratinocyte stem cells to small temperature variations

Fahd Azzabi Zouraq

Being in the layer at the interface of the body with the outside world, keratinocyte stem cells constantly adapt and respond to environmental cues such as temperature and oxygen. However, until now, experiments involving temperature have only examined cold and heat shocks. Here, our work illustrates that small variations of temperature (~1°C) are enough to affect colony size of keratinocyte stem cells and alter expression of some genes involved in various signalling pathways. The promoter regions of the temperature sensitive genes share a sequence located in some Alu short interspersed nuclear elements (Alu SINEs). This sequence termed TORRID contains several IFHL-like motifs (IFHL motif is related to ribogenesis in yeast) and interestingly a minimal TORRID sequence might be enough for a gene to acquire temperature sensitivity. Our findings demonstrated that the expression of temperature sensitive genes is regulated through the mammalian target of rapamycin (mTOR) pathway. The transcription factors ILF1 and RAP1, and histone deacetylase II (HDAC2) –human orthologs of yeast Fhl1p, Rap1 and Rpd3 respectively– associate with TORRID sequences. Small variations of temperature were enough to alter this association. Moreover, mTOR can translocate to the nucleus and associate with the TORRID sequences in a rapamycin and temperature dependent manner. Furthermore, Raptor and Rictor, members of the mTOR complexes 1 and 2 are also involved in this binding. Hence, our results reveal not only that TOR transcriptional activity is evolutionary conserved from yeast to human, but also that a small variation in temperature represents a new input to the mTOR pathway and fine-tune regulation of transcription through TORRID sequences. These results shed new light on the role of Alus in gene regulation.

EPFL2008

Expression of cre recombinase in pigment cells

Friedrich Beermann

Conditional gene targeting using the Cre/loxP system enables specific deletion of a gene in a tissue of interest. For application of Cre-mediated recombination in pigment cells, Cre expression has to be targeted to pigment cells in transgenic mice. So far, no pigment cell-specific Cre transgenic line has been reported and we present and discuss our first results on use of Cre recombinase in pigment cells. A construct was generated where Cre recombinase is controlled by the promoter of the mouse dopachrome tautomerase (Dct) gene. The construct was functionally tested in vitro and introduced into mice. Following breeding to two reporter mouse strains, we detected Cre recombinase activity in telencephalon, melanoblasts, and retinal pigment epithelium (RPE). Our data demonstrate the feasibility of pigment cell-specific Cre/loxP-mediated recombination.

2002