In addition to coordinating immune and inflammatory responses, NF-kappaB/Rel transcription factors control cell survival. Normally, NF-kappaB dimers are sequestered in the cytoplasm by binding to inhibitory IkappaB proteins, and can be activated rapidly by signals that induce the sequential phosphorylation and proteolysis of IkappaBs. Activation of NF-kappaB antagonizes apoptosis or programmed cell death by numerous triggers, including the ligand engagement of 'death receptors' such as tumour-necrosis factor (TNF) receptor. The anti-apoptotic activity of NF-kappaB is also crucial to oncogenesis and to chemo- and radio-resistance in cancer. Cytoprotection by NF-kappaB involves the activation of pro-survival genes; however, its basis remains poorly understood. Here we report that NF-kappaB complexes downregulate the c-Jun amino-terminal kinase (JNK) cascade, thus establishing a link between the NF-kappaB and the JNK pathways. This link involves the transcriptional upregulation of gadd45beta/myd118 (ref. 4), which downregulates JNK signalling induced by the TNF receptor (TNF-R). This NF-kappaB-dependent inhibition of the JNK pathway is central to the control of cell death. Our findings define a protective mechanism that is mediated by NF-kappaB complexes and establish a role for the persistent activation of JNK in the apoptotic response to TNF-alpha.

During inflammation, NF-kappaB transcription factors antagonize apoptosis induced by tumor necrosis factor (TNF)alpha. This antiapoptotic activity of NF-kappaB involves suppressing the accumulation of reactive oxygen species (ROS) and controlling the activation of the c-Jun N-terminal kinase (JNK) cascade. However, the mechanism(s) by which NF-kappaB inhibits ROS accumulation is unclear. We identify ferritin heavy chain (FHC)--the primary iron storage factor--as an essential mediator of the antioxidant and protective activities of NF-kappaB. FHC is induced downstream of NF-kappaB and is required to prevent sustained JNK activation and, thereby, apoptosis triggered by TNFalpha. FHC-mediated inhibition of JNK signaling depends on suppressing ROS accumulation and is achieved through iron sequestration. These findings establish a basis for the NF-kappaB-mediated control of ROS induction and identify a mechanism by which NF-kappaB suppresses proapoptotic JNK signaling. Our results suggest modulation of FHC or, more broadly, of iron metabolism as a potential approach for anti-inflammatory therapy.

Ribosomes are specialized entities that participate in regulation of gene expression through their rRNAs carrying ribozyme activity. Ribosome biogenesis is overactivated in p53-inactivated cancer cells, although involvement of p53 on ribosome quality is unknown. Here, we show that p53 represses expression of the rRNA methyl-transferase fibrillarin (FBL) by binding directly to FBL. High levels of FBL are accompanied by modifications of the rRNA methylation pattern, impairment of translational fidelity, and an increase of internal ribosome entry site (IRES)-dependent translation initiation of key cancer genes. FBL overexpression contributes to tumorigenesis and is associated with poor survival in patients with breast cancer. Thus, p53 acts as a safeguard of protein synthesis by regulating FBL and the subsequent quality and intrinsic activity of ribosomes.

Hhex is required for early development of the liver. A null mutation of Hhex results in a failure to form the liver bud and embryonic lethality. Therefore, Hhex null mice are not informative as to whether this gene is required during later stages of hepatobiliary morphogenesis. To address this question, we derived Hhex conditional null mice using the Cre-loxP system and two different Cre transgenics (Foxa3-Cre and Alfp-Cre). Deletion of Hhex in the hepatic diverticulum (Foxa3-Cre;Hhex(d2,3/-)) led to embryonic lethality and resulted in a small and cystic liver with loss of Hnf4alpha and Hnf6 expression in early hepatoblasts. In addition, the gall bladder was absent and the extrahepatic bile duct could not be identified. Loss of Hhex in the embryonic liver (Alfp-Cre;Hhex(d2,3/-)) caused irregular development of intrahepatic bile ducts and an absence of Hnf1beta in many (cystic) biliary epithelial cells, which resulted in a slow, progressive form of polycystic liver disease in adult mice. Thus, we have shown that Hhex is required during multiple stages of hepatobiliary development. The altered expression of Hnf4alpha, Hnf6 and Hnf1beta in Hhex conditional null mice suggests that Hhex is an essential component of the genetic networks regulating hepatoblast differentiation and intrahepatic bile duct morphogenesis.

No abstract

Nucleolin is a multi-functional nucleolar protein that is required for ribosomal RNA gene (rRNA) transcription in vivo, but the mechanism by which nucleolin modulates RNA polymerase I (RNAPI) transcription is not well understood. Nucleolin depletion results in an increase in the heterochromatin mark H3K9me2 and a decrease in H4K12Ac and H3K4me3 euchromatin histone marks in rRNA genes. ChIP-seq experiments identified an enrichment of nucleolin in the ribosomal DNA (rDNA) coding and promoter region. Nucleolin is preferentially associated with unmethylated rRNA genes and its depletion leads to the accumulation of RNAPI at the beginning of the transcription unit and a decrease in UBF along the coding and promoter regions. Nucleolin is able to affect the binding of transcription termination factor-1 on the promoter-proximal terminator T0, thus inhibiting the recruitment of TIP5 and HDAC1 and the establishment of a repressive heterochromatin state. These results reveal the importance of nucleolin for the maintenance of the euchromatin state and transcription elongation of rDNA.

We recently reported that megalin is subjected to regulated intramembrane proteolysis (RIP) and includes 1) protein kinase C (PKC)-regulated, metalloprotease-mediated ectodomain shedding producing a membrane-bound megalin COOH-terminal fragment (MCTF) and 2) gamma-secretase-mediated cleavage of the MCTF producing a soluble megalin intracellular domain (MICD). Based on studies of RIP of other receptors, the MICD is predicted to target to the nucleus and regulate gene expression. To determine whether RIP of megalin regulates proximal tubule gene expression, we stably expressed the transfected MCTF (tMCTF) or transfected MICD (tMICD) in opossum kidney proximal tubule (OKP) cells and examined the resulting phenotype. Immunoblotting and immunocytochemical analysis of tMCTF cells showed the tMCTF was expressed and constitutively processed by gamma-secretase. Analysis of specific protein expression in tMCTF- and tMICD-transfected cells using Western blot showed endogenous megalin and Na(+)/H(+) exchanger 3 (NHE3) protein expression to be dramatically lower than that of control cells. Expression of other proteins including myosin VI, beta-adaptin, and the Na-K-ATPase appeared unchanged. Analysis of specific mRNA expression using quantitative real-time PCR showed megalin and NHE3 mRNA levels were significantly lower in tMCTF- and tMICD-transfected cells compared with controls. Inhibition of gamma-secretase activity in tMCTF cells resulted in an 8- to 10-fold recovery of megalin mRNA within 4 h. These data show that the COOH-terminal domain of megalin regulates expression of specific proteins in OKP cells and provides the first evidence that RIP of megalin may be part of a signaling pathway linking protein absorption and gene expression in proximal tubule.

BackgroundVertebrate heterochromatin contains a non-allelic variant of the histone H2A called macroH2A1, which has the characteristic of being three times the size of the canonical H2A. The macroH2A1 C-terminal extension can recruit onto chromatin the poly-ADP-ribose polymerase (PARP)1, which is crucial for DNA repair. This led to the speculation that macroH2A1 could be essential for genome surveillance; however, no experimental evidence supported this hypothesis. Because macroH2A1 has been found to be enriched on the inactive X-chromosome in females, it is thought to play a role in sex chromosome dosage compensation through its ability to regulate gene expression. However, more genetic data are needed to further understand the function of macroH2A1 in mammals.ResultsDeletion of the murine gene H2afy, which encodes for macroH2A1, resulted in lipid accumulation in liver. Hepatic steatosis caused by H2afy disruption occurred specifically in homozygous mutant females. The metabolic disorder constantly affected half of the number of homozygote females. Given the mixed genetic background of the mutants, an unreported genetic modifier is likely to influence the penetrance of the phenotype. In addition, the X-linked thyroxine-binding globulin (Tbg) gene was specifically upregulated in steatotic livers. Chromatin immunoprecitation indicated that macroH2A1 is enriched at the Tbg promoter in wild-type female animals, indicating that increased Tbg expression in H2afy null mutants is likely to be a direct consequence of the absence of macroH2A1. Furthermore, male mice, which are not prone to the metabolic disorder, had a reduced level of macroH2A1 incorporated into the Tbg promoter.ConclusionsBecause TBG is the main carrier of the thyroid hormone T4, which regulates energy metabolism, we propose that overexpression of TBG is responsible for the fat accumulation observed in H2afy-deficient liver. Moreover, our results suggest that the sexual dimorphism of the steatotic phenotype is probably due to the different incorporation of macroH2A1 in males and females. In combination with previous studies, our data demonstrate a role for macroH2A1 in regulating homeostasis in a sex-dependent manner, subject to genetic background.