Genes to Cells

The AAA⁺ superfamily of ATPases, which contain a homologous ATPase module, are found in all kingdoms of living organisms where they participate in diverse cellular processes including membrane fusion, proteolysis and DNA replication. Recent structural studies have revealed that they usually form ring‐shaped oligomers, which are crucial for their ATPase activities and mechanisms of action. These ring‐shaped oligomeric complexes are versatile in their mode of action, which collectively seem to involve some form of disruption of molecular or macromolecular structure; unfolding of proteins, disassembly of protein complexes, unwinding of DNA, or alteration of the state of DNA–protein complexes. Thus, the AAA⁺ proteins represent a novel type of molecular chaperone. Comparative analyses have also revealed significant similarities and differences in structure and molecular mechanism between AAA⁺ ATPases and other ring‐shaped ATPases.

DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b cooperatively regulate cytosine methylation in CpG dinucleotides in mammalian genomes, providing an epigenetic basis for gene silencing and maintenance of genome integrity. Proper CpG methylation is required for the normal growth of various somatic cell types, indicating its essential role in the basic cellular function of mammalian cells. Previous studies using Dnmt1^−/– or Dnmt3a^−/–Dnmt3b^−/– ES cells, however, have shown that undifferentiated embryonic stem (ES) cells can tolerate hypomethylation for their proliferation. In an attempt to investigate the effects of the complete loss of CpG DNA methyltransferase function, we established mouse ES cells lacking all three of these enzymes by gene targeting. Despite the absence of CpG methylation, as demonstrated by genome‐wide methylation analysis, these triple knockout (TKO) ES cells grew robustly and maintained their undifferentiated characteristics. TKO ES cells retained pericentromeric heterochromatin domains marked with methylation at Lys9 of histone H3 and heterochromatin protein‐1, and maintained their normal chromosome numbers. Our results indicate that ES cells can maintain stem cell properties and chromosomal stability in the absence of CpG methylation and CpG DNA methyltransferases.

The superoxide‐producing NAD(P)H oxidase Nox4 was initially identified as an enzyme that is highly expressed in the kidney and is possibly involved in oxygen sensing and cellular senescence. Although the oxidase is also abundant in vascular endothelial cells, its role remains to be elucidated. Here we show that Nox4 preferentially localizes to the nucleus of human umbilical vein endothelial cells (HUVECs), by immunocytochemistry and immunoelectron microscopy using three kinds of affinity‐purified antibodies raised against distinct immunogens from human Nox4. Silencing of Nox4 by RNA interference (RNAi) abrogates nuclear signals given with the antibodies, confirming the nuclear localization of Nox4. The nuclear fraction of HUVECs exhibits an NAD(P)H‐dependent superoxide‐producing activity in a manner dependent on Nox4, which activity can be enhanced upon cell stimulation with phorbol 12‐myristate 13‐acetate. This stimulant also facilitates gene expression as estimated in the present transfection assay of HUVECs using a reporter regulated by the Maf‐recognition element MARE, a DNA sequence that constitutes a part of oxidative stress response. Both basal and stimulated transcriptional activities are impaired by RNAi‐mediated Nox4 silencing. Thus Nox4 appears to produce superoxide in the nucleus of HUVECs, thereby regulating gene expression via a mechanism for oxidative stress response.

Background: The mononegavirus superfamily (Mononegavirales) comprises three families, Rhabdoviridae, Paramyxoviridae and Filoviridae. These viruses possess a single stranded negative sense RNA as the genome. Recent success in the recovery of infectious virus from a transfected cDNA of mononegaviruses including Sendai virus, a prototypic paramyxovirus, is opening the possibility of their genetic engineering. However, infectious viruses have been recovered only by initiating the infectious cycle with cDNA directing the synthesis of antigenomic positive sense (+) RNA. Starting with genomic negative sense (−) RNA has been unsuccessful. Furthermore, the recovery efficiency has often been extremely low.

Results: We describe here an analogous system that allows recovery of Sendai virus at a high rate, from cells in which the transfected cDNA and plasmids to support the synthesis of viral nucleocapsid protein and RNA polymerases are coexpressed by vaccinia virus‐driven bacteriophage T7 polymerase. Our system was able to recover the virus from cDNA directing not only (+)RNA but also (−)RNA. Moreover, using this system, we succeeded in recovery of the virus by transfection of in vitro synthesized (+)RNA or (−)RNA. This improved virus recovery appeared to be accomplished by supplying the supporting plasmids at an optimal ratio and by minimizing the cytopathic effect of the vaccinia virus by specific inhibitors. In addition, it was probably critical that our cDNAs were constructed to generate viral authentic RNAs without adding T7 promoter‐specific nucleotides to the 5′ ends. An immediate application of the system was demonstrated by the creation of a candidate vaccine strain with a predetermined attenuating mutation in the cleavage‐activation site of the viral fusion glycoprotein.

Conclusion: We have established methods which greatly improve the recovery of Sendai virus from cDNA. There is essentially no absolute obstacle to recovery of the virus from the (−)RNA template. Even the complete full length RNA chain in the naked form appears to be properly encapsidated to become a functional template.

Communication between cell surface receptors and nuclear transcription factors is of primary importance to multicellular organisms. Since there are numerous molecules involved in this process, their mutual interaction forms complex networks of informational regulation, which is still under extensive investigation. The RBP‐Jκ transcription factor interacts directly with the Notch receptor involved in cell lineage commitment, implicating the presence of a uniquely simple communication strategy between the surface receptor and the nucleus.

Autophagy is a self‐eating system conserved among eukaryotes, in which cellular components including organelles are entrapped into a double membrane structure called the autophagosome and then degraded by lysosomal hydrolases. In addition to its role in supplying amino acids in response to nutrient starvation, autophagy is involved in quality control to maintain cell health. Thus, inactivation of autophagy causes the formation of cytoplasmic protein inclusions, which comprise misfolded proteins and the accumulation of many degenerated organelles, resulting in liver injury, diabetes, myopathy and neurodegeneration. Furthermore, although autophagy has been considered nonselective, increasing evidence points to the selectivity of autophagy in sorting vacuolar enzymes and removal of aggregate‐prone proteins and unwanted organelles. Such selectivity allows diverse cellular regulation, similar to the ubiquitin proteasome pathway. In this review, we discuss the physiological roles of selective autophagy and their molecular mechanisms.

Background: Activating transcription factor‐4 (ATF4)—also termed CREB2, C/ATF, and TAXREB67—is a basic‐leucine zipper (bZip) transcription factor that belongs to the ATF/CREB family. In addition to its own family members, ATF4 can also form heterodimers with other related but distinct bZIP proteins such as the C/EBP, AP‐1 and Maf families, which may give rise to a variety of combinatorial diversity in gene regulation. In order to assess the in vivo essential role of ATF4, we have generated mice lacking ATF4 by gene targeting.

Results: ATF4‐deficient mice exhibited severe microphthalmia. Although ATF4‐deficient eyes revealed a normal gross lens structure up to embryonic day 14.5, later on the ATF4‐deficient lens, degenerated due to apoptosis without the formation of lens secondary fibre cells. Retinal development was normal in the mutant mice. The lens‐specific expression of ATF4 in the mutant mice led not only to the recovery of lens secondary fibres but also to the induction of hyperplasia of these fibres.

Conclusion: These results demonstrated that ATF4 is essential for the later stages of lens fibre cell differentiation.

Pluripotency of embryonic stem (ES) cells is maintained by a network consisting of multiple transcription factors, including Oct3/4, Sox2, Nanog, Klf4 and Sall4. Among these factors, the forced expressions of Oct3/4, Sox2 and Klf4 are sufficient to reprogram fibroblasts into induced pluripotent stem (iPS) cells. The current study analyzed the role of Sall4 during the generation of ES cells and iPS cells. The mouse Sall4 gene was deleted by homologous recombination. Sall4‐null embryos died shortly after implantation, as has been reported. ES‐like cell lines can be established from Sall4‐null blastocysts, albeit with a lower efficiency and a slower time course. The knockdown of Sall4 significantly decreased the efficiency of iPS cell generation from mouse fibroblasts. Furthermore, retroviral transduction of Sall4 significantly increased the efficiency of iPS cell generation in mouse and some human fibroblast lines. These results demonstrated that Sall4 plays positive roles in the generation of pluripotent stem cells from blastocysts and fibroblasts.