Jay C. Vary

The lta4h locus modulates susceptibility to mycobacterial infection in zebrafish and humans.

Exposure to Mycobacterium tuberculosis produces varied early outcomes, ranging from resistance to infection to progressive disease. Here we report results from a forward genetic screen in zebrafish larvae that identify multiple mutant classes with distinct patterns of innate susceptibility to Mycobacterium marinum. A hypersusceptible mutant maps to the lta4h locus encoding leukotriene A(4) hydrolase, which catalyzes the final step in the synthesis of leukotriene B(4) (LTB(4)), a potent chemoattractant and proinflammatory eicosanoid. lta4h mutations confer hypersusceptibility independent of LTB(4) reduction, by redirecting eicosanoid substrates to anti-inflammatory lipoxins. The resultant anti-inflammatory state permits increased mycobacterial proliferation by limiting production of tumor necrosis factor. In humans, we find that protection from both tuberculosis and multibacillary leprosy is associated with heterozygosity for LTA4H polymorphisms that have previously been correlated with differential LTB(4) production. Our results suggest conserved roles for balanced eicosanoid production in vertebrate resistance to mycobacterial infection.

Host Genotype-Specific Therapies Can Optimize the Inflammatory Response to Mycobacterial Infections

Susceptibility to tuberculosis is historically ascribed to an inadequate immune response that fails to control infecting mycobacteria. In zebrafish, we find that susceptibility to Mycobacterium marinum can result from either inadequate or excessive acute inflammation. Modulation of the leukotriene A(4) hydrolase (LTA4H) locus, which controls the balance of pro- and anti-inflammatory eicosanoids, reveals two distinct molecular routes to mycobacterial susceptibility converging on dysregulated TNF levels: inadequate inflammation caused by excess lipoxins and hyperinflammation driven by excess leukotriene B(4). We identify therapies that specifically target each of these extremes. In humans, we identify a single nucleotide polymorphism in the LTA4H promoter that regulates its transcriptional activity. In tuberculous meningitis, the polymorphism is associated with inflammatory cell recruitment, patient survival and response to adjunctive anti-inflammatory therapy. Together, our findings suggest that host-directed therapies tailored to patient LTA4H genotypes may counter detrimental effects of either extreme of inflammation.

Targeted disruption of the Kcnq1 gene produces a mouse model of Jervell and Lange– Nielsen Syndrome

KCNQ1 encodes KCNQ1, which belongs to a family of voltage-dependent K+ ion channel proteins. KCNQ1 associates with a regulatory subunit, KCNE1, to produce the cardiac repolarizing current, IKs. Loss-of-function mutations in the human KCNQ1 gene have been linked to Jervell and Lange–Nielsen Syndrome (JLNS), a disorder characterized by profound bilateral deafness and a cardiac phenotype. To generate a mouse model for JLNS, we created a line of transgenic mice that have a targeted disruption in the Kcnq1 gene. Behavioral analysis revealed that the Kcnq1−/− mice are deaf and exhibit a shaker/waltzer phenotype. Histological analysis of the inner ear structures of Kcnq1−/− mice revealed gross morphological anomalies because of the drastic reduction in the volume of endolymph. ECGs recorded from Kcnq1−/− mice demonstrated abnormal T- and P-wave morphologies and prolongation of the QT and JT intervals when measured in vivo, but not in isolated hearts. These changes are indicative of cardiac repolarization defects that appear to be induced by extracardiac signals. Together, these data suggest that Kcnq1−/− mice are a potentially valuable animal model of JLNS.

Hairpin-bisulfite PCR: Assessing epigenetic methylation patterns on complementary strands of individual DNA molecules

Epigenetic inheritance, the transmission of gene expression states from parent to daughter cells, often involves methylation of DNA. In eukaryotes, cytosine methylation is a frequent component of epigenetic mechanisms. Failure to transmit faithfully a methylated or an unmethylated state of cytosine can lead to altered phenotypes in plants and animals. A central unresolved question in epigenetics concerns the mechanisms by which a locus maintains, or changes, its state of cytosine methylation. We developed “hairpin-bisulfite PCR” to analyze these mechanisms. This method reveals the extent of methylation symmetry between the complementary strands of individual DNA molecules. Using hairpin-bisulfite PCR, we determined the fidelity of methylation transmission in the CpG island of the FMR1 gene in human lymphocytes. For the hypermethylated CpG island of this gene, characteristic of inactive-X alleles, we estimate a maintenance methylation efficiency of ≈0.96 per site per cell division. For de novo methylation efficiency (Ed), remarkably different estimates were obtained for the hypermethylated CpG island (Ed = 0.17), compared with the hypomethylated island on the active-X chromosome (Ed < 0.01). These results clarify the mechanisms by which the alternative hypomethylated and hypermethylated states of CpG islands are stably maintained through many cell divisions. We also analyzed a region of human L1 transposable elements. These L1 data provide accurate methylation patterns for the complementary strand of each repeat sequence analyzed. Hairpin-bisulfite PCR will be a powerful tool in studying other processes for which genetic or epigenetic information differs on the two complementary strands of DNA.

Yeast Isw1p Forms Two Separable Complexes In Vivo

ABSTRACT There are several classes of ATP-dependent chromatin remodeling complexes, which modulate the structure of chromatin to regulate a variety of cellular processes. The budding yeast, Saccharomyces cerevisiae, encodes two ATPases of the ISWI class, Isw1p and Isw2p. Previously Isw1p was shown to copurify with three other proteins. Here we identify these associated proteins and show that Isw1p forms two separable complexes in vivo (designated Isw1a and Isw1b). Biochemical assays revealed that while both have equivalent nucleosome-stimulated ATPase activities, Isw1a and Isw1b differ in their abilities to bind to DNA and nucleosomal substrates, which possibly accounts for differences in specific activities in nucleosomal spacing and sliding. In vivo, the two Isw1 complexes have overlapping functions in transcriptional regulation of some genes yet distinct functions at others. In addition, these complexes show different contributions to cell growth at elevated temperatures.

Human TOLLIP regulates TLR2 and TLR4 signaling and its polymorphisms are associated with susceptibility to tuberculosis

Tuberculosis, one of the leading causes of death worldwide, stimulates inflammatory responses with beneficial and pathologic consequences. The regulation and nature of an optimal inflammatory response to Mycobacterium tuberculosis remains poorly understood in humans. Insight into mechanisms of negative regulation of the TLR-mediated innate immune response to M. tuberculosis could provide significant breakthroughs in the design of new vaccines and drugs. We hypothesized that TOLLIP and its common variants negatively regulate TLR signaling in human monocytes and are associated with susceptibility to tuberculosis. Using short hairpin RNA knockdown of TOLLIP in peripheral blood human monocytes, we found that TOLLIP suppresses TNF and IL-6 production after stimulation with TLR2 and TLR4 ligands. In contrast, secretion of the anti-inflammatory cytokine IL-10 was induced by TOLLIP. We also discovered two common polymorphisms that are associated with either decreased levels of mRNA expression (rs3750920) or increased IL-6 production (rs5743899) in a sample of 56 healthy volunteers. Furthermore, in a case-population study in Vietnam with 760 cord blood samples and 671 TB case patients, we found that SNPs rs3750920 and rs5743899 were associated with susceptibility to tuberculosis (p = 7.03 × 10−16 and 6.97 × 10−7, respectively). These data demonstrate that TOLLIP has an anti-inflammatory effect on TLR signaling in humans and that TOLLIP deficiency is associated with an increased risk of tuberculosis. To our knowledge, these data also show the first associations of TOLLIP polymorphisms with any infectious disease. These data also implicate an unexpected mechanism of negative regulation of TLR signaling in human tuberculosis pathogenesis.

Isw1 functions in parallel with the NuA4 and Swr1 complexes in stress-induced gene repression

ABSTRACT The packaging of DNA into chromatin allows eukaryotic cells to organize and compact their genomes but also creates an environment that is generally repressive to nuclear processes that depend upon DNA accessibility. There are several classes of enzymes that modulate the primary structure of chromatin to regulate various DNA-dependent processes. The biochemical activities of the yeast Isw1 ATP-dependent chromatin-remodeling enzyme have been well characterized in vitro, but little is known about how these activities are utilized in vivo. In this work, we sought to discern genetic backgrounds that require Isw1 activity for normal growth. We identified a three-way genetic interaction among Isw1, the NuA4 histone acetyltransferase complex, and the Swr1 histone replacement complex. Transcription microarray analysis revealed parallel functions for these three chromatin-modifying factors in the regulation of TATA-containing genes, including the repression of a large number of stress-induced genes under normal growth conditions. In contrast to a recruitment-based model, we find that the NuA4 and Swr1 complexes act throughout the genome while only a specific subset of the genome shows alterations in transcription.

Assembly of Yeast Chromatin Using ISWI Complexes

Publisher Summary This chapter describes the in vitro reconstitution of evenly spaced nucleosomal arrays, using a system consisting of recombinant yeast histones, recombinant yeast Nap1, and a member of the yeast ISWI class of ATP dependent chromatin-remodeling factors. This system produces a clean substrate, similar to the ACF method; however, the availability of histone mutants from S. cerevisiae allows reconstitution with a wide variety of mutant nucleosomes, all of which can be expressed recombinantly. In addition, these recombinant histones are devoid of posttranslational modifications, resulting in a homogeneous population, which may be uniformly modified if so desired. This system provides a great degree of experimental flexibility in its application. The purification and assembly protocols described in this chapter are the result of many refinements of previously existing techniques and additional increases in efficiency will likely be achieved by careful titrations and modifications of these methods if larger quantities or specific activities are required.

Unusual granulomatous variant of scleromyxedema

Scleromyxedema is notable for significant morbidity and mortality. A generalized eruption of waxy papules in the absence of thyroid disease with histologic findings of mucin deposition, increased fibroblast proliferation, and fibrosis are the characteristic features of scleromyxedema. We report a case of scleromyxedema that, on histology, was associated with interstitial granuloma annulare-like features. Based on our literature review, this is a rare presentation of this disease. Familiarity with the histologic aspects of scleromyxedema, as described in this report, can help to improve the accuracy of this diagnosis, particularly in atypical presentations.

From the Cover: Hairpin-bisulfite PCR: Assessing epigenetic methylation patterns on complementary strands of individual DNA molecules

Epigenetic inheritance, the transmission of gene expression states from parent to daughter cells, often involves methylation of DNA. In eukaryotes, cytosine methylation is a frequent component of epigenetic mechanisms. Failure to transmit faithfully a methylated or an unmethylated state of cytosine can lead to altered phenotypes in plants and animals. A central unresolved question in epigenetics concerns the mechanisms by which a locus maintains, or changes, its state of cytosine methylation. We developed “hairpin-bisulfite PCR” to analyze these mechanisms. This method reveals the extent of methylation symmetry between the complementary strands of individual DNA molecules. Using hairpin-bisulfite PCR, we determined the fidelity of methylation transmission in the CpG island of the FMR1 gene in human lymphocytes. For the hypermethylated CpG island of this gene, characteristic of inactive-X alleles, we estimate a maintenance methylation efficiency of ≈0.96 per site per cell division. For de novo methylation efficiency (Ed), remarkably different estimates were obtained for the hypermethylated CpG island (Ed = 0.17), compared with the hypomethylated island on the active-X chromosome (Ed < 0.01). These results clarify the mechanisms by which the alternative hypomethylated and hypermethylated states of CpG islands are stably maintained through many cell divisions. We also analyzed a region of human L1 transposable elements. These L1 data provide accurate methylation patterns for the complementary strand of each repeat sequence analyzed. Hairpin-bisulfite PCR will be a powerful tool in studying other processes for which genetic or epigenetic information differs on the two complementary strands of DNA.