Regulation of H2O2 Stress-responsive Genes through a Novel Transcription Factor in the Protozoan Pathogen Entamoeba histolytica

Pearson, Richard; Morf, Laura; Singh, Upinder

doi:10.1074/jbc.m112.423467

Cited by 41 publications

(56 citation statements)

References 52 publications

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“…In this manner, multiple genes including Rhomboid protease 1 (ROM1), Myb-transcription factor, and an H 2 O 2 -responsive transcription factor were down-regulated (47). These genes also displayed long term silencing despite removal of the trigger plasmid.…”

Section: Resultsmentioning

confidence: 90%

Dimethylated H3K27 Is a Repressive Epigenetic Histone Mark in the Protist Entamoeba histolytica and Is Significantly Enriched in Genes Silenced via the RNAi Pathway

Foda

Singh

2015

Journal of Biological Chemistry

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Background:Entamoeba histolytica has an RNAi-mediated TGS pathway but the epigenetic marks are not known. Results: H3K27Me2 is an inducible repressive histone modification enriched in loci silenced by small RNAs. Conclusion: Transcriptional gene silencing and chromatin modification are coupled to maintain prolonged gene silencing. Significance: This work identifies the first repressive histone mark in Entamoeba and links it to RNAi.

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Section: Resultsmentioning

confidence: 90%

Dimethylated H3K27 Is a Repressive Epigenetic Histone Mark in the Protist Entamoeba histolytica and Is Significantly Enriched in Genes Silenced via the RNAi Pathway

Foda

Singh

2015

Journal of Biological Chemistry

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“…In order to identify potential regulatory motifs in the core promoter or immediate downstream regions of a gene, the MEME program (http://meme.nbcr.net/meme/) was used as previously described (Hackney et al, 2007; Pearson et al, 2013). In brief, 150 nt upstream from the ATG start codon of all annotated transcripts were analyzed to identify conserved core promoter motifs.…”

Section: Methodsmentioning

confidence: 99%

“…EMSAs were performed as previously described (Pearson et al, 2013). The oligonucleotides used in EMSAs were as follows (mutations underlined) – (i) Ei CPM-GL wild type (WT) sense: 5’ -acgtcagtAGGCGAACTACAAAAGATcagt- 3’, (ii) Ei CPM-GL WT anti-sense: 5’-acgtactgATCTTTTGTAGTTCGCCTactg- 3’; (iii) Ei CPM-GL Mutant sense: 5’ -acgtcagtAGGC CG A TA A T A GT AGATcagt- 3’, (iv) Ei CPM-GL Mutant anti-sense: 5’ -acgtactgATCT AC T A T TA T CG GCCTactg- 3’; (v) E. invadens 3’- U-Rich Motif ( Ei 3’-URM) WT sense: 5’ -acgtcagtTGCATTTGTTTAAAcagt- 3’, (vi) Ei 3’-URM WT anti-sense: 5’ -acgtactgTTTAAACAAATGCAactg- 3’; (vii) Ei 3’-URM Mutant sense: 5’ -acgtcagtTGCACAGATATAAAcagt- 3’, (viii) Ei 3’-URM Mutant anti-sense: 5’ -acgtactgTTTA T A TCTG TGCAactg- 3’.…”

Section: Methodsmentioning

confidence: 99%

“…Complementary overlapping probes were annealed and labeled using [ 32 P] α-ATP and Klenow fragment (Invitrogen, USA) (Hackney et al, 2007). Binding reactions was carried out as described previously (Pearson et al, 2013). Briefly, binding reaction was set in a total volume of 20 μl, which included 2 μl of 10X EMSA binding buffer (10 mM Tris-HCl, pH 7.9, 50 mM NaCl, 1 mM EDTA, 3% glycerol, 0.05% milk powder and 0.05 mg of bromophenol blue), 5 μg of nuclear extract, 2 μg of poly (dI-dC), and 50 fmol of labeled probe.…”

Section: Methodsmentioning

confidence: 99%

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Regulation of gene expression in the protozoan parasite Entamoeba invadens: identification of core promoter elements and promoters with stage-specific expression patterns

Manna

Ehrenkaufer

Singh

2014

International Journal for Parasitology

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Developmental switching between life-cycle stages is a common feature among many pathogenic organisms. Entamoeba histolytica is an important human pathogen and is a leading parasitic cause of death globally. During its life cycle, Entamoeba converts between cysts (essential for disease transmission) and trophozoites (responsible for tissue invasion). Despite being central to its biology, the triggers that are involved in the developmental pathways of this parasite are not well understood. In order to define the transcriptional network associated with stage conversion we used Entamoeba invadens which serves as a model system for Entamoeba developmental biology, and performed RNA sequencing at different developmental time points . In this study RNA-Seq data was utilized to define basal transcriptional control elements as well as to identify promoters which regulate stage-specific gene expression patterns. We discovered that the 5’ and 3’ untranslated regions of E. invadens genes are short, a median of 20 nucleotides (nt) and 26 nt respectively. Bioinformatics analysis of DNA sequences proximate to the start and stop codons identified two conserved motifs: (i) E. invadens Core Promoter Motif - GAAC-Like (EiCPM-GL) (GAACTACAAA), and (ii) E. invadens 3’- U-Rich Motif (Ei3’-URM) (TTTGTT) in the 5’ and 3’ flanking regions, respectively. Electrophoretic mobility shift assays demonstrated that both motifs specifically bind nuclear protein(s) from E. invadens trophozoites. Additionally, we identified select genes with stage-specific expression patterns and analyzed the ability of each gene promoter to drive a luciferase reporter gene during the developmental cycle. This approach confirmed three trophozoite-specific, four encystation-specific and two excystation-specific promoters. This work lays the framework for use of stage-specific promoters to express proteins of interest in a particular life-cycle stage, adding to the molecular toolbox for genetic manipulation of E. invadens and allowing further dissection of factors controlling Entamoeba developmental biology.

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“…Fusion of 132 bp of the coding region of a "trigger gene" to a full-length coding region of another gene results in generation of antisense sRNAs to the fused gene and subsequent gene silencing. This technique has been successfully used to downregulate the expression of several amebic genes, including virulence genes and transcription factors (20,42). However, not all genes are amenable to silencing, as we noted that RNAi pathway genes, including genes that encode Argonaute (Ago2-1, Ago2-2, and Ago 2-3) and RNase III, were not silenced via the trigger method despite generation of functional sRNAs to the trigger-fused gene (43).…”

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confidence: 96%

Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens

et al. 2016

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b Entamoeba histolytica, a protozoan parasite, is an important human pathogen and a leading parasitic cause of death. The organism has two life cycle stages, trophozoites, which are responsible for tissue invasion, and cysts, which are involved in pathogen transmission. Entamoeba invadens is the model system to study Entamoeba developmental biology, as high-grade regulated encystation and excystation are readily achievable. However, the lack of gene-silencing tools in E. invadens has limited the molecular studies that can be performed. Using the endogenous RNA interference (RNAi) pathway in Entamoeba, we developed an RNAi-based trigger gene-silencing approach in E. invadens. We demonstrate that a gene's coding region that has abundant antisense small RNAs (sRNAs) can trigger silencing of a gene that is fused to it. The trigger fusion leads to the generation of abundant antisense sRNAs that map to the target gene, with silencing occurring independently of trigger location at the 5= or 3= end of a gene. Gene silencing is stably maintained during development, including encystation and excystation. We have used this approach to successfully silence two E. invadens genes: a putative rhomboid protease gene and a SHAQKY family Myb gene. The Myb gene is upregulated during oxidative stress and development, and its downregulation led, as predicted, to decreased viability under oxidative stress and decreased cyst formation. Thus, the RNAi trigger silencing method can be used to successfully investigate the molecular functions of genes in E. invadens. Dissection of the molecular basis of Entamoeba stage conversion is now possible, representing an important technical advance for the system.

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Regulation of H2O2 Stress-responsive Genes through a Novel Transcription Factor in the Protozoan Pathogen Entamoeba histolytica

Cited by 41 publications

References 52 publications

Dimethylated H3K27 Is a Repressive Epigenetic Histone Mark in the Protist Entamoeba histolytica and Is Significantly Enriched in Genes Silenced via the RNAi Pathway

Dimethylated H3K27 Is a Repressive Epigenetic Histone Mark in the Protist Entamoeba histolytica and Is Significantly Enriched in Genes Silenced via the RNAi Pathway

Regulation of gene expression in the protozoan parasite Entamoeba invadens: identification of core promoter elements and promoters with stage-specific expression patterns

Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens

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