RhoV, a Potential Notch Target Gene in Murine Skin, Does not Play an Essential Role in Epidermis Development and Homeostasis

The evolutionarily conserved Notch signaling cascade plays a pivotal role in the regulation of many fundamental processes such as stem cell maintenance, proliferation, and differentiation. Recently, we aimed to identify downstream target genes regulated by Notch1 during epidermal differentiation. For this purpose an Affymetrix mouse gene chip array of 14.000 genes was performed in both Notch1 gain and loss of function experiments using murine primary keratinocytes. Thirteen genes positively or negatively regulated, based on expression levels and relevance of the involved signaling pathways, were further validated by real-time PCR. Among these, we decided to focus on the Rho GTPase RhoV because it showed robust up- and down-regulation under both gain- and loss-of-function conditions. Therefore, we generated different transgenic mice expressing either a dominant active or a dominant negative form of RhoV specifically in the skin. Morphological and histological analyses did not reveal any overt skin phenotype in transgenic mice. In addition, challenging the system by performing wound healing assays demonstrated that the healing process in transgenic mice occurred normally and that the migration and/or differentiation of keratinocytes was unimpaired. Taken together, our data suggest that gain or loss of RhoV function does not perturb skin development and homeostasis, and does not affect the wound healing process. In addition to the investigation of the potential Notch target gene RhoV, we sought to generate both a floxed Jag2 gene targeted mouse line and a Notch2-specifc reporter mouse. The former was intended to enable conditional deletion of the Notch ligand Jagged2 and therefore allow its role to be studied in a loss-of-function context. The Notch2 reporter mouse is a tool enabling the in vivo study of cells that receive a Notch2-mediated signal. The strategy employs elements of a Tet-Off system and allows cells receiving a Notch2 specific signal to be identified in any tissue at any given time point.

The Cell Differentiation Status Influences the Outcome of Notch-Induced Malignancy

Caroline Poisson

The Notch signaling cascade is a well conserved pathway that regulates many aspects of embryonic development and plays a crucial role in the differentiation process and tissue homeostasis in multiple organs of adult species. Several human pathological disorders, including cancer, arise as a consequence of the deregulation of the Notch signaling pathway. Notch deregulation, up to date, has been best characterized in acute T-cell lymphoblastic leukemia (T-ALL). Indeed, in T-ALL pediatric patients activating Notch1 mutations have been identified in more than 50% of the cases. Increased knowledge of the mechanisms underlying T-ALL development and maintenance is clearly needed as one out of 5 patients with T-ALL has a very poor prognosis. Moreover, the high toxicity of chemotherapeutic regimens has been associated with elevated risks of developing severe secondary health problems. In the present study, we characterized the role of Notch in T-ALL induction and maintenance. Using transgenic mouse models, we could demonstrate that the oncogenic potential of Notch is dependent on RBP-Jκ transcriptional activity. Indeed, the canonical Notch signaling pathway, mediated by the RBP-Jκ transcription factor, is essential for both Notch-mediated T-ALL induction and maintenance, as well as for the induction of a Notch-mediated lymphoproliferative disorder. Moreover, constitutive overexpression of Notch1 at discrete developmental stages within the hematopoietic system, led us to identify a correlation between the differentiation status of a cell, within the hierarchy of the hematopoietic tree, and its sensitivity to Notch1-induced transformation. We demonstrated that forced Notch1 expression in hematopoietic stem cells was not sufficient to induce T-ALL. Nevertheless, aberrant Notch1 expression in the bone marrow resulted in the development of a lethal lymphoproliferative disorder. Overexpression of Notch1 in thmus at the DN2 to DN3 stage of T-cell delvelopment generated an aggressive transplantable T-ALL with important metastatic potential. This suggested that progenitors already committed to the T-cell lineage are highly susceptible to Notch-mediated transformation, which is relevant to the human disease. Nevertheless, forced Notch1 expression after the β-selection checkpoint in the thymys, was no longer sufficient to induce transformation of late DN3 to DN4 thymocytes. We therefore established a murine genetic system that allowed us to dissect the mechanisms of aberrant Notch1 signaling in a lymphoproliferative disorder and a true T-ALL model. We were able to assess the genetic signature that is involved in the transplantability and metastatic potential of leukemic cells and generated a list of candidate genes implicated in these processes. In addition, we correlated genes identified in our murine models with genes associated as well in human T-ALL profiles to identify genes implicated in T-ALL pathogenesis in both mouse and human.

EPFL2011

IRF6 is a mediator of Notch pro-differentiation and tumour suppressive function in keratinocytes

Matteo Di Piazza, Piotr Dziunycz, Freddy Radtke

While the pro-differentiation and tumour suppressive functions of Notch signalling in keratinocytes are well established, the underlying mechanisms remain poorly understood. We report here that interferon regulatory factor 6 (IRF6), an IRF family member with an essential role in epidermal development, is induced in differentiation through a Notch-dependent mechanism and is a primary Notch target in keratinocytes and keratinocyte-derived SCC cells. Increased IRF6 expression contributes to the impact of Notch activation on growth/differentiation-related genes, while it is not required for induction of 'canonical' Notch targets like p21 WAF1/Cip1, Hes1 and Hey1. Down-modulation of IRF6 counteracts differentiation of primary human keratinocytes in vitro and in vivo, promoting ras-induced tumour formation. The clinical relevance of these findings is illustrated by the strikingly opposite pattern of expression of Notch1 and IRF6 versus epidermal growth factor receptor in a cohort of clinical SCCs, as a function of their grade of differentiation. Thus, IRF6 is a primary Notch target in keratinocytes, which contributes to the role of this pathway in differentiation and tumour suppression. The EMBO Journal (2011) 30, 4571-4585. doi:10.1038/emboj.2011.325; Published online 9 September 2011 Subject Categories: signal transduction; molecular biology of disease

Nature Publishing Group2011

KRAB/KAP1 Regulation of Embryonic Stem Cell Pluripotent Self-renewal

Andrea Corsinotti

KRAB-ZFPs constitute the largest family of transcription factors (TFs) encoded by the mouse genome and are known to interact with the co-repressor KAP1. KRAB/KAP1-mediated regulation is essential for several development- and ESC- specific functions, in particular regulation of pluripotent self-renewal. We performed an expression analysis in mouse pluripotent ESCs and differentiated cells. A large number of KRAB-ZFPs was expressed in pluripotent cells and many of them were differentially regulated in other cell types, suggesting that a set of KRAB-ZFPs can be involved in ESC-specific functions. We identified a set of candidate genes, including Zfp459, which show a pluripotency-specific expression pattern. Zfp459 depletion in ESCs induced transcriptional perturbation of pluripotency- and differentiation-associated genes, even if their ability to self-renew or to differentiate was not impaired, and its expression was not required for the formation of blastocysts ex vivo. To understand the precise role of Zfp459 in ESCs further experiments for the identification of its targets are still required. We used a lentiviral vector (LV) to over-express in ESCs ZFP57, which had been previously demonstrated to play a role in regulating genomic imprinting, in fusion with HA-tags. ChIP-seq experiments using a HA antibody demonstrated that ZFP57-HA binds all the known and predicted imprinted loci and identified a hexanucleotide consensus in most of the ZFP57 binding sites co-occupied also by KAP1 and its associated factor SetDB1. We also demonstrated that our LV based expression system could be exploited to perform ChIP-seq analyses in ESCs of TFs, such as Zfp459, for which specific antibodies are not available. Finally, when we cultured ESCs in different conditions, we observed that KAP1 recruitment upstream of Nanog, which had been previously observed, was dependent on the MAPK and GSK3 signaling pathways. We hypothesized that this could be due to the presence in these conditions of ESCs expressing heterogeneous Nanog levels. However, when Nanoghigh and Nanoglow cells were sorted, both the populations shown a comparable enrichment for KAP1 binding upstream of Nanog. The precise role for the KAP1-mediated regulation of Nanog in ESCs and its dependence on extrinsic signaling pathways remains unclear, even if we hypothesize that it can depend on the mono-/bi-allelic expression of Nanog in ESCs and during early development.