The A-type lamins are constituents of the nuclear lamina in differentiated cells and have been proposed to play an important role in nuclear organization. In this study, we isolated and characterized a genomic clone containing the putative promoter region of the rat lamin A gene. Sequence analysis of about 2 kb of this region combined with primer extension data revealed the presence of a TATA box at -33, a GC box at -101, and AP1 motifs at -7, -424, and -1677. Deletion analysis of the promoter fragments in three mammalian cell lines indicated that a 221-bp segment of the proximal promoter containing the GC box and AP1 motif at -7 was sufficient to give high levels of luciferase activity in reporter gene assays. Mutations in these two motifs resulted in considerable loss of reporter gene activity. Analysis by electrophoretic mobility shift assays (EMSAs) has provided evidence for specific binding of the AP1 and Sp1 family of transcription factors to the promoter, a conclusion supported by DNase I footprinting data. This characterization of the 5' promoter region of the lamin A gene should afford a basis for the further clarification of the mechanism of regulation of this important gene during growth and development.

Lamins are major structural proteins of the nucleus and are essential for nuclear integrity and organization of nuclear functions. Mutations in the human lamin genes lead to highly degenerative genetic diseases that affect a number of different tissues such as muscle, adipose or neuronal tissues, or cause premature ageing syndromes. New findings on the role of lamins in cellular signalling pathways, as well as in ubiquitin-mediated proteasomal degradation, have given important insights into possible mechanisms of pathogenesis.

The A-type lamins are localized in the interior of the nucleus as well as on the nuclear periphery. In this study, we have characterized a monoclonal antibody LA-2F9 produced against recombinant rat lamin A which stains a subpopulation of various cell types in a pattern of small nucleoplasmic foci that are unusually susceptible to mild detergent/salt extraction. The specific reactivity of mAb LA-2F9 towards lamins was confirmed by immunoblotting of HeLa and C3H10T(1/2) whole cell lysates and nuclear lysates. The epitope for LA-2F9 was narrowed down to amino acid residues 268-278 (SAKLDNARQSA). To check whether the appearance of lamin foci was cell-cycle-dependent, C3H10T(1/2) cells were serum-starved and then refed to trigger cells to enter the G(1) phase of the cell-cycle. The intensity of staining increased 3.5-fold within 6 h of refeeding, when the maximum number of cells were labeled with LA-2F9. We also checked whether the LA-2F9 foci colocalized with nuclear proteins known to be distributed in small foci such as hnRNPs, snRNPs, SC-35, and p80 coilin, but did not find evidence of colocalization. Our studies suggest that LA-2F9 has a novel and specific reactivity towards detergent-susceptible lower order lamin structures that are likely to be assembly intermediates.

Lamin A is a major component of the nuclear lamina that is expressed in various types of differentiated cells. We have analysed previously the putative promoter sequences of the gene and shown that the rat lamin A proximal promoter contains two essential motifs, a GC box that can bind to Sp1 and Sp3, and an AP-1 motif that can bind to c-Jun and c-Fos. In this study we have investigated the role of Sp1 and Sp3 in transactivation of the promoter. Functional analysis of the promoter in Drosophila SL2 cells has demonstrated that it is inactive in the absence of Sp proteins. Activation by expression of Sp3 is more pronounced than that by Sp1 although both proteins can bind to the GC box in vitro; activation clearly depends on an intact GC box as deduced from mutant analysis. Promoter activity in SL2 cells also requires an intact AP-1 motif, which can bind to endogenous Drosophila Jun and Fos proteins. Furthermore, overexpression of c-Jun and c-Fos results in fourfold activation of the promoter in PCC-4 embryonal carcinoma cells. Our demonstration that activation of the lamin A proximal promoter is mediated by Sp3 and AP-1 transcription factors affords a basis for further studies on the regulation of this important gene during development and disease.Keywords: AP-1 transcription factor; Lamin A promoter; nuclear lamina; Sp3 transcription factor.The lamins are components of the nuclear lamina, a filamentous network of proteins underlying the inner nuclear membrane. The lamina plays an important role in nuclear assembly and disassembly during mitosis and has been proposed to be involved in the spatial organization of the nucleus [1±3]. Two major kinds of lamins are present in higher vertebrates. The B-type lamins (B1 and B2) are found in nearly all somatic cells, whereas the A-type lamins (A and C) are expressed primarily in differentiated cells, in a stage-specific manner depending upon the cell lineage (reviewed in [4]). Lamins A and C are alternatively spliced products of the LMNA gene, whereas lamins B1 and B2 are encoded by separate genes. Germ cells contain additional splice variants of the lamin A and B2 genes. The differential regulation of lamin expression in different tissues has important implications for lamin function, as mutations in the LMNA gene have recently been shown to lead to complex disease phenotypes, affecting mainly muscle, cardiac and adipose tissues [5±7].The expression of the LMNA gene is primarily under transcriptional control [8±10]. Maximal levels of lamin A and C RNAs are observed in dividing, differentiated cells, and these are reduced to barely detectable amounts in quiescent cells [8]. Hence the expression of the gene depends on both the growth and the differentiation status of the cell. As an initial approach to understanding the mechanism of regulation of the LMNA gene, we have focussed our attention on the activation of the rat lamin A proximal promoter. We have reported previously the isolation of the 5 H upstream region of the rat LMNA gene, and the characterization of the minimal...

SummaryC3G (RAPGEF1), engaged in multiple signaling pathways, is essential for the early development of the mouse. In this study, we have examined its role in mouse embryonic stem cell self-renewal and differentiation. C3G null cells generated by CRISPR mediated knock-in of a targeting vector exhibited enhanced clonogenicity and long-term self-renewal. They did not differentiate in response to LIF withdrawal when compared to the wild type ES cells and were defective for lineage commitment upon teratoma formation in vivo. Gene expression analysis of C3G KO cells showed misregulated expression of a large number of genes compared with WT cells. They express higher levels of self-renewal factors like KLF4 and ESRRB and show high STAT3 activity, and very low ERK activity compared to WT cells. Reintroduction of C3G expression in a KO line partially reverted expression of ESRRB, and KLF4, and ERK activity similar to that seen in WT cells. The expression of self-renewal factors was persistent for a longer time, and induction of lineage-specific markers was not seen when C3G KO cells were induced to form embryoid bodies. C3G KO cells showed poor adhesion and significantly reduced levels of pFAK, pPaxillin, and Integrin-β1, in addition to downregulation of the cluster of genes involved in cell adhesion, compared to WT cells. Our results show that C3G is essential for the regulation of STAT3, ERK, and adhesion signaling, to maintain pluripotency of mouse embryonic stem cells and enable their lineage commitment for differentiation. Graphical abstract