Allon Canaan

EBNA1 regulates cellular gene expression by binding cellular promoters

Epstein–Barr virus (EBV) is associated with several types of lymphomas and epithelial tumors including Burkitts lymphoma (BL), HIV-associated lymphoma, posttransplant lymphoproliferative disorder, and nasopharyngeal carcinoma. EBV nuclear antigen 1 (EBNA1) is expressed in all EBV associated tumors and is required for latency and transformation. EBNA1 initiates latent viral replication in B cells, maintains the viral genome copy number, and regulates transcription of other EBV-encoded latent genes. These activities are mediated through the ability of EBNA1 to bind viral-DNA. To further elucidate the role of EBNA1 in the host cell, we have examined the effect of EBNA1 on cellular gene expression by microarray analysis using the B cell BJAB and the epithelial 293 cell lines transfected with EBNA1. Analysis of the data revealed distinct profiles of cellular gene changes in BJAB and 293 cell lines. Subsequently, chromatin immune-precipitation revealed a direct binding of EBNA1 to cellular promoters. We have correlated EBNA1 bound promoters with changes in gene expression. Sequence analysis of the 100 promoters most enriched revealed a DNA motif that differs from the EBNA1 binding site in the EBV genome.

Werner protein protects nonproliferating cells from oxidative DNA damage

ABSTRACT Werner syndrome, caused by mutations of the WRN gene, mimics many changes of normal aging. Although roles for WRN protein in DNA replication, recombination, and telomere maintenance have been suggested, the pathology of rapidly dividing cells is not a feature of Werner syndrome. To identify cellular events that are specifically vulnerable to WRN deficiency, we used RNA interference (RNAi) to knockdown WRN or BLM (the RecQ helicase mutated in Bloom syndrome) expression in primary human fibroblasts. Withdrawal of WRN or BLM produced accelerated cellular senescence phenotype and DNA damage response in normal fibroblasts, as evidenced by induction of γH2AX and 53BP1 nuclear foci. After WRN depletion, the induction of these foci was seen most prominently in nondividing cells. Growth in physiological (3%) oxygen or in the presence of an antioxidant prevented the development of the DNA damage foci in WRN-depleted cells, whereas acute oxidative stress led to inefficient repair of the lesions. Furthermore, WRN RNAi-induced DNA damage was suppressed by overexpression of the telomere-binding protein TRF2. These conditions, however, did not prevent the DNA damage response in BLM-ablated cells, suggesting a distinct role for WRN in DNA homeostasis in vivo. Thus, manifestations of Werner syndrome may reflect an impaired ability of slowly dividing cells to limit oxidative DNA damage.

FAT10/Diubiquitin-Like Protein-Deficient Mice Exhibit Minimal Phenotypic Differences

ABSTRACT The FAT10 gene encodes a diubiquitin-like protein containing two tandem head-to-tail ubiquitin-like domains. There is a high degree of similarity between murine and human FAT10 sequences at both the mRNA and protein levels. In various cell lines, FAT10 expression was shown to be induced by gamma interferon or by tumor necrosis factor alpha. In addition, FAT10 expression was found to be up-regulated in some Epstein-Barr virus-infected B-cell lines, in activated dendritic cells, and in several epithelial tumors. However, forced expression of FAT10 in cultured cells was also found to produce apoptotic cell death. Overall, these findings suggest that FAT10 may modulate cellular growth or cellular viability. Here we describe the steps to generate, by genetic targeting, a FAT10 gene knockout mouse model. The FAT10 knockout homozygous mice are viable and fertile. No gross lesions or obvious histological differences were found in these mutated mice. Examination of lymphocyte populations from spleen, thymus, and bone marrow did not reveal any abnormalities. However, flow cytometry analysis demonstrated that the lymphocytes of FAT10 knockout mice were, on average, more prone to spontaneous apoptotic death. Physiologically, these mice demonstrated a high level of sensitivity toward endotoxin challenge. These findings indicate that FAT10 may function as a survival factor.

The Ubiquitin-Like Protein FAT10 Mediates NF-κB Activation

NF-kappaB is a central mediator of innate immunity and contributes to the pathogenesis of several renal diseases. FAT10 is a TNF-alpha-inducible ubiquitin-like protein with a putative role in immune response, but whether FAT10 participates in TNF-alpha-induced NF-kappaB activation is unknown. Here, using renal tubular epithelial cells (RTECs) derived from FAT10(-/-) and FAT10(+/+) mice, we observed that FAT10 deficiency abrogated TNF-alpha-induced NF-kappaB activation and reduced the induction of NF-kappaB-regulated genes. Despite normal IkBalpha degradation and polyubiquitination, FAT10 deficiency impaired TNF-alpha-induced IkBalpha degradation and nuclear translocation of p65 in RTECs, suggesting defective proteasomal degradation of polyubiquitinated IkBalpha. In addition, FAT10 deficiency reduced the expression of the proteasomal subunit low molecular mass polypeptide 2 (LMP2). Transduction of FAT10(-/-) RTECs with FAT10 restored LMP2 expression, TNF-alpha-induced IkBalpha degradation, p65 nuclear translocation, and NF-kappaB activation. Furthermore, LMP2 transfection restored IkBalpha degradation in FAT10(-/-) RTECs. In humans, common types of chronic kidney disease associated with tubulointerstitial upregulation of FAT10. These data suggest that FAT10 mediates NF-kappaB activation and may promote tubulointerstitial inflammation in chronic kidney diseases.

The ubiquitin-like modifier FAT10 decorates autophagy-targeted Salmonella and contributes to Salmonella resistance in mice.

ABSTRACT Bacterial invasion of eukaryotic cells is counteracted by cell-autonomous innate immune mechanisms including xenophagy. The decoration of cytosolic bacteria by ubiquitylation and binding of galectin-8 leads to recruitment of autophagy adaptors like p62 (also known as SQSTM1), NDP52 (also known as CALCOCO2) and optineurin, which initiate the destruction of bacteria by xenophagy. Here, we show that the functionally barely characterized IFN&ggr;- and TNF&agr;-inducible ubiquitin-like modifier FAT10 (also known as ubiquitin D, UBD), which binds to the autophagy adaptor p62, but has not been shown to associate with pathogens before, is recruited to cytosolic Salmonella Typhimurium in human cells. FAT10-decorated S. Typhimurium were simultaneously decorated with ubiquitin, p62, NDP52 and the autophagy marker LC3B (MAP1LC3B). FAT10 colocalized with p62-positive microdomains on S. Typhimurium, whereas colocalization with NDP52 was only partial. A kinetic analysis revealed an early, but only transient, decoration of bacteria by FAT10, which resembled that of p62. Although bacterial replication was not detectably altered in FAT10-depleted or overexpressing cells in vitro, survival experiments revealed that NRAMP1-transgenic mice that were FAT10-deficient had a higher susceptibility to orally inoculated S. Typhimurium bacteria than NRAMP1-transgenic mice that were wild-type for FAT10. Taken together, our data suggest a role for FAT10 in the intracellular defense against bacteria.

Extended lifespan and reduced adiposity in mice lacking the FAT10 gene.

Significance For the first time we describe a physiological role for HLA-F adjacent transcript 10 (FAT10) in metabolism, obesity, and aging in mammals. We show that FAT10 knockout prevents the development of age-associated obesity in mice while extending lifespan and vigor without the appearance of deleterious developmental effects. In addition, we did not observe an increase in cancer incidence. If the role of FAT10 in humans is similar to mice, then targeting of FAT10 may hold promising therapeutic impact for the treatment of various diseases including obesity and obesity-related diseases and aging associated diseases. The HLA-F adjacent transcript 10 (FAT10) is a member of the ubiquitin-like gene family that alters protein function/stability through covalent ligation. Although FAT10 is induced by inflammatory mediators and implicated in immunity, the physiological functions of FAT10 are poorly defined. We report the discovery that FAT10 regulates lifespan through pleiotropic actions on metabolism and inflammation. Median and overall lifespan are increased 20% in FAT10ko mice, coincident with elevated metabolic rate, preferential use of fat as fuel, and dramatically reduced adiposity. This phenotype is associated with metabolic reprogramming of skeletal muscle (i.e., increased AMP kinase activity, β-oxidation and -uncoupling, and decreased triglyceride content). Moreover, knockout mice have reduced circulating glucose and insulin levels and enhanced insulin sensitivity in metabolic tissues, consistent with elevated IL-10 in skeletal muscle and serum. These observations suggest novel roles of FAT10 in immune metabolic regulation that impact aging and chronic disease.

Homogeneous assay for detection of active Epstein-Barr nuclear antigen 1 by thrombin activity modulation.

Epstein-Barr virus (EBV) has been associated with several malignancies as Burkitts lymphoma, nasopharyngeal carcinoma, and Hodgkins disease. In those diseases, Epstein-Barr nuclear antigen 1 (EBNA-1) is constitutively expressed. Here, we reported an innovative system to detect active EBNA-1 protein in a homogeneous assay. The system is based on the modulation of thrombin activity by a self-complementary single stranded DNA (scssDNA), which was designed and synthesized to mimic the palindromic target sites of EBNA-1 in the EBV genome. This model system showed a limit of detection of 3.75 ng mL(-1) of active EBNA-1 protein with a dynamic detection range from 3.75 to 250 ng mL(-1) with a correlation coefficient of 0.997. This new homogeneous assay for active EBNA-1 protein detection and quantification provides a very useful tool for rapid screening of EBNA-1 blockers in biomedical research.