Insights into the identification and evolutionary conservation of key genes in the transcriptional circuits of meiosis initiation and commitment in budding yeast.

Initiation of meiosis in budding yeast does not commit the cells for meiosis. Thus, two distinct signaling cascades may differentially regulate meiosis initiation and commitment in budding yeast. To distinguish between the role of these signaling cascades, we reconstructed protein-protein interaction networks and gene regulatory networks with upregulated genes in meiosis initiation and commitment. Analyzing the integrated networks, we identified four master regulators (MRs) [Ume6p, Msn2p, Met31p, Ino2p], three transcription factors (TFs), and 279 target genes (TGs) unique for meiosis initiation, and three MRs [Ndt80p, Aro80p, Rds2p], 11 TFs, and 948 TGs unique for meiosis commitment. Functional enrichment analysis of these distinct members from the transcriptional cascades for meiosis initiation and commitment revealed that nutritional cues rewire gene expression for initiating meiosis and chromosomal recombination commits cells to meiosis. As meiotic chromosomal recombination is highly conserved in eukaryotes, we compared the evolutionary rate of unique members in the transcriptional cascade of two meiotic phases of Saccharomyces cerevisiae with members of the phylum Ascomycota, revealing that the transcriptional cascade governing chromosomal recombination during meiosis commitment has experienced greater purifying selection pressure (P value = 0.0013, 0.0382, 0.0448, 0.0369, 0.02967, 0.04937, 0.03046, 0.03357 and < 0.00001 for Ashbya gossypii, Yarrowia lipolytica, Debaryomyces hansenii, Aspergillus fumigatus, Neurospora crassa, Kluyveromyces lactis, Schizosaccharomyces pombe, Schizosaccharomyces cryophilus, and Schizosaccharomyces octosporus, respectively). This study demarcates crucial players driving meiosis initiation and commitment and demonstrates their differential rate of evolution in budding yeast.

CRISPR/Cas9-Mediated Genome Engineering Reveals the Contribution of the 26S Proteasome to the Extremophilic Nature of the Yeast Debaryomyces hansenii.

The marine yeast Debaryomyces hansenii is of high importance in the food, chemical, and medical industries. D. hansenii is also a popular model for studying molecular mechanisms of halo- and osmotolerance. The absence of genome editing technologies hampers D. hansenii research and limits its biotechnological application. We developed novel and efficient single- and dual-guide CRISPR systems for markerless genome editing of D. hansenii. The single-guide system allows high-efficiency (up to 95%) mutation of genes or regulatory elements. The dual-guide system is applicable for efficient deletion of genomic loci. We used these tools to study transcriptional regulation of the 26S proteasome, an ATP-dependent protease complex whose proper function is vital for all cells and organisms. We developed a genetic approach to control the activity of the 26S proteasome by deregulation of its essential subunits. The mutant strains were sensitive to geno- and proteotoxic stresses as well as high salinity and osmolarity, suggesting a contribution of the proteasome to the extremophilic properties of D. hansenii. The developed CRISPR systems allow efficient D. hansenii genome engineering, providing a genetic way to control proteasome activity, and should advance applications of this yeast.

Thymocyte selection-associated high-mobility group box as a potential diagnostic marker differentiating hypopigmented mycosis fungoides from early vitiligo: A pilot study.

BACKGROUND: Hypopigmented mycosis fungoides is a rare variant of mycosis fungoides that may mimic many benign inflammatory hypopigmented dermatoses, and as yet there is no identified marker to differentiate between them. AIM: The aim of this study was to study the expression of thymocyte selection-associated high-mobility group box (TOX) in hypopigmented mycosis fungoides and one of its inflammatory mimickers (early active vitiligo) to assess its potential as a differentiating diagnostic marker. METHODS: A case-control study was done using immunohistochemical analysis of TOX expression in 15 patients with hypopigmented mycosis fungoides and 15 patients with early active vitiligo. Immunohistochemical analysis was done via a semi-quantitative method and an image analysis method. RESULTS: Hypopigmented mycosis fungoides showed a statistically significant higher expression of TOX than early active vitiligo. The expression of TOX was positive in a majority of hypopigmented mycosis fungoides cases (14 cases, 93.3%), while only one case (6.7%) of vitiligo was weakly positive. TOX also displayed 93.3% sensitivity and specificity, with a cut-off value of 1.5. LIMITATIONS: This was a pilot study testing hypopigmented mycosis fungoides against only a single benign inflammatory mimicker (early vitiligo). Other benign mimickers were not included. CONCLUSION: Our findings showed that TOX expression can differentiate hypopigmented mycosis fungoides from early active vitiligo which is one of its benign inflammatory mimickers, with a high degree of sensitivity and specificity.

The actinobacterial WhiB-like (Wbl) family of transcription factors.

The WhiB-like (Wbl) family of proteins are exclusively found in Actinobacteria. Wbls have been shown to play key roles in virulence and antibiotic resistance in Mycobacteria and Corynebacteria, reflecting their importance during infection by the human pathogens Mycobacterium tuberculosis, Mycobacterium leprae and Corynebacterium diphtheriae. In the antibiotic-producing Streptomyces, several Wbls have important roles in the regulation of morphological differentiation, including WhiB, a protein that controls the initiation of sporulation septation and the founding member of the Wbl family. In recent years, genome sequencing has revealed the prevalence of Wbl paralogues in species throughout the Actinobacteria. Wbl proteins are small (generally ~80-140 residues) and each contains four invariant cysteine residues that bind an O2 - and NO-sensitive [4Fe-4S] cluster, raising the question as to how they can maintain distinct cellular functions within a given species. Despite their discovery over 25 years ago, the Wbl protein family has largely remained enigmatic. Here I summarise recent research in Mycobacteria, Corynebacteria and Streptomyces that sheds light on the biochemical function of Wbls as transcription factors and as potential sensors of O2 and NO. I suggest that Wbl evolution has created diversity in protein-protein interactions, [4Fe-4S] cluster-sensitivity and the ability to bind DNA.

NFκB transcription factor (p65) immunohistochemistry in leprosy dermal microvasculature.

Leprosy caused by Mycobacterium leprae is characterized by a spectrum of clinical manifestations that are determined by the predominant immunological profile of the host. The recruitment of leukocytes to the sites of injury can influence the development of these profiles. Cell adhesion molecules such as ICAM-1, VCAM-1 and CD62E participate in this process and their expression is regulated by transcriptions factors such as NFκB. To correlate the expression of cell adhesion molecules and NFκB (p65) in leprosy lesions, 30 skin biopsies of patients with leprosy [16 with the tuberculoid (TT) or borderline tuberculoid (BT) forms and 14 with the lepromatous (LL) or borderline lepromatous (BL) forms] were analyzed by immunohistochemistry. A larger mean number of cells expressing VCAM-1 (BT/TT: 18.28 ± 1.4; BL/LL: 10.67 ± 1.2; p = 0.0002), ICAM-1 (BT/TT: 9.92 ± 1.1; BL/LL: 5.87 ± 1.0; p = 0.0084) and CD62E (BT/TT: 13.0 ± 1.5; BL/LL: 2.58 ± 0.3; p = 0.0001) were observed in BT and TT lesions. The mean number of cells expressing NFκB was similar in the two clinical forms (BT/TT: 2.21 ± 2.7; BL/LL: 2.35 ± 3.1;p = 0.9285). No significant correlation was observed between expression of the transcription factor and adhesion molecules analyzed. The synthesis of ICAM-1, VCAM-1 and CD62E depends on the activation of NFκB, which acts synergistically with other transcription factors. Adequate activation of intracellular signaling pathways results in the production of endothelial adhesion molecules, contributing to the recruitment of cells to the site of injury and thus eliciting an effective inflammatory response in the elimination of the bacillus.

Functional analysis of Debaryomyces hansenii Rpn4 on a genetic background of Saccharomyces cerevisiae.

The transcription factor ScRpn4 coordinates the expression of Saccharomyces cerevisiae proteasomal genes. ScRpn4 orthologues are found in a number of other Saccharomycetes yeasts. Their functions, however, have not yet been characterised experimentally in vivo . We expressed the Debaryomyces hansenii DEHA2D12848 gene encoding an ScRpn4 orthologue (DhRpn4), in an S. cerevisiae strain lacking RPN4 . We showed that DhRpn4 activates transcription of proteasomal genes using ScRpn4 binding site and provides resistance to various stresses. The 43-238 aa segment of DhRpn4 contains an unique portable transactivation domain. Similar to the ScRpn4 N-terminus, this domain lacks a compact structure Moreover, upon overexpression in D. hansenii , DhRpn4 upregulates protesomal genes. Thus, we show that DhRpn4 is the activator for proteasomal genes.

Mycobacterial secretion systems ESX-1 and ESX-5 play distinct roles in host cell death and inflammasome activation.

During infection of humans and animals, pathogenic mycobacteria manipulate the host cell causing severe diseases such as tuberculosis and leprosy. To understand the basis of mycobacterial pathogenicity, it is crucial to identify the molecular virulence mechanisms. In this study, we address the contribution of ESX-1 and ESX-5--two homologous type VII secretion systems of mycobacteria that secrete distinct sets of immune modulators--during the macrophage infection cycle. Using wild-type, ESX-1- and ESX-5-deficient mycobacterial strains, we demonstrate that these secretion systems differentially affect subcellular localization and macrophage cell responses. We show that in contrast to ESX-1, the effector proteins secreted by ESX-5 are not required for the translocation of Mycobacterium tuberculosis or Mycobacterium marinum to the cytosol of host cells. However, the M. marinum ESX-5 mutant does not induce inflammasome activation and IL-1ß activation. The ESX-5 system also induces a caspase-independent cell death after translocation has taken place. Importantly, by means of inhibitory agents and small interfering RNA experiments, we reveal that cathepsin B is involved in both the induction of cell death and inflammasome activation upon infection with wild-type mycobacteria. These results reveal distinct roles for two different type VII secretion systems during infection and shed light on how virulent mycobacteria manipulate the host cell in various ways to replicate and spread.

Identification of a repressor of a truncated denitrification pathway in Moraxella catarrhalis.

Growth of Moraxella catarrhalis in a biofilm resulted in marked upregulation of two open reading frames (ORFs), aniA and norB, predicted to encode a nitrite reductase and a nitric oxide reductase, respectively (W. Wang, L. Reitzer, D. A. Rasko, M. M. Pearson, R. J. Blick, C. Laurence, and E. J. Hansen, Infect. Immun. 75:4959-4971, 2007). An ORF designated nsrR, which was located between aniA and norB, was shown to encode a predicted transcriptional regulator. Inactivation of nsrR resulted in increased expression of aniA and norB in three different M. catarrhalis strains, as measured by both DNA microarray analysis and quantitative reverse transcriptase PCR. Provision of a wild-type nsrR gene in trans in an nsrR mutant resulted in decreased expression of the AniA protein. DNA microarray analysis revealed that two other ORFs (MC ORF 683 and MC ORF 1550) were also consistently upregulated in an nsrR mutant. Consumption of both nitrite and nitric oxide occurred more rapidly with cells of an nsrR mutant than with wild-type cells. However, growth of nsrR mutants was completely inhibited by a low level of sodium nitrite. This inhibition of growth by nitrite was significantly reversed by introduction of an aniA mutation into the nsrR mutant and was completely reversed by the presence of a wild-type nsrR gene in trans. NsrR regulation of the expression of aniA was sensitive to nitrite, whereas NsrR regulation of norB was sensitive to nitric oxide.

Heterologous expression implicates a GATA factor in regulation of nitrogen metabolic genes and ion homeostasis in the halotolerant yeast Debaryomyces hansenii.

The yeast Debaryomyces hansenii has a remarkable capacity to proliferate in salty and alkaline environments such as seawater. A screen for D. hansenii genes able to confer increased tolerance to high pH when overexpressed in Saccharomyces cerevisiae yielded a single gene, named here DhGZF3, encoding a putative negative GATA transcription factor related to S. cerevisiae Dal80 and Gzf3. Overexpression of this gene in wild-type S. cerevisiae increased caffeine and rapamycin tolerance, blocked growth in low glucose concentrations and nonfermentable carbon sources, and resulted in lithium- and sodium-sensitive cells. Sensitivity to salt could be attributed to a reduced cation efflux, most likely because of a decrease in expression of the ENA1 Na(+)-ATPase gene. Overexpression of DhGZF3 did not affect cell growth in a gat1 mutant but was lethal in the absence of Gln3. These are positive factors that oppose both Gzf3 and Dal80. Genome-wide transcriptional profiling of wild-type cells overexpressing DhGZF3 shows decreased expression of a number of genes that are usually induced in poor nitrogen sources. In addition, the entire pathway leading to Lys biosynthesis was repressed, probably as a result of a decrease in the expression of the specific Lys14 transcription factor. In conclusion, our results demonstrate that DhGzf3 can play a role as a negative GATA transcription factor when expressed in S. cerevisiae and that it most probably represents the only member of this family in D. hansenii. These findings also point to the GATA transcription factors as relevant elements for alkaline-pH tolerance.

The Escherichia coli biotin regulatory system: a transcriptional switch.

The ability of any organism to survive depends, in part, on mechanisms that enable it to modify its patterns of gene expression in response to extra- and intracellular signals. In the classical response mechanisms, a small molecule signal impinges on either an extra- or intracellular receptor, and through a series of events the signal is ultimately transmitted to transcription regulatory proteins. An alternative to this classical mechanism is provided by multi-functional transcription factors. These proteins function directly in transcription as well as in at least one additional cellular process. An example of this class of proteins includes the dimerization cofactor of hepatocyte nuclear factor (DcoH), which serves as an enzyme involved in regeneration of the tetra-hydrobiopterin cofactor and as a factor that stabilizes the dimerization of the hepatocyte nuclear transcription factor (Mendel DB, Khavari PA, Conley PB, Graves MK, Hansen LP, Admon A, et al. Characterization of a cofactor that regulates dimerization of a mammalian homeodomain protein. Science 1991;254:1762-7; Citron BA, Davis MD, Milstien S, Gutierrez J, Mendel DB, Crabtree GR. Identity of 4a-carbinolamine dehydratase, a component of the phenylalanine hydroxylation system, and DCoH, a transregulator of homeodomain proteins. Proc Natl Acad Sci U S A 1992;89:11891-4). Another example is the protein PutA, a redox enzyme involved in proline utilization and a regulator of transcription of the genes involved in proline utilization (Ostrovsky de Spicer P, Maloy S. Puta protein, a membrane-associated flavin dehydrogenase, acts as a redox-dependent transcriptional regulator. Proc Natl Acad Sci U S A 1993;90:4295-8). While several proteins of this class have been identified, their mechanisms of functional switching remain to be elucidated.

The mycelium-associated Streptomyces reticuli catalase-peroxidase, its gene and regulation by FurS.

During early stages of growth, Streptomyces reticuli synthesizes a hyphae-associated, haem-containing enzyme which exhibits catalase and peroxidase activities with broad substrate specificity (CpeB). The purified dimeric enzyme (160 kDa) consists of two identical subunits. Using anti-CpeB antibodies and an expression- as well as a mini-library, the corresponding cpeB gene was identified and sequenced. It encodes a protein of 740 aa with a molecular mass of 81.3 kDa. The deduced protein shares the highest level of amino acid identity with KatG from Caulobacter crescentus and Mycobacterium tuberculosis, and PerA from Bacillus stearothermophilus. Streptomyces lividans transformants carrying cpeB and the upstream-located furS gene with its regulatory region on the bifunctional vector pWHM3 produced low or enhanced levels of CpeB in the presence or absence of Fe ions, respectively. An in-frame deletion of the major part of furS induces increased CpeB synthesis. The data imply that FurS regulates the transcription of cpeB. The deduced FurS protein is rich in histidine residues, contains a putative N-terminally situated helix-turn-helix motif and has a molecular mass of 15.1 kDa. It shares only 29% amino acid identity with the Escherichia coli ferric uptake regulator (Fur) protein, but about 64% with FurA deduced from the genomic sequences of several mycobacteria. The predicted secondary structures of FurS and FurA are highly similar and considerably divergent from those of the E. coli Fur. In contrast to some Gram-negative bacteria, within several mycobacteria an intact furA gene or a furA pseudogene is upstream of a catalase-peroxidase (katG) gene predicted to encode a functional or a non-functional (Mycobacterium leprae) enzyme. Thus the data obtained for Streptomyces reticuli are expected to serve as an additional model to elucidate the regulation of mycobacterial catalase-peroxidase genes.

The Epstein-Barr virus-encoded nuclear antigen EBNA-5 accumulates in PML-containing bodies.

EBNA-5 is one of the Epstein-Barr virus (EBV)-encoded nuclear proteins required for immortalization of human B lymphocytes. In the nuclei of EBV-transformed lymphoblastoid cell lines EBNA-5 is preferentially targetted to distinct nuclear foci. Previously we have shown (W.Q. Jiang, L. Szekely, V. Wendel-Hansen, N. Ringertz, G. Klein, and A. Rosen, Exp. Cell Res. 197:314-318, 1991) that the same foci also contained the retinoblastoma (Rb) protein. Using a similar double immunofluorescence technique, we now show that these foci colocalize with nuclear bodies positive for PML, the promyelocytic leukemia-associated protein. Artificial spreading of the chromatin by exposure to the forces of fluid surface tension disrupts this colocalization gradually, suggesting that the bodies consist of at least two subcomponents. Heat shock or metabolic stress induced by high cell density leads to the release of EBNA-5 from the PML-positive nuclear bodies and induces it to translocate to the nucleoli. In addition to their presence in nuclear bodies, both proteins are occasionally present in nuclear aggregates and doughnut-like structures in which PML is concentrated in an outer shell. Nuclear bodies with prominent PML staining are seen in resting B lymphocytes. This staining pattern does not change upon EBV infection. In freshly infected cells EBNA-5 antigens are first distributed throughout the nucleoplasm. After a few days intensely staining foci develop. These foci coincide with PML-positive nuclear bodies. At a later stage and in established lymphoblastoid cell lines EBNA-5 is almost exclusively present in the PML-positive nuclear foci. The colocalization is restricted to EBV-infected human lymphoblasts. The data presented indicate that the distinct EBNA-5 foci are not newly formed structures but the result of translocation of the viral protein to a specialized domain present already in the nuclei of uninfected cells.

Identification of a transcriptional enhancer upstream from the bovine thyroglobulin gene.

The DNA sequences corresponding to a DNaseI-hypersensitive region identified previously in bovine thyroglobulin gene chromatin (Hansen et al. (1988) Eur. J. Biochem. 178, 387-393) exhibited the properties of a transcriptional enhancer in a transient assay in primary cultured dog thyrocytes, but did not so in transfected HeLa cells. By contrast to the thyroglobulin proximal promoter, the enhancer element did not require cyclic AMP stimulation of the thyrocytes to be active. Using a bi-directional deletion approach, the minimal region displaying enhancer activity has been localized between positions -1906 and -1744 relative to the thyroglobulin gene transcription start. DNA-footprinting experiments revealed the presence of several binding sites for the thyroid-specific transcription factor TTF-1 within the enhancer sequence.