Impaired nuclear functions lead to increased senescence and inefficient differentiation in human myoblasts with a dominant p.R545C mutation in the LMNA gene

We have studied myoblasts from a patient with a severe autosomal dominant Emery-Dreifuss muscular dystrophy (AD-EDMD) caused by an arginine 545 to cystein point mutation (p.R545C) in the carboxy-terminal domain of the lamin A/C gene. This mutation has pleiotropic cellular effects on these myoblasts as demonstrated by nuclear structural defects, exhibiting lobulations which increase with cell passages in culture. The organization of both lamin A/C and its inner nuclear membrane partner emerin are altered, eventually showing a honeycomb pattern upon immunofluorescence microscopy. In addition, the distribution of histone H3 trimethylated at lysine 27 and of phosphorylated RNA polymerase II, markers of inactive and active chromatin domains, respectively, are altered suggesting an impact on gene expression. Patient myoblasts also presented a high index of senescence in ex vivo culture. Moreover, our data show for the first time in an AD-EDMD context that the 20S core particle of the proteasome was inactivated. With cell passages, the 20S core protein progressively accumulated into discrete nuclear foci that largely colocalized with promyelocytic leukemia (PML) bodies while p21 accumulated throughout the nuclear compartment. Proteasome inactivation has been linked to normal cellular ageing. Our data indicate that it may also contribute to premature senescence in AD-EDMD patient myoblasts. Finally, when transferred to low-serum medium, patient myoblasts were deficient in ex vivo differentiation, as assessed by the absence of myotube formation and myogenin induction. Altogether, these data suggest that the LMNA mutation p.R545C impairs both proliferation and differentiation capacities of myoblasts as part of the pathogenesis of AD-EDMD.     

Transcriptional activation of interleukin-8 by beta-catenin-Tcf4

Nuclear translocation of beta-catenin and its association with Tcf/Lef factors are key steps in transduction of the Wnt signal, which is aberrantly activated in a variety of human cancers. In a search for new beta-catenin-Tcf target genes, we analyzed beta-catenin-induced alterations of gene expression in primary human hepatocytes, after transduction of either dominant stable beta-catenin or its truncated, transactivation-deficient counterpart by means of a lentiviral vector. cDNA microarray analysis revealed a limited set of up-regulated genes, including known Wnt targets such as matrilysin and keratin-1. In this screen, we identified the CXC chemokine interleukin 8 (IL-8) as a direct target of beta-catenin-Tcf4. IL-8 is constitutively expressed in various cancers, and it has been implicated in tumor progression through its mitogenic, motogenic, and angiogenic activities. The IL-8 promoter contains a unique consensus Tcf/Lef site that is critical for IL-8 activation by beta-catenin. We show here that the p300 coactivator was required for efficient transactivation of beta-catenin on this promoter. Ectopic expression of beta-catenin in hepatoma cells promoted IL-8 secretion, which stimulated endothelial cell migration. These data define IL-8 as a Wnt target and suggest that IL-8 induction by beta-catenin might be implicated in developmental and tumorigenic processes.     

HP1gamma associates with euchromatin and heterochromatin in mammalian nuclei and chromosomes

Heterochromatin protein 1 (HP1) is a nonhistone chromosomal protein, first identified in Drosophila, that plays a dose-dependent role in gene silencing. Three orthologs, HP1alpha, HP1beta, and HP1gamma, have been characterized in mammals. While HP1alpha and HP1beta have been unambiguously localized in heterochromatin by immunocytochemical methods, HP1gamma has been found either exclusively associated with euchromatin or present in both euchromatin and heterochromatin. Here, using an antibody directed against a peptide epitope at the carboxyl-terminal end of the molecule, we localize HP1gamma in both euchromatin and heterochromatin compartments of interphase nuclei, as well as in the pericentromeric chromatin and arms of mitotic chromosomes of 3T3 cells. This dual location was also observed in nuclei expressing HP1gamma as a fusion protein with green fluorescent protein. In contrast, when the distribution of HP1gamma was analyzed with antibodies directed against an amino-terminal epitope, the protein was detectable in euchromatin and not in heterochromatin, except for transient heterochromatin staining during the late S phase, when the heterochromatin undergoes replication. These data suggest that the controversial immunolocalization of HP1gamma in chromatin is due to the use of antibodies directed against topologically distinct epitopes, those present at the amino-terminal end of the molecule being selectively masked in nonreplicative heterochromatin.     

Subcellular localization of SREBP1 depends on its interaction with the C-terminal region of wild-type and disease related A-type lamins

Lamins A and C are nuclear intermediate filament proteins expressed in most differentiated somatic cells. Previous data suggested that prelamin A, the lamin A precursor, accumulates in some lipodystrophy syndromes caused by mutations in the lamin A/C gene, and binds and inactivates the sterol regulatory element binding protein 1 (SREBP1). Here we show that, in vitro, the tail regions of prelamin A, lamin A and lamin C bind a polypeptide of SREBP1. Such interactions also occur in HeLa cells, since expression of lamin tail regions impedes nucleolar accumulation of the SREBP1 polypeptide fused to a nucleolar localization signal sequence. In addition, the tail regions of A-type lamin variants that occur in Dunnigan-type familial partial lipodystrophy of (R482W) and Hutchison Gilford progeria syndrome (?607–656) bind to the SREBP1 polypeptide in vitro, and the corresponding FLAG-tagged full-length lamin variants co-immunoprecipitate the SREBP1 polypeptide in cells. Overexpression of wild-type A-type lamins and variants favors SREBP1 polypeptide localization at the intranuclear periphery, suggesting its sequestration. Our data support the hypothesis that variation of A-type lamin protein level and spatial organization, in particular due to disease-linked mutations, influences the sequestration of SREBP1 at the nuclear envelope and thus contributes to the regulation of SREBP1 function.     

Reducing greenhouse gas emissions and adapting agricultural management for climate change in developing countries: providing the basis for action

Agriculture in developing countries has attracted increasing attention in international negotiations within the United Nations Framework Convention on Climate Change for both adaptation to climate change and greenhouse gas mitigation. However, there is limited understanding about potential complementarity between management practices that promote adaptation and mitigation, and limited basis to account for greenhouse gas emission reductions in this sector. The good news is that the global research community could provide the support needed to address these issues through further research linking adaptation and mitigation. In addition, a small shift in strategy by the Intergovernmental Panel on Climate Change (IPCC) and ongoing assistance from agricultural organizations could produce a framework to move the research and development from concept to reality. In turn, significant progress is possible in the near term providing the basis for UNFCCC negotiations to move beyond discussion to action for the agricultural sector in developing countries.