scholarly journals DNA topoisomerase IIIβ promotes cyst generation by inducing cyst wall protein gene expression in Giardia lamblia

Open Biology ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 190228
Author(s):  
Chin-Hung Sun ◽  
Shih-Che Weng ◽  
Jui-Hsuan Wu ◽  
Szu-Yu Tung ◽  
Li-Hsin Su ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cyst Wall ◽  
Giardia Lamblia ◽  
Drug Targets ◽  
Cyst Formation ◽  
Dna Topoisomerase ◽  
Catalytic Core ◽  
Mutant Proteins ◽  
Cleavage Activity

Giardia lamblia causes waterborne diarrhoea by transmission of infective cysts. Three cyst wall proteins are highly expressed in a concerted manner during encystation of trophozoites into cysts. However, their gene regulatory mechanism is still largely unknown. DNA topoisomerases control topological homeostasis of genomic DNA during replication, transcription and chromosome segregation. They are involved in a variety of cellular processes including cell cycle, cell proliferation and differentiation, so they may be valuable drug targets. Giardia lamblia possesses a type IA DNA topoisomerase (TOP3β) with similarity to the mammalian topoisomerase IIIβ. We found that TOP3β was upregulated during encystation and it possessed DNA-binding and cleavage activity. TOP3β can bind to the cwp promoters in vivo using norfloxacin-mediated topoisomerase immunoprecipitation assays. We also found TOP3β can interact with MYB2, a transcription factor involved in the coordinate expression of cwp1-3 genes during encystation. Interestingly, overexpression of TOP3β increased expression of cwp1 - 3 and myb2 genes and cyst formation. Microarray analysis confirmed upregulation of cwp1-3 and myb2 genes by TOP3β. Mutation of the catalytically important Tyr residue, deletion of C-terminal zinc ribbon domain or further deletion of partial catalytic core domain reduced the levels of cleavage activity, cwp1-3 and myb2 gene expression, and cyst formation. Interestingly, some of these mutant proteins were mis-localized to cytoplasm. Using a CRISPR/Cas9 system for targeted disruption of top3β gene, we found a significant decrease in cwp1-3 and myb2 gene expression and cyst number. Our results suggest that TOP3β may be functionally conserved, and involved in inducing Giardia cyst formation.

scholarly journals A Novel Spo11 Homologue Functions as a Positive Regulator in Cyst Differentiation in Giardia lamblia

2021 ◽  
Vol 22 (21) ◽  
pp. 11902
Author(s):  
Yu-Chien Chen ◽  
Szu-Yu Tung ◽  
Chia-Wei Huang ◽  
Soo-Wah Gan ◽  
Bo-Chi Lin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cyst Wall ◽  
Giardia Lamblia ◽  
Genetic Material ◽  
Cyst Formation ◽  
Positive Regulator ◽  
Double Stranded Dna ◽  
Recombination Processes

Giardia lamblia persists in a dormant state with a protective cyst wall for transmission. It is incompletely known how three cyst wall proteins (CWPs) are coordinately synthesized during encystation. Meiotic recombination is required for sexual reproduction in animals, fungi, and plants. It is initiated by formation of double-stranded breaks by a topoisomerase-like Spo11. It has been shown that exchange of genetic material in the fused nuclei occurs during Giardia encystation, suggesting parasexual recombination processes of this protozoan. Giardia possesses an evolutionarily conserved Spo11 with typical domains for cleavage reaction and an upregulated expression pattern during encystation. In this study, we asked whether Spo11 can activate encystation process, like other topoisomerases we previously characterized. We found that Spo11 was capable of binding to both single-stranded and double-stranded DNA in vitro and that it could also bind to the cwp promoters in vivo as accessed in chromatin immunoprecipitation assays. Spo11 interacted with WRKY and MYB2 (named from myeloblastosis), transcription factors that can activate cwp gene expression during encystation. Interestingly, overexpression of Spo11 resulted in increased expression of cwp1-3 and myb2 genes and cyst formation. Mutation of the Tyr residue for the active site or two conserved residues corresponding to key DNA-binding residues for Arabidopsis Spo11 reduced the levels of cwp1-3 and myb2 gene expression and cyst formation. Targeted disruption of spo11 gene with CRISPR/Cas9 system led to a significant decrease in cwp1-3 and myb2 gene expression and cyst number. Our results suggest that Spo11 acts as a positive regulator for Giardia differentiation into cyst.

scholarly journals A Novel Multiprotein Bridging Factor 1-Like Protein Induces Cyst Wall Protein Gene Expression and Cyst Differentiation in Giardia lamblia

2021 ◽  
Vol 22 (3) ◽  
pp. 1370
Author(s):  
Shao-Wei Huang ◽  
Zi-Qi Lin ◽  
Szu-Yu Tung ◽  
Li-Hsin Su ◽  
Chun-Che Ho ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Cyst Wall ◽  
Giardia Lamblia ◽  
Giardia Cyst ◽  
Cell Nuclei ◽  
Gene Encoding ◽  
Genome Database ◽  
Cell Growth And Differentiation

The capacity to synthesize a protective cyst wall is critical for infectivity of Giardia lamblia. It is of interest to know the mechanism of coordinated synthesis of three cyst wall proteins (CWPs) during encystation, a differentiation process. Multiprotein bridging factor 1 (MBF1) gene family is a group of transcription coactivators that bridge various transcription factors. They are involved in cell growth and differentiation in yeast and animals, or in stress response in fungi and plants. We asked whether Giardia has MBF1-like genes and whether their products influence gene expression. BLAST searches of the Giardia genome database identified one gene encoding a putative MBF1 protein with a helix-turn-helix domain. We found that it can specifically bind to the AT-rich initiator promoters of the encystation-induced cwp1-3 and myb2 genes. MBF1 localized to cell nuclei and cytoplasm with higher expression during encystation. In addition, overexpression of MBF1 induced cwp1-3 and myb2 gene expression and cyst generation. Mutation of the helixes in the helix-turn-helix domain reduced cwp1-3 and myb2 gene expression and cyst generation. Chromatin immunoprecipitation assays confirmed the binding of MBF1 to the promoters with its binding sites in vivo. We also found that MBF1 can interact with E2F1, Pax2, WRKY, and Myb2 transcription factors that coordinately up-regulate the cwp genes during encystation. Using a CRISPR/Cas9 system for targeted disruption of mbf1 gene, we found a downregulation of cwp1-3 and myb2 genes and decrease of cyst generation. Our results suggest that MBF1 is functionally conserved and positively regulates Giardia cyst differentiation.

The C-terminal domain of Saccharomyces cerevisiae DNA topoisomerase II

1994 ◽  
Vol 14 (5) ◽  
pp. 3197-3207
Author(s):  
P R Caron ◽  
P Watt ◽  
J C Wang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Localization ◽  
Topoisomerase Ii ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
Mutant Proteins ◽  
Terminal Domain ◽  
Catalytically Active

A set of carboxy-terminal deletion mutants of Saccharomyces cerevisiae DNA topoisomerase II were constructed for studying the functions of the carboxyl domain in vitro and in vivo. The wild-type yeast enzyme is a homodimer with 1,429 amino acid residues in each of the two polypeptides; truncation of the C terminus to Ile-1220 has little effect on the function of the enzyme in vitro or in vivo, whereas truncations extending beyond Gln-1138 yield completely inactive proteins. Several mutant enzymes with C termini in between these two residues were found to be catalytically active but unable to complement a top2-4 temperature-sensitive mutation. Immunomicroscopy results suggest that the removal of a nuclear localization signal in the C-terminal domain is likely to contribute to the physiological dysfunction of these proteins; the ability of these mutant proteins to relax supercoiled DNA in vivo shows, however, that at least some of the mutant proteins are present in the nuclei in a catalytically active form. In contrast to the ability of the catalytically active mutant proteins to relax supercoiled intracellular DNA, all mutants that do not complement the temperature-dependent lethality and high frequency of chromosomal nondisjunction of top2-4 were found to lack decatenation activity in vivo. The plausible roles of the DNA topoisomerase II C-terminal domain, in addition to providing a signal for nuclear localization, are discussed in the light of these results.

scholarly journals Nucleosomes represent a physical barrier for cleavage activity of DNA topoisomerase I in vivo

2008 ◽  
Vol 409 (3) ◽  
pp. 651-656 ◽  
Author(s):  
Francesca Di Felice ◽  
Francesco Chiani ◽  
Giorgio Camilloni
Keyword(s):  
Topoisomerase I ◽  
Basic Question ◽  
Dna Topoisomerase I ◽  
Physical Barrier ◽  
Dna Topoisomerase ◽  
Histone Octamer ◽  
Cleavage Activity ◽  
Cellular Dna

DNA topoisomerase I together with the other cellular DNA topoisomerases releases the torsional stress from DNA caused by processes such as replication, transcription and recombination. Despite the well-defined knowledge of its mechanism of action, DNA topoisomerase I in vivo activity has been only partially characterized. In fact the basic question concerning the capability of the enzyme to cleave and rejoin DNA wrapped around a histone octamer remains still unanswered. By studying both in vivo and in vitro the cleavage activity of DNA topoisomerase I in the presence of camptothecin on a repeated trinucleotide sequence, (TTA)35, lying in chromosome XIII of Saccharomyces cerevisiae, we can conclude that nucleosomes represent a physical barrier for the enzyme activity.

scholarly journals DNA Topoisomerase II Is Involved in Regulation of Cyst Wall Protein Genes and Differentiation in Giardia lamblia

2013 ◽  
Vol 7 (5) ◽  
pp. e2218 ◽  
Author(s):  
Bo-Chi Lin ◽  
Li-Hsin Su ◽  
Shih-Che Weng ◽  
Yu-Jiao Pan ◽  
Nei-Li Chan ◽  
...  
Keyword(s):  
Cyst Wall ◽  
Giardia Lamblia ◽  
Topoisomerase Ii ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase

scholarly journals Novel Gal3 proteins showing altered Gal80p binding cause constitutive transcription of Gal4p-activated genes in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (5) ◽  
pp. 2566-2575 ◽  
Author(s):  
T E Blank ◽  
M P Woods ◽  
C M Lebo ◽  
P Xin ◽  
J E Hopper
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Strong Support ◽  
Constitutive Expression ◽  
Mutant Proteins ◽  
Binding Characteristics ◽  
Isolation And Characterization ◽  
In Cells

Gal4p-mediated activation of galactose gene expression in Saccharomyces cerevisiae normally requires both galactose and the activity of Gal3p. Recent evidence suggests that in cells exposed to galactose, Gal3p binds to and inhibits Ga180p, an inhibitor of the transcriptional activator Gal4p. Here, we report on the isolation and characterization of novel mutant forms of Gal3p that can induce Gal4p activity independently of galactose. Five mutant GAL3(c) alleles were isolated by using a selection demanding constitutive expression of a GAL1 promoter-driven HIS3 gene. This constitutive effect is not due to overproduction of Gal3p. The level of constitutive GAL gene expression in cells bearing different GAL3(c) alleles varies over more than a fourfold range and increases in response to galactose. Utilizing glutathione S-transferase-Gal3p fusions, we determined that the mutant Gal3p proteins show altered Gal80p-binding characteristics. The Gal3p mutant proteins differ in their requirements for galactose and ATP for their Gal80p-binding ability. The behavior of the novel Gal3p proteins provides strong support for a model wherein galactose causes an alteration in Gal3p that increases either its ability to bind to Gal80p or its access to Gal80p. With the Gal3p-Gal80p interaction being a critical step in the induction process, the Gal3p proteins constitute an important new reagent for studying the induction mechanism through both in vivo and in vitro methods.

scholarly journals Evolutionarily Conserved Long Non-coding RNA Regulates Gene Expression in Cytokine Storm During COVID-19

2021 ◽  
Vol 8 ◽  
Author(s):  
Olanrewaju B. Morenikeji ◽  
Kahleel Bernard ◽  
Ellis Strutton ◽  
Madeleine Wallace ◽  
Bolaji N. Thomas
Keyword(s):  
Gene Expression ◽  
Drug Targets ◽  
Regulatory Elements ◽  
Cytokine Storm ◽  
Cellular Processes ◽  
Non Coding Rna ◽  
Evolutionarily Conserved ◽  
Multiple Species

Coronavirus is a family of viruses including alpha-, beta-, gamma-, delta-coronaviruses. Only alpha- and betacoronaviruses have been observed to infect humans. Past outbreaks of SARS-CoV and MERS-CoV, both betacoronavirus, are the result of a spillover from animals. Recently, a new strain termed SARS-CoV-2 emerged in December 2019 in Wuhan, China. Severe cases of COVID-19, the disease caused by SARS-CoV-2, lead to acute respiratory distress syndrome (ARDS). One contributor to the development of ARDS is cytokine storm, an overwhelming inflammatory immune response. Long non-coding RNAs (lncRNAs) are genetic regulatory elements that, among many functions, alter gene expression and cellular processes. lncRNAs identified to be pertinent in COVID-19 cytokine storm have the potential to serve as disease markers or drug targets. This project aims to computationally identify conserved lncRNAs potentially regulating gene expression in cytokine storm during COVID-19. We found 22 lncRNAs that can target 10 cytokines overexpressed in COVID-19 cytokine storm, 8 of which targeted two or more cytokine storm cytokines. In particular, the lncRNA non-coding RNA activated by DNA damage (NORAD), targeted five out of the ten identified cytokine storm cytokines, and is evolutionarily conserved across multiple species. These lncRNAs are ideal candidates for further in vitro and in vivo analysis.

scholarly journals Correction: DNA Topoisomerase II Is Involved in Regulation of Cyst Wall Protein Genes and Differentiation in Giardia lamblia

2017 ◽  
Vol 11 (1) ◽  
pp. e0005326
Author(s):  
Bo-Chi Lin ◽  
Li-Hsin Su ◽  
Shih-Che Weng ◽  
Yu-Jiao Pan ◽  
Nei-Li Chan ◽  
...  
Keyword(s):  
Cyst Wall ◽  
Giardia Lamblia ◽  
Topoisomerase Ii ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase
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