Epigenetic control of rDNA loci in response to intracellular energy status.

Intracellular energy balance is important for cell survival. In eukaryotic cells, the most energy-consuming process is ribosome biosynthesis, which adapts to changes in intracellular energy status. However, the mechanism that links energy status and ribosome biosynthesis is largely unknown. Here, we describe eNoSC, a protein complex that senses energy status and controls rRNA transcription. eNoSC contains Nucleomethylin, which binds histone H3 dimethylated Lys9 in the rDNA locus, in a complex with SIRT1 and SUV39H1. Both SIRT1 and SUV39H1 are required for energy-dependent transcriptional repression, suggesting that a change in the NAD(+)/NADH ratio induced by reduction of energy status could activate SIRT1, leading to deacetylation of histone H3 and dimethylation at Lys9 by SUV39H1, thus establishing silent chromatin in the rDNA locus. Furthermore, eNoSC promotes restoration of energy balance by limiting rRNA transcription, thus protecting cells from energy deprivation-dependent apoptosis. These findings provide key insight into the mechanisms of energy homeostasis in cells.

PPARgamma ligands suppress the feedback loop between E2F2 and cyclin-E1.

PPARgamma is a nuclear hormone receptor that plays a key role in the induction of peroxisome proliferation. A number of studies showed that PPARgamma ligands suppress cell cycle progression; however, the mechanism remains to be determined. Here, we showed that PPARgamma ligand troglitazone inhibited G1/S transition in colon cancer cells, LS174T. Troglitazone did not affect on either expression of CDK inhibitor (p18) or Wnt signaling pathway, indicating that these pathways were not involved in the troglitazone-dependent cell cycle arrest. GeneChip and RT-PCR analyses revealed that troglitazone decreased mRNA levels of cell cycle regulatory factors E2F2 and cyclin-E1 whose expression is activated by E2F2. Down-regulation of E2F2 by troglitazone results in decrease of cyclin-E1 transcription, which could inhibit phosphorylation of Rb protein, and consequently evoke the suppression of E2F2 transcriptional activity. Thus, we propose that troglitazone suppresses the feedback loop containing E2F2, cyclin-E1, and Rb protein.

Regulation of clathrin-mediated endocytosis by p53.

The p53 gene encodes a multi-functional protein to prevent tumorigenesis. Although there have been many reports of the nuclear functions of p53, little is known about the cytosolic functions of p53. Here, we found that p53 is present in cytosol as well as nuclei under unstressed conditions and binds to clathrin heavy chain (CHC). CHC is known to play a role in receptor-mediated endocytosis. Based on our findings, we examined the effect of p53 on clathrin-mediated endocytosis of epidermal growth factor receptor (EGFR). Surprisingly, p53 co-localized with CHC at the plasma membrane in response to EGF stimulation. In cells with ablated p53 expression by RNAi, EGFR internalization was delayed and intracellular signaling from EGFR was altered. Thus, our findings provide evidence that cytosolic p53 may participate in the regulation of clathrin-mediated endocytosis to control the correct signaling from EGFR.

Requirement of clathrin heavy chain for p53-mediated transcription.

The p53 protein is a transcription factor that activates various genes responsible for growth arrest and/or apoptosis in response to DNA damage. Here, we report that clathrin heavy chain (CHC) binds to p53 and contributes to p53-mediated transcription. CHC is known to be a cytosolic protein that functions as a vesicle transporter. We found, however, that CHC exists not only in cytosol but also in nuclei. CHC expression enhances p53-dependent transactivation, whereas the reduction of CHC expression by RNA interference (RNAi) attenuates its transcriptional activity. Moreover, CHC binds to the p53-responsive promoter in vivo and stabilizes p53-p300 interaction to promote p53-mediated transcription. Thus, nuclear CHC is required for the transactivation of p53 target genes and plays a distinct role from clathrin-mediated endocytosis.

Augmentation of NK cell-mediated cytotoxicity to tumor cells by inhibitory NK cell receptor blockers.

NK cells monitor expression of MHC class I by inhibitory receptors and preferentially kill cells that lose or down-regulate MHC class I expression. One possible mechanism by which tumor cells evade NK cell killing is continued expression of appropriate MHC class I ligands to engage inhibitory receptors on NK cells. We show here that small-mol.-wt blockers against the mouse inhibitory NK cell receptor Ly49A enhance NK cell killing of such tumor cells. We identified Ly49A-binding peptides by selecting phages with the capacity to bind recombinant Ly49A expressed in Escherichia coli from a phage display random peptide library. The Ly49A-binding peptides could also bind Ly49A expressed on mammalian cells. Importantly, the Ly49A-binding peptides blocked Ly49A recognition of its MHC class I ligands H-2Dd and H-2Dk. Moreover, blockade of Ly49A by the peptides enhanced cytotoxicity of Ly49A+ NK cells towards H-2Dd-expressing tumor cells. These results clearly indicate effectiveness of small-mol.-wt blockers of inhibitory NK cell receptors in enhancing NK cell-mediated killing of tumor cells that are otherwise resistant because of MHC class I expression.