Clemencia Colmenares

Interferon action and apoptosis are defective in mice devoid of 2′,5′-oligoadenylate-dependent RNase L

2′,5′‐Oligoadenylate‐dependent RNase L functions in the interferon‐inducible, RNA decay pathway known as the 2‐5A system. To determine the physiological roles of the 2‐5A system, mice were generated with a targeted disruption of the RNase L gene. The antiviral effect of interferon α was impaired in RNase L−/− mice providing the first evidence that the 2‐5A system functions as an antiviral pathway in animals. In addition, remarkably enlarged thymuses in the RNase L−/− mice resulted from a suppression of apoptosis. There was a 2‐fold decrease in apoptosis in vivo in the thymuses and spleens of RNase L−/− mice. Furthermore, apoptosis was substantially suppressed in RNase L−/− thymocytes and fibroblasts treated with different apoptotic agents. These results suggest that both interferon action and apoptosis can be controlled at the level of RNA stability by RNase L. Another implication is that the 2‐5A system is likely to contribute to the antiviral activity of interferon by inducing apoptosis of infected cells.

Organogenesis relies on SoxC transcription factors for the survival of neural and mesenchymal progenitors

During organogenesis, neural and mesenchymal progenitor cells give rise to many cell lineages, but their molecular requirements for self-renewal and lineage decisions are incompletely understood. In this study, we show that their survival critically relies on the redundantly acting SoxC transcription factors Sox4, Sox11 and Sox12. The more SoxC alleles that are deleted in mouse embryos, the more severe and widespread organ hypoplasia is. SoxC triple-null embryos die at midgestation unturned and tiny, with normal patterning and lineage specification, but with massively dying neural and mesenchymal progenitor cells. Specific inactivation of SoxC genes in neural and mesenchymal cells leads to selective apoptosis of these cells, suggesting SoxC cell-autonomous roles. Tead2 functionally interacts with SoxC genes in embryonic development, and is a direct target of SoxC proteins. SoxC genes therefore ensure neural and mesenchymal progenitor cell survival, and function in part by activating this transcriptional mediator of the Hippo signalling pathway.

Loss of the SKI proto-oncogene in individuals affected with 1p36 deletion syndrome is predicted by strain-dependent defects in Ski-/- mice

Experiments involving overexpression of Ski have suggested that this gene is involved in neural tube development and muscle differentiation. In agreement with these findings, Ski−/− mice display a cranial neural tube defect that results in exencephaly and a marked reduction in skeletal muscle mass. Here we show that the penetrance and expressivity of the phenotype changes when the null mutation is backcrossed into the C57BL6/J background, with the principal change involving a switch from a neural tube defect to midline facial clefting. Other defects, including depressed nasal bridge, eye abnormalities, skeletal muscle defects and digit abnormalities, show increased penetrance in the C57BL6/J background. These phenotypes are interesting because they resemble some of the features observed in individuals diagnosed with 1p36 deletion syndrome, a disorder caused by monosomy of the short arm of human chromosome 1p (refs. 6–9). These similarities prompted us to re-examine the chromosomal location of human SKI and to determine whether SKI is included in the deletions of 1p36. We found that human SKI is located at distal 1p36.3 and is deleted in all of the individuals tested so far who have this syndrome. Thus, SKI may contribute to some of the phenotypes common in 1p36 deletion syndrome, and particularly to facial clefting.

Selective restoration of male fertility in mice lacking angiotensin-converting enzymes by sperm-specific expression of the testicular isozyme.

Although angiotensin-converting enzyme (ACE) has been studied primarily in the context of its role in blood pressure regulation, this widely distributed enzyme has many other physiological functions. The ACE gene encodes two isozymes. The somatic isozyme is expressed in many tissues, including vascular endothelial cells, renal epithelial cells, and testicular Leydig cells, whereas the testicular or germinal angiotensin-converting enzyme is expressed only in sperm. The ACE gene knockout mice lack both isozymes and they exhibit low blood pressure, kidney dysfunctions, and male infertility. Here, we report the use of a sperm-specific promoter and interbreeding of transgenic and gene knockout mice for generating a mouse strain that expressed ACE only in sperm. The experimental mice maintained the kidney defects of ACE-/- mice, but unlike the knockout strain, the males were fertile. Thus, we established that the role of ACE in male fertility is completely dependent on its exclusive expression in sperm. Our study clearly demonstrated how transgenic and knockout techniques can be combined for ascribing a specific physiological function to the expression of a multifunctional protein in a given tissue.

Increased susceptibility to tumorigenesis of ski-deficient heterozygous mice

The c-ski proto-oncogene product (c-Ski) acts as a co-repressor and binds to other co-repressors N-CoR/SMRT and mSin3A which form a complex with histone deacetylase (HDAC). c-Ski mediates the transcriptional repression by a number of repressors, including nuclear hormone receptors and Mad. c-Ski also directly binds to, and recruits the HDAC complex to Smads, leading to inhibition of tumor growth factor-β (TGF-β) signaling. This is consistent with the function of ski as an oncogene. Here we show that loss of one copy of c-ski increases susceptibility to tumorigenesis in mice. When challenged with a chemical carcinogen, c-ski heterozygous mice showed an increased level of tumor formation relative to wild-type mice. In addition, c-ski-deficient mouse embryonic fibroblasts (MEFs) had increased proliferative capacity, whereas overexpression of c-Ski suppressed the proliferation. Furthermore, the introduction of activated Ki-ras into c-ski-deficient MEFs resulted in neoplastic transformation. These findings demonstrate that c-ski acts as a tumor suppressor in some types of cells. The level of cdc25A mRNA, which is down regulated by two tumor suppressor gene products, Rb and Mad, was upregulated in c-ski-deficient MEFs, whereas it decreased by overexpressing c-Ski in MEFs. This is consistent with the fact that c-Ski acts as a co-repressor of Mad and Rb. These results support the view that the decreased activities of Mad and Rb in ski-deficient cells at least partly contribute to enhanced proliferation and susceptibility to tumorigenesis. Human c-ski gene was mapped to a region close to the p73 tumor suppressor gene at the 1p36.3 locus, which is already known to contain multiple uncharacterized tumor suppressor genes.

Identification of a core functional and structural domain of the v-Ski oncoprotein responsible for both transformation and myogenesis.

The v-ski oncogene promotes cellular transformation and myogenic differentiation. In quail embryo fibroblasts the two properties are displayed simultaneously and terminal muscle differentiation occurs only among cells already transformed by v-ski. To understand how the two phenotypes are derived from a single gene, we have undertaken to identify functionally important regions in v-ski and to test whether these regions can promote one phenotype without the other. We have generated both random and targeted mutations in v-ski and evaluated the effects of these mutations on expression, intracellular location, transformation, and myogenesis. Among a total of 26 mutants analysed, we have not found complete separation of the myogenic and transforming properties. Mutations in the region of v-Ski encoded by exon 1 of c-ski frequently abolish both its transformation and muscle differentiation activities, whereas mutations outside of this region are always tolerated. When expressed in cells from a minigene containing only the exon 1 sequence, the protein displays the transforming and myogenic activities similar to v-Ski. These results argue that the amino acid sequence encoded by exon 1 contains the core functional domain of the oncoprotein. To determine whether this functional domain has a structural counterpart, we have fragmented the v-Ski protein by limited proteolysis and found a single proteolytically stable domain spanning the entire exon 1-encoded region. Physical studies of the polypeptide encoded by exon 1 confirms that it folds into a compact, globular protein. The finding that both the transforming and myogenic properties of v-Ski are inseparable by mutation and are contained in a single domain suggests that they are derived from the same function.

The Protooncogene Ski Controls Schwann Cell Proliferation and Myelination

Schwann cell proliferation and subsequent differentiation to nonmyelinating and myelinating cells are closely linked processes. Elucidating the molecular mechanisms that control these events is key to the understanding of nerve development, regeneration, nerve-sheath tumors, and neuropathies. We define the protooncogene Ski, an inhibitor of TGF-beta signaling, as an essential component of the machinery that controls Schwann cell proliferation and myelination. Functional Ski overexpression inhibits TGF-beta-mediated proliferation and prevents growth-arrested Schwann cells from reentering the cell cycle. Consistent with these findings, myelinating Schwann cells upregulate Ski during development and remyelination after injury. Myelination is blocked in myelin-competent cultures derived from Ski-deficient animals, and genes encoding myelin components are downregulated in Ski-deficient nerves. Conversely, overexpression of Ski in Schwann cells causes an upregulation of myelin-related genes. The myelination-regulating transcription factor Oct6 is involved in a complex modulatory relationship with Ski. We conclude that Ski is a crucial signal in Schwann cell development and myelination.

Protooncogene Ski cooperates with the chromatin-remodeling factor Satb2 in specifying callosal neurons

First insights into the molecular programs orchestrating the progression from neural stem cells to cortical projection neurons are emerging. Loss of the transcriptional regulator Ski has been linked to the human 1p36 deletion syndrome, which includes central nervous system defects. Here, we report critical roles for Ski in the maintenance of the neural stem cell pool and the specification of callosal neurons. Ski-deficient callosal neurons lose their identity and ectopically express the transcription factor Ctip2. The misspecified callosal neurons largely fail to form the corpus callosum and instead redirect their axons toward subcortical targets. We identify the chromatin-remodeling factor Satb2 as a partner of Ski, and show that both proteins are required for transcriptional repression of Ctip2 in callosal neurons. We propose a model in which Satb2 recruits Ski to the Ctip2 locus, and Ski attracts histone deacetylases, thereby enabling the formation of a functional nucleosome remodeling and deacetylase repressor complex. Our findings establish a central role for Ski–Satb2 interactions in regulating transcriptional mechanisms of callosal neuron specification.

Genetic and molecular control of folate-homocysteine metabolism in mutant mice

Abstract. Hyperhomocysteinemia adversely affects fundamental aspects of fetal development, adulthood, and aging, but the role of elevated homocysteine levels in these birth defects and adult diseases remains unclear. Mouse models are valuable for investigating the causes and consequences of hyperhomocysteinemia. We used a phenotype-based approach to identify mouse mutants for studying the relation between single gene mutations, homocysteine levels as a measure of the status of homocysteine metabolism, and gene expression profiles as a way to assess the impact of protein deficiency in mutant mice on steady-state transcription levels of genes in the folate-homocysteine pathways. These mutants were selected based on their propensity to produce phenotypes that are reminiscent of those associated with anomalies in folate-homocysteine metabolism in humans. We report identification of new, single-gene mouse models of homocysteinemia and characterization of their molecular and physiological impact on folate-homocysteine metabolism. Mutations in several genes involved in the hedgehog and WNT signal transduction pathways, as well as a gene involved in lipid metabolism, resulted in elevated homocysteine levels and altered expression profiles of folate-homocysteine metabolism genes. These results begin to unravel the complex relations between elevation of a single amino acid in the blood and the diverse birth defects and adult diseases associated with hyperhomocysteinemia.

Chromosomal instability in mouse embryonic fibroblasts null for the transcriptional co-repressor Ski

Ski is a transcriptional regulator that has been considered an oncoprotein given its ability to induce oncogenic transformation in avian model systems. However, studies in mouse and in some human tumor cells have also indicated a tumor suppressor activity for this protein. We found that Ski−/− mouse embryo fibroblasts exhibit high levels of genome instability, namely aneuploidy, consistent with a tumor suppressor function for Ski. Time‐lapse microscopy revealed lagging chromosomes and chromatin/chromosome bridges as the major cause of micronuclei (MN) formation and the subsequent aneuploidy. Although these cells arrested in mitosis after treatment with spindle disrupting drugs and exhibited a delayed metaphase/anaphase transition, spindle assembly checkpoint (SAC) was not sufficient to prevent chromosome missegregation, consistent with a weakened SAC. Our in vivo analysis also showed dynamic metaphase plate rearrangements with switches in polarity in cells arrested in metaphase. Importantly, after ectopic expression of Ski the cells that displayed this metaphase arrest died directly during metaphase or after aberrant cell division, relating SAC activation and mitotic cell death. This increased susceptibility to undergo mitosis‐associated cell death reduced the number of MN‐containing cells. The presented data support a new role for Ski in the mitotic process and in maintenance of genetic stability, providing insights into the mechanism of tumor suppression mediated by this protein. J. Cell. Physiol. 227: 278–287, 2012.