[Show abstract][Hide abstract] ABSTRACT: Notch is a family of transmembrane receptors that participate in the regulation of cell differentiation, proliferation and stemness. Notch pathway activation has also been found associated to different human cancers including primary cutaneous T-cell lymphomas (CTCL). The elucidation of the mechanisms driving Notch activation in these particular diseases has remained elusive. We here studied the possibility that DNA methylation at Notch pathways gene promoters, and/or deregulation of Notch-associated microRNAs contribute to activate Notch in Mycosis Fungoides (MF). By genome-wide DNA methylation analysis, we failed to detect any consistent methylation at the Notch1, the Notch-ligand Jagged1 or the Notch-target Hes1 gene promoters, but found a significant methylation of the Notch-related microRNAs, particularly miR-200c and miR-124. Downregulation of miR-200c is associated with overexpression of Jagged1, concomitant to Notch1 activation. CTCL cell lines were infected with lentiviral vector encoding for miR-200c and, ectopic expression of miR-200c in CTCL lines resulted in Jagged1 protein downregulation associated with a reduction in the levels of active Notch1. Our study deciphers a epigenetic mechanism regulating Notch pathway in MF that might contribute to the future design of more specific therapeutic strategies.Journal of Investigative Dermatology accepted article preview online, 24 August 2015. doi:10.1038/jid.2015.328.
Full-text · Article · Aug 2015 · Journal of Investigative Dermatology
[Show abstract][Hide abstract] ABSTRACT: Notch1 is required to generate the earliest embryonic hematopoietic stem cells (HSCs); however since Notch-deficient embryos die early in gestation, additional functions for Notch in embryonic HSC biology have not been described. We used two complementary genetic models to address this important biological question. Unlike Notch1-deficient mice, mice lacking the conserved Notch1 transcriptional activation domain (TAD) show attenuated Notch1 function in vivo and survive until late gestation, succumbing to multiple cardiac abnormalities. Notch1 TAD-deficient HSCs emerge and successfully migrate to the fetal liver but are decreased in frequency by embryonic day 14.5. In addition, TAD-deficient fetal liver HSCs fail to compete with wild-type HSCs in bone marrow transplant experiments. This phenotype is independently recapitulated by conditional knockout of Rbpj, a core Notch pathway component. In vitro analysis of Notch1 TAD-deficient cells shows that the Notch1 TAD is important to properly assemble the Notch1/Rbpj/Maml trimolecular transcription complex. Together, these studies reveal an essential role for the Notch1 TAD in fetal development and identify important cell-autonomous functions for Notch1 signaling in fetal HSC homeostasis.
Full-text · Article · Mar 2014 · Genes & development
[Show abstract][Hide abstract] ABSTRACT: Previous studies have identified Notch as a key regulator of hematopoietic stem cell (HSC) development, but the underlying downstream mechanisms remain unknown. The Notch target Hes1 is widely expressed in the aortic endothelium and hematopoietic clusters, though Hes1-deficient mice show no overt hematopoietic abnormalities. We now demonstrate that Hes is required for the development of HSC in the mouse embryo, a function previously undetected as the result of functional compensation by de novo expression of Hes5 in the aorta/gonad/mesonephros (AGM) region of Hes1 mutants. Analysis of embryos deficient for Hes1 and Hes5 reveals an intact arterial program with overproduction of nonfunctional hematopoietic precursors and total absence of HSC activity. These alterations were associated with increased expression of the hematopoietic regulators Runx1, c-myb, and the previously identified Notch target Gata2. By analyzing the Gata2 locus, we have identified functional RBPJ-binding sites, which mutation results in loss of Gata2 reporter expression in transgenic embryos, and functional Hes-binding sites, which mutation leads to specific Gata2 up-regulation in the hematopoietic precursors. Together, our findings show that Notch activation in the AGM triggers Gata2 and Hes1 transcription, and next HES-1 protein represses Gata2, creating an incoherent feed-forward loop required to restrict Gata2 expression in the emerging HSCs.
Full-text · Article · Dec 2012 · Journal of Experimental Medicine
[Show abstract][Hide abstract] ABSTRACT: Understanding how hematopoietic stem cells (HSCs) are generated and the signals that control this process is a crucial issue for regenerative medicine applications that require in vitro production of HSC. HSCs emerge during embryonic life from an endothelial-like cell population that resides in the aorta-gonad-mesonephros (AGM) region. We show here that β-catenin is nuclear and active in few endothelial nonhematopoietic cells closely associated with the emerging hematopoietic clusters of the embryonic aorta during mouse development. Importantly, Wnt/β-catenin activity is transiently required in the AGM to generate long-term HSCs and to produce hematopoietic cells in vitro from AGM endothelial precursors. Genetic deletion of β-catenin from the embryonic endothelium stage (using VE-cadherin-Cre recombinase), but not from embryonic hematopoietic cells (using Vav1-Cre), precludes progression of mutant cells toward the hematopoietic lineage; however, these mutant cells still contribute to the adult endothelium. Together, those findings indicate that Wnt/β-catenin activity is needed for the emergence but not the maintenance of HSCs in mouse embryos.
Full-text · Article · Jul 2012 · Journal of Experimental Medicine
[Show abstract][Hide abstract] ABSTRACT: Hematopoiesis is the process that generates all the cell types of the blood, which are responsible for oxygen transport and immune defense. It has been now more than 50 years from the demonstration that blood cells derive from a common ancestor called Hematopoietic Stem Cell (HSC) McCulloch and Till (1960). Thus, the hematopoietic process relies on the unlimited and distinctive self-renewal ability of HSC, which in the adult mammalian organisms reside in the bone marrow, but their generation occurs during embryonic life. Questions still remain about how HSCs acquire and maintain the features of self-renewal and pluripotency that define stem-cell populations. Notch is a crucial signaling pathway involved in the generation of cell diversity and stem-cell maintenance in different systems. In some cases, Notch prevents differentiation, while in other contexts Notch directly participates in promoting cell differentiation. In the following sections, we will review what is known about the role of Notch in HSC establishment and hematopoietic cell lineage specification.
Full-text · Article · Jun 2012 · Current topics in microbiology and immunology
[Show abstract][Hide abstract] ABSTRACT: Notch activation is a current event in T Acute Lymphoblastic Leukemia (T-ALL) but the downstream elements that are able to support Notch-dependent leukemias are not well characterized. We have recently shown that the Notch-Hes1-CYLD-NFkB axis is crucial in the maintenance of T-ALL, but detailed evaluation of the contribution of each one of these elements is still missing. Here we use a Notch1-induced leukemia in vivo model to study the effect of silencing the Notch-target gene, Hes1, or over-expressing the Hes1-target, CYLD. We here show that both strategies completely abolish the ability of constitutive active Notch1 to generate T-ALL.
[Show abstract][Hide abstract] ABSTRACT: It was previously shown that the NF-κB pathway is downstream of oncogenic Notch1 in T cell acute lymphoblastic leukemia (T-ALL). Here, we visualize Notch-induced NF-κB activation using both human T-ALL cell lines and animal models. We demonstrate that Hes1, a canonical Notch target and transcriptional repressor, is responsible for sustaining IKK activation in T-ALL. Hes1 exerts its effects by repressing the deubiquitinase CYLD, a negative IKK complex regulator. CYLD expression was found to be significantly suppressed in primary T-ALL. Finally, we demonstrate that IKK inhibition is a promising option for the targeted therapy of T-ALL as specific suppression of IKK expression and function affected both the survival of human T-ALL cells and the maintenance of the disease in vivo.