Multifarious Transcriptional Regulation of Adhesion Protein Gene
 ap65
 -
 1
 by a Novel Myb1 Protein in the Protozoan Parasite
 Trichomonas vaginalis

Ong, Shiou-Jeng; Hsu, Heng-Ming; Liu, Hsing-Wei; Chu, Chien-Hsin; Tai, Jung-Hsiang

doi:10.1128/ec.5.2.391-399.2006

Cited by 46 publications

(88 citation statements)

References 37 publications

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“…A T. vaginalis stable cell line transfected with the expression plasmid pAP65-1-m(MRE-1)-ha-myb3/TUBneo expressed hemagglutinin (HA)-Myb3 to a level detected in only a small fraction of cells by an immunofluorescence assay (IFA) using a 100ϫ dilution of a mouse anti-HA antibody (29). To generate a plasmid with a higher HA-tagged Myb3 (HA-Myb3) expression level than that conferred by pAPm(MRE-1)-ha-myb3, the sequence encompassing the ap65-2.1 promoter was amplified from pAP65-2.1-ha-myb2/TUBneo (30) by a PCR using the primer pair pAP65-2.1-sac2-5= and ap65-2.1-hind3-3= (see Table S1 in the supplemental material).…”

Section: Methodsmentioning

confidence: 99%

“…T. vaginalis was fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min and permeabilized in 0.2% Triton X-100 in PBS for 15 min at room temperature prior to the IFA using a mouse anti-HA monoclonal antibody (400ϫ; HA-7; Sigma) and fluorescein isothiocyanate (FITC)-conjugated mouse immunoglobulin G (IgG), as previously described (29). The nuclear DNA was stained with DAPI (4=,6-diamidino-2-phenylindole).…”

Section: Methodsmentioning

confidence: 99%

“…To date, multiple parasitic factors that contribute to cytoadherence, including some carbohydrate moieties and several reputed adhesion proteins on plasma membranes, have been identified (1-6, 16, 26). Serial studies on inducible transcription of the adhesion protein gene, ap65-1, revealed that its transcription is critically coregulated by three Myb proteins, Myb1, Myb2, and Myb3, through competitive entries of multiple Myb recognition elements in the distal promoter region in a space-and time-dependent manner (18,(28)(29)(30)38), suggesting that these Myb proteins are differentially regulated in response to various stimuli from host environments. Myb proteins are ubiquitous in eukaryotes that share a conserved DNA-binding domain (DBD) composed of one to three repeat sequences termed R1, R2R3, and R1R2R3.…”

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confidence: 99%

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Iron-Inducible Nuclear Translocation of a Myb3 Transcription Factor in the Protozoan Parasite Trichomonas vaginalis

Hsu

Indra

et al. 2012

Eukaryot Cell

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cIn Trichomonas vaginalis, a novel nuclear localization signal spanning the folded R2R3 DNA-binding domain of a Myb2 protein was previously identified. To study whether a similar signal is used for nuclear translocation by other Myb proteins, nuclear translocation of Myb3 was examined in this report. When overexpressed, hemagglutinin-tagged Myb3 was localized to nuclei of transfected cells, with a cellular distribution similar to that of endogenous Myb3. Fusion to a bacterial tetracycline repressor, R2R3, of Myb3 that spans amino acids (aa) 48 to 156 was insufficient for nuclear translocation of the fusion protein, unless its C terminus was extended to aa 167. The conserved isoleucine in helix 2 of R2R3, which is important for Myb2's structural integrity in maintaining DNA-binding activity and nuclear translocation, was also vital for the former activity of Myb3, but less crucial for the latter. Sequential nuclear influx and efflux of Myb3, which require further extension of the nuclear localization signal to aa 180, were immediately induced after iron repletion. Sequence elements that regulate nuclear translocation with cytoplasmic retention, nuclear influx, and nuclear efflux were identified within the C-terminal tail. These results suggest that the R2R3 DNAbinding domain also serves as a common module for the nuclear translocation of both Myb2 and Myb3, but there are intrinsic differences between the two nuclear localization signals.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Iron-Inducible Nuclear Translocation of a Myb3 Transcription Factor in the Protozoan Parasite Trichomonas vaginalis

Hsu

Indra

et al. 2012

Eukaryot Cell

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“…XP_001317116) or Rpb3 (GenBank accession no. XP_001313008) tagged with double-HA epitope at the C terminus were used for ChIP assays as previously described (Ong et al 2006), except washes were conducted twice with each of the following buffers: (1) 1% Triton X-100, 1 mM EDTA, 50 mM HEPES (pH 7.5), 150 mM NaCl, 0.1% deoxycholate; (2) 1% Triton X-100, 1 mM EDTA, 50 mM HEPES (pH 7.5), 500 mM NaCl, 0.1% deoxycholate; (3) 0.5% NP-40, 1 mM EDTA, 10 mM Tris-HCl (pH 8.0), 250 mM LiCl, 0.5% deoxycholate. For negative controls, either wild-type cells were included or the primary anti-HA antibody was omitted for the transfected cell samples.…”

Section: Chromatin Immunoprecipitation (Chip) Analysismentioning

confidence: 99%

Spliceosomal snRNAs in the unicellular eukaryote Trichomonas vaginalis are structurally conserved but lack a 5′-cap structure

Simões-Barbosa

Meloni²,

Wohlschlegel³

et al. 2008

RNA

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Few genes in the divergent eukaryote Trichomonas vaginalis have introns, despite the unusually large gene repertoire of this human-infective parasite. These introns are characterized by extended conserved regulatory motifs at the 59 and 39 boundaries, a feature shared with another divergent eukaryote, Giardia lamblia, but not with metazoan introns. This unusual characteristic of T. vaginalis introns led us to examine spliceosomal small nuclear RNAs (snRNAs) predicted to mediate splicing reactions via interaction with intron motifs. Here we identify T. vaginalis U1, U2, U4, U5, and U6 snRNAs, present predictions of their secondary structures, and provide evidence for interaction between the U2/U6 snRNA complex and a T. vaginalis intron. Structural models predict that T. vaginalis snRNAs contain conserved sequences and motifs similar to those found in other examined eukaryotes. These data indicate that mechanisms of intron recognition as well as coordination of the two catalytic steps of splicing have been conserved throughout eukaryotic evolution. Unexpectedly, we found that T. vaginalis spliceosomal snRNAs lack the 59 trimethylguanosine cap typical of snRNAs and appear to possess unmodified 59 ends. Despite the lack of a cap structure, U1, U2, U4, and U5 genes are transcribed by RNA polymerase II, whereas the U6 gene is transcribed by RNA polymerase III.

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“…Two transcription activators, Myb2 and Myb3, and a Myb1 repressor were demonstrated to coregulate inducible transcription of the ap65-1 gene (15,34,35,44), which encodes a 65-kDa malic enzyme that may also serve as an adhesion protein under iron-replete conditions (21,31). The parasite genome encodes a super family of Myb proteins comprising Ͼ400 members (3), most of which share a conserved R2R3 DNA-binding domain (DBD), which comprises six ␣-helices spanning ϳ100 amino acid (aa) residues (19,28,33), and highly variable N and C termini.…”

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A Highly Organized Structure Mediating Nuclear Localization of a Myb2 Transcription Factor in the Protozoan Parasite Trichomonas vaginalis

et al. 2011

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Nuclear proteins usually contain specific peptide sequences, referred to as nuclear localization signals (NLSs), for nuclear import. These signals remain unexplored in the protozoan pathogen, Trichomonas vaginalis. The nuclear import of a Myb2 transcription factor was studied here using immunodetection of a hemagglutinin-tagged Myb2 overexpressed in the parasite. The tagged Myb2 was localized to the nucleus as punctate signals. With mutations of its polybasic sequences, 48KKQK51 and 61KR62, Myb2 was localized to the nucleus, but the signal was diffusive. When fused to a C-terminal non-nuclear protein, the Myb2 sequence spanning amino acid (aa) residues 48 to 143, which is embedded within the R2R3 DNA-binding domain (aa 40 to 156), was essential and sufficient for efficient nuclear import of a bacterial tetracycline repressor (TetR), and yet the transport efficiency was reduced with an additional fusion of a firefly luciferase to TetR, while classical NLSs from the simian virus 40 T-antigen had no function in this assay system. Myb2 nuclear import and DNA-binding activity were substantially perturbed with mutation of a conserved isoleucine (I74) in helix 2 to proline that altered secondary structure and ternary folding of the R2R3 domain. Disruption of DNA-binding activity alone by point mutation of a lysine residue, K51, preceding the structural domain had little effect on Myb2 nuclear localization, suggesting that nuclear translocation of Myb2, which requires an ordered structural domain, is independent of its DNA binding activity. These findings provide useful information for testing whether myriad Mybs in the parasite use a common module to regulate nuclear import.

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Multifarious Transcriptional Regulation of Adhesion Protein Gene ap65 - 1 by a Novel Myb1 Protein in the Protozoan Parasite Trichomonas vaginalis

Cited by 46 publications

References 37 publications

Iron-Inducible Nuclear Translocation of a Myb3 Transcription Factor in the Protozoan Parasite Trichomonas vaginalis

Iron-Inducible Nuclear Translocation of a Myb3 Transcription Factor in the Protozoan Parasite Trichomonas vaginalis

Spliceosomal snRNAs in the unicellular eukaryote Trichomonas vaginalis are structurally conserved but lack a 5′-cap structure

A Highly Organized Structure Mediating Nuclear Localization of a Myb2 Transcription Factor in the Protozoan Parasite Trichomonas vaginalis

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