Victor L. Boyartchuk

Histone acetyltransferase Rtt109 is required for Candida albicans pathogenesis

Candida albicans is a ubiquitous opportunistic pathogen that is the most prevalent cause of hospital-acquired fungal infections. In mammalian hosts, C. albicans is engulfed by phagocytes that attack the pathogen with DNA-damaging reactive oxygen species (ROS). Acetylation of histone H3 lysine 56 (H3K56) by the fungal-specific histone acetyltransferase Rtt109 is important for yeast model organisms to survive DNA damage and maintain genome integrity. To assess the importance of Rtt109 for C. albicans pathogenicity, we deleted the predicted homolog of Rtt109 in the clinical C. albicans isolate, SC5314. C. albicans rtt109−/− mutant cells lack acetylated H3K56 (H3K56ac) and are hypersensitive to genotoxic agents. Additionally, rtt109−/− mutant cells constitutively display increased H2A S129 phosphorylation and elevated DNA repair gene expression, consistent with endogenous DNA damage. Importantly, C. albicans rtt109−/− cells are significantly less pathogenic in mice and more susceptible to killing by macrophages in vitro than are wild-type cells. Via pharmacological inhibition of the host NADPH oxidase enzyme, we show that the increased sensitivity of rtt109−/− cells to macrophages depends on the host’s ability to generate ROS, providing a mechanistic link between the drug sensitivity, gene expression, and pathogenesis phenotypes. We conclude that Rtt109 is particularly important for fungal pathogenicity, suggesting a unique target for therapeutic antifungal compounds.

Genetic variation in Mon1a affects protein trafficking and modifies macrophage iron loading in mice

We undertook a quantitative trait locus (QTL) analysis in mice to identify modifier genes that might influence the severity of human iron disorders. We identified a strong QTL on mouse chromosome 9 that differentially affected macrophage iron burden in C57BL/10J and SWR/J mice. A C57BL/10J missense allele of an evolutionarily conserved gene, Mon1a, cosegregated with the QTL in congenic mouse lines. We present evidence that Mon1a is involved in trafficking of ferroportin, the major mammalian iron exporter, to the surface of iron-recycling macrophages. Differences in amounts of surface ferroportin correlate with differences in cellular iron content. Mon1a is also important for trafficking of cell-surface and secreted molecules unrelated to iron metabolism, suggesting that it has a fundamental role in the mammalian secretory apparatus.

Loss of p53 and Ink4a/Arf cooperate in a cell autonomous fashion to induce metastasis of hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. HCC patients frequently present with disease that has metastasized to other regions of the liver, the portal vein, lymph nodes, or lungs, leading to poor prognoses. Therefore, model systems that allow exploration of the molecular mechanisms underlying metastasis in this disease are greatly needed. We describe here a metastatic HCC model generated after the somatic introduction of the mouse polyoma virus middle T antigen to mice with liver-specific deletion of the Trp53 tumor suppressor locus and show the cell autonomous effect of p53 loss of function on HCC metastasis. We additionally find that cholangiocarcinoma also develops in these mice, and some tumors display features of both HCC and cholangiocarcinoma, suggestive of origin from liver progenitor cells. Concomitant loss of the Ink4a/Arf tumor suppressor locus accelerates tumor formation and metastasis, suggesting potential roles for the p16 and p19 tumor suppressors in this process. Significantly, tumor cell lines isolated from tumors lacking both Trp53 and Ink4a/Arf display enhanced invasion activity in vitro relative to those lacking Trp53 alone. Thus, our data illustrate a new model system amenable for the analysis of HCC metastasis.

The Host Resistance Locus sst1 Controls Innate Immunity to Listeria monocytogenes Infection in Immunodeficient Mice

Epidemiological, clinical, and experimental approaches have convincingly demonstrated that host resistance to infection with intracellular pathogens is significantly influenced by genetic polymorphisms. Using a mouse model of infection with virulent Mycobacterium tuberculosis (MTB), we have previously identified the sst1 locus as a genetic determinant of host resistance to tuberculosis. In this study we demonstrate that susceptibility to another intracellular pathogen, Listeria monocytogenes, is also influenced by the sst1 locus. The contribution of sst1 to anti-listerial immunity is much greater in immunodeficient scid mice, indicating that this locus controls innate immunity and becomes particularly important when adaptive immunity is significantly depressed. Similar to our previous observations using infection with MTB, the resistant allele of sst1 prevents formation of necrotic infectious lesions in vivo. We have shown that macrophages obtained from sst1-resistant congenic mice possess superior ability to kill L. monocytogenes in vitro. The bactericidal effect of sst1 is dependent on IFN-γ activation and reactive oxygen radical production by activated macrophages after infection, but is independent of NO production. It is possible that there is a single gene that controls common IFN-dependent macrophage function, which is important in the pathogenesis of infections caused by both MTB and L. monocytogenes. However, host resistance to the two pathogens may be controlled by two different polymorphic genes encoded within the sst1 locus. The polymorphic gene(s) encoded within the sst1 locus that controls macrophage interactions with the two intracellular pathogens remains to be elucidated.

Listeria monocytogenes Infection Induces Prosurvival Metabolic Signaling in Macrophages

ABSTRACT Host cells use metabolic signaling through the LXRα nuclear receptor to defend against Listeria monocytogenes infection. 25-Hydroxycholesterol is a natural ligand of LXRs that is produced by the enzyme cholesterol 25-hydroxylase (CH25H). We found that expression of Ch25h is upregulated following L. monocytogenes infection in a beta interferon (IFN-β)-dependent fashion. Moreover, increased Ch25h expression promotes survival of L. monocytogenes-infected cells and increases sensitivity of the host to infection. We determined that expression of Cd5l, a prosurvival gene, is controlled by CH25H. In addition, we found that CD5L inhibits activation of caspase-1, promoting survival of infected macrophages. Our results reveal a mechanism by which an intracellular pathogen can prolong survival of infected cells, thus providing itself with a protected environment in which to replicate.

The Histone Demethylase Jhdm1a Regulates Hepatic Gluconeogenesis

Hepatic gluconeogenesis is required for maintaining blood glucose homeostasis; yet, in diabetes mellitus, this process is unrestrained and is a major contributor to fasting hyperglycemia. To date, the impacts of chromatin modifying enzymes and chromatin landscape on gluconeogenesis are poorly understood. Through catalyzing the removal of methyl groups from specific lysine residues in the histone tail, histone demethylases modulate chromatin structure and, hence, gene expression. Here we perform an RNA interference screen against the known histone demethylases and identify a histone H3 lysine 36 (H3K36) demethylase, Jhdm1a, as a key negative regulator of gluconeogenic gene expression. In vivo, silencing of Jhdm1a promotes liver glucose synthesis, while its exogenous expression reduces blood glucose level. Importantly, the regulation of gluconeogenesis by Jhdm1a requires its demethylation activity. Mechanistically, we find that Jhdm1a regulates the expression of a major gluconeogenic regulator, C/EBPα. This is achieved, at least in part, by its USF1-dependent association with the C/EBPα promoter and its subsequent demethylation of dimethylated H3K36 on the C/EBPα locus. Our work provides compelling evidence that links histone demethylation to transcriptional regulation of gluconeogenesis and has important implications for the treatment of diabetes.

The Classical Pink-Eyed Dilution Mutation Affects Angiogenic Responsiveness

Angiogenesis is the process by which new blood vessels are formed from existing vessels. Mammalian populations, including humans and mice, harbor genetic variations that alter angiogenesis. Angiogenesis-regulating gene variants can result in increased susceptibility to multiple angiogenesis-dependent diseases in humans. Our efforts to dissect the complexity of the genetic diversity that regulates angiogenesis have used laboratory animals due to the availability of genome sequence for many species and the ability to perform high volume controlled breeding. Using the murine corneal micropocket assay, we have observed more than ten-fold difference in angiogenic responsiveness among various mouse strains. This degree of difference is observed with either bFGF or VEGF induced corneal neovascularization. Ongoing mapping studies have identified multiple loci that affect angiogenic responsiveness in several mouse models. In this study, we used F2 intercrosses between C57BL/6J and the 129 substrains 129P1/ReJ and 129P3/J, as well as the SJL/J strain, where we have identified new QTLs that affect angiogenic responsiveness. In the case of AngFq5, on chromosome 7, congenic animals were used to confirm the existence of this locus and subcongenic animals, combined with a haplotype-based mapping approach that identified the pink-eyed dilution mutation as a candidate polymorphism to explain AngFq5. The ability of mutations in the pink-eyed dilution gene to affect angiogenic response was demonstrated using the p-J allele at the same locus. Using this allele, we demonstrate that pink-eyed dilution mutations in Oca2 can affect both bFGF and VEGF-induced corneal angiogenesis.

Genetic control of the innate immune response to Borrelia hermsii influences the course of relapsing fever in inbred strains of mice.

ABSTRACT Host susceptibility to infection is controlled in large measure by the genetic makeup of the host. Spirochetes of the genus Borrelia include nearly 40 species of vector-borne spirochetes that are capable of infecting a wide range of mammalian hosts, causing Lyme disease and relapsing fever. Relapsing fever is associated with high-level bacteremia, as well as hematologic manifestations, such as thrombocytopenia (i.e., low platelet numbers) and anemia. To facilitate studies of genetic control of susceptibility to Borrelia hermsii infection, we performed a systematic analysis of the course of infection using immunocompetent and immunocompromised inbred strains of mice. Our analysis revealed that sensitivity to B. hermsii infections is genetically controlled. In addition, whereas the role of adaptive immunity to relapsing fever-causing spirochetes is well documented, we found that innate immunity contributes significantly to the reduction of bacterial burden. Similar to human infection, the progression of the disease in mice was associated with thrombocytopenia and anemia. Histological and fluorescence in situ hybridization (FISH) analysis of infected tissues indicated that red blood cells (RBCs) were removed by tissue-resident macrophages, a process that could lead to anemia. Spirochetes in the spleen and liver were often visualized associated with RBCs, lending support to the hypothesis that direct interaction of B. hermsii spirochetes with RBCs leads to clearance of bacteria from the bloodstream by tissue phagocytes.