A distinct isoform of lymphoid enhancer binding factor 1 (LEF1) epigenetically restricts EBV reactivation to maintain viral latency.

As a human tumor virus, EBV is present as a latent infection in its associated malignancies where genetic and epigenetic changes have been shown to impede cellular differentiation and viral reactivation. We reported previously that levels of the Wnt signaling effector, lymphoid enhancer binding factor 1 (LEF1) increased following EBV epithelial infection and an epigenetic reprogramming event was maintained even after loss of the viral genome. Elevated LEF1 levels are also observed in nasopharyngeal carcinoma and Burkitt lymphoma. To determine the role played by LEF1 in the EBV life cycle, we used in silico analysis of EBV type 1 and 2 genomes to identify over 20 Wnt-response elements, which suggests that LEF1 may bind directly to the EBV genome and regulate the viral life cycle. Using CUT&RUN-seq, LEF1 was shown to bind the latent EBV genome at various sites encoding viral lytic products that included the immediate early transactivator BZLF1 and viral primase BSLF1 genes. The LEF1 gene encodes various long and short protein isoforms. siRNA depletion of specific LEF1 isoforms revealed that the alternative-promoter derived isoform with an N-terminal truncation (ΔN LEF1) transcriptionally repressed lytic genes associated with LEF1 binding. In addition, forced expression of the ΔN LEF1 isoform antagonized EBV reactivation. As LEF1 repression requires histone deacetylase activity through either recruitment of or direct intrinsic histone deacetylase activity, siRNA depletion of LEF1 resulted in increased histone 3 lysine 9 and lysine 27 acetylation at LEF1 binding sites and across the EBV genome. Taken together, these results indicate a novel role for LEF1 in maintaining EBV latency and restriction viral reactivation via repressive chromatin remodeling of critical lytic cycle factors.

Epstein-Barr virus stably confers an invasive phenotype to epithelial cells through reprogramming of the WNT pathway.

Epstein-Barr virus (EBV)-associated carcinomas, such as nasopharyngeal carcinoma (NPC), exhibit an undifferentiated and metastatic phenotype. To determine viral contributions involved in the invasive phenotype of EBV-associated carcinomas, EBV-infected human telomerase-immortalized normal oral keratinocytes (NOK) were investigated. EBV-infected NOK were previously shown to undergo epigenetic reprogramming involving CpG island hypermethylation and delayed responsiveness to differentiation. Here, we show that EBV-infected NOK acquired an invasive phenotype that was epigenetically retained after viral loss. The transcription factor lymphoid enhancer factor 1 (LEF1) and the secreted ligand WNT5A, expressed in NPC, were increased in EBV-infected NOK with sustained expression for more than 20 passages after viral loss. Increased LEF1 levels involved four LEF1 variants, and EBV-infected NOK showed a lack of responsiveness to ß-catenin activation. Although forced expression of WNT5A and LEF1 enhanced the invasiveness of parental NOK, LEF1 knockdown reversed the invasive phenotype of EBV-infected NOK in the presence of WNT5A. Viral reprogramming of LEF1 and WNT5A was observed several passages after EBV infection, suggesting that LEF1 and WNT5A may provide a selective advantage to virally-infected cells. Our findings suggest that EBV epigenetically reprogrammed epithelial cells with features of basal, wound healing keratinocytes, with LEF1 contributing to the metastatic phenotype of EBV-associated carcinomas.

Saccharomyces cerevisiae Mhr1 can bind Xho I-induced mitochondrial DNA double-strand breaks in vivo.

Mitochondrial DNA (mtDNA) double-strand break (DSB) repair is essential for maintaining mtDNA integrity, but little is known about the proteins involved in mtDNA DSB repair. Here, we utilize Saccharomyces cerevisiae as a eukaryotic model to identify proteins involved in mtDNA DSB repair. We show that Mhr1, a protein known to possess homologous DNA pairing activity in vitro, binds to mtDNA DSBs in vivo, indicating its involvement in mtDNA DSB repair. Our data also indicate that Yku80, a protein previously implicated in mtDNA DSB repair, does not compete with Mhr1 for binding to mtDNA DSBs. In fact, C-terminally tagged Yku80 could not be detected in yeast mitochondrial extracts. Therefore, we conclude that Mhr1, but not Yku80, is a potential mtDNA DSB repair factor in yeast.

Evidence for double-strand break mediated mitochondrial DNA replication in Saccharomyces cerevisiae.

The mechanism of mitochondrial DNA (mtDNA) replication in Saccharomyces cerevisiae is controversial. Evidence exists for double-strand break (DSB) mediated recombination-dependent replication at mitochondrial replication origin ori5 in hypersuppressive ρ- cells. However, it is not clear if this replication mode operates in ρ+ cells. To understand this, we targeted bacterial Ku (bKu), a DSB binding protein, to the mitochondria of ρ+ cells with the hypothesis that bKu would bind persistently to mtDNA DSBs, thereby preventing mtDNA replication or repair. Here, we show that mitochondrial-targeted bKu binds to ori5 and that inducible expression of bKu triggers petite formation preferentially in daughter cells. bKu expression also induces mtDNA depletion that eventually results in the formation of ρ0 cells. This data supports the idea that yeast mtDNA replication is initiated by a DSB and bKu inhibits mtDNA replication by binding to a DSB at ori5, preventing mtDNA segregation to daughter cells. Interestingly, we find that mitochondrial-targeted bKu does not decrease mtDNA content in human MCF7 cells. This finding is in agreement with the fact that human mtDNA replication, typically, is not initiated by a DSB. Therefore, this study provides evidence that DSB-mediated replication is the predominant form of mtDNA replication in ρ+ yeast cells.

Regulation of mitochondrial structure and function by protein import: A current review.

By generating the majority of a cell's ATP, mitochondria permit a vast range of reactions necessary for life. Mitochondria also perform other vital functions including biogenesis and assembly of iron-sulfur proteins, maintenance of calcium homeostasis, and activation of apoptosis. Accordingly, mitochondrial dysfunction has been linked with the pathology of many clinical conditions including cancer, type 2 diabetes, cardiomyopathy, and atherosclerosis. The ongoing maintenance of mitochondrial structure and function requires the import of nuclear-encoded proteins and for this reason, mitochondrial protein import is indispensible for cell viability. As mitochondria play central roles in determining if cells live or die, a comprehensive understanding of mitochondrial structure, protein import, and function is necessary for identifying novel drugs that may destroy harmful cells while rescuing or protecting normal ones to preserve tissue integrity. This review summarizes our current knowledge on mitochondrial architecture, mitochondrial protein import, and mitochondrial function. Our current comprehension of how mitochondrial functions maintain cell homeostasis and how cell death occurs as a result of mitochondrial stress are also discussed.

Exposure of Mycobacterium marinum to low-shear modeled microgravity: effect on growth, the transcriptome and survival under stress.

Waterborne pathogenic mycobacteria can form biofilms, and certain species can cause hard-to-treat human lung infections. Astronaut health could therefore be compromised if the spacecraft environment or water becomes contaminated with pathogenic mycobacteria. This work uses Mycobacterium marinum to determine the physiological changes in a pathogenic mycobacteria grown under low-shear modeled microgravity (LSMMG). M. marinum were grown in high aspect ratio vessels (HARVs) using a rotary cell culture system subjected to LSMMG or the control orientation (normal gravity, NG) and the cultures used to determine bacterial growth, bacterium size, transcriptome changes, and resistance to stress. Two exposure times to LSMMG and NG were examined: bacteria were grown for ~40 h (short), or 4 days followed by re-dilution and growth for ~35 h (long). M. marinum exposed to LSMMG transitioned from exponential phase earlier than the NG culture. They were more sensitive to hydrogen peroxide but showed no change in resistance to gamma radiation or pH 3.5. RNA-Seq detected significantly altered transcript levels for 562 and 328 genes under LSMMG after short and long exposure times, respectively. Results suggest that LSMMG induced a reduction in translation, a downregulation of metabolism, an increase in lipid degradation, and increased chaperone and mycobactin expression. Sigma factor H (sigH) was the only sigma factor transcript induced by LSMMG after both short and long exposure times. In summary, transcriptome studies suggest that LSMMG may simulate a nutrient-deprived environment similar to that found within macrophage during infection. SigH is also implicated in the M. marinum LSMMG transcriptome response.

Iron status, anemia, and plasma erythropoietin levels in acute and chronic mouse models of colitis.

BACKGROUND: Approximately one-half of patients with inflammatory bowel disease (IBD) suffer from anemia, with the most prevalent cause being iron deficiency. Accompanying the anemia are increases in erythropoietin, a plasma protein that can initiate the feedback production of new red blood cells. Anemia also occurs in animal models that are used to investigate the mechanisms of IBD; however, the extent to which iron deficiency produces the anemia in these animal models is unknown. Also unknown in the different animal models of IBD is whether the anemia upregulates the production of erythropoietin or, alternatively, whether a decrease in erythropoietin contributes to the induction of anemia. METHODS: Two mouse models of colitis were used in this study: (1) acute 6-day ingestion of dextran sodium sulfate and (2) T-cell transfer into lymphopenic recipient mice. Measurements included indices of colitis severity, hematocrit, blood hemoglobin, plasma erythropoietin, serum iron concentration, plasma iron-binding capacities, transferrin saturation, and tissue iron concentrations. RESULTS: Both models of colitis induced significant decreases in hematocrit, blood hemoglobin, and transferrin saturation, with the spleen and liver showing a decrease in iron content in the T-cell transfer model. Additionally, both models of colitis demonstrated significant increases in plasma erythropoietin and plasma iron-binding capacities. CONCLUSIONS: The measurements of iron, whether in acute (dextran sodium sulfate) or chronic (T-cell transfer) models of colitis, were generally consistent with iron-deficient anemia, with large increases in erythropoietin indicative of tissue hypoxia. These changes in animal models of colitis are similar to those found in human IBD.