Regulation of nuclear translocation of the Myb1 transcription factor by TvCyclophilin 1 in the protozoan parasite Trichomonas vaginalis.

In Trichomonas vaginalis, a Myb1 protein was previously demonstrated to repress transcription of an iron-inducible ap65-1 gene. In this study, a human cyclophilin A homologue, TvCyclophilin 1 (TvCyP1), was identified as a Myb1-binding protein using a bacterial two-hybrid library screening system. The recombinant TvCyP1 exhibited typical peptidyl-prolyl isomerase activity with kcat/Km of ∼7.1 µm(-1) s(-1). In a pulldown assay, the His-tagged Myb1 interacted with a GST-TvCyP1 fusion protein, which had an enzymatic proficiency half that of recombinant TvCyP1. Both the enzymatic proficiency of GST-TvCyP1 and its binding to His-Myb1 were eliminated by mutation of Arg(63) in the catalytic motif or inhibited by cyclosporin A. TvCyP1 was primarily localized to the hydrogenosomes by immunofluorescence assay, but it was also co-purified with Myb1 in certain vesicle fractions from differential and gradient centrifugations. Transgenic cells overexpressing HA-TvCyP1 had a higher level of nuclear Myb1 but a much lower level of Myb1 associated with the vesicles than control and those overexpressing HA-TvCyP1(R63A). Myb1 was detected at a much higher level in the HA-TvCyP1 protein complex than in the HA-TvCyP1(R63A) protein complex immunoprecipitated from P15 and P100, but not S100, fractions of postnuclear lysates. A TvCyP1-binding motif, (105)YGPKWNK(111), was identified in Myb1 in which Gly(106) and Pro(107) were essential for its binding to TvCyP1. Mutation of Gly(106) and Pro(107), respectively, in HA-Myb1 resulted in cytoplasmic retention and elevated nuclear translocation of the overexpressed protein. These results suggest that TvCyP1 may induce the release of Myb1 that is restrained to certain cytoplasmic vesicles prior to its nuclear translocation.

Iron-inducible nuclear translocation of a Myb3 transcription factor in the protozoan parasite Trichomonas vaginalis.

In 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 DNA-binding 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.

A highly organized structure mediating nuclear localization of a Myb2 transcription factor in the protozoan parasite Trichomonas vaginalis.

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.

Transcriptional regulation of an iron-inducible gene by differential and alternate promoter entries of multiple Myb proteins in the protozoan parasite Trichomonas vaginalis.

Iron-inducible transcription of a malic enzyme gene (also reputed to be ap65-1) in Trichomonas vaginalis was previously shown to involve a Myb1 repressor and a Myb2 activator, each of which may preferentially select two closely spaced promoter sites, MRE-1/MRE-2r, which comprises overlapping promoter elements, and MRE-2f. In the present study, an iron-inducible approximately 32-kDa Myb3 nuclear protein was demonstrated to bind only the MRE-1 element. Changes in the iron supply, which produced antagonistic effects on the levels of Myb2 and Myb3 expression, also resulted in temporal and alternate entries of Myb2 and Myb3 into the ap65-1 promoter. Repression or activation of basal and iron-inducible ap65-1 transcription was detected in transfected cells when Myb3 was, respectively, substantially knocked down or overexpressed. In the latter case, increased Myb3 promoter entry was detected with concomitant decrease in Myb2 promoter entry under specific conditions, while Myb3 promoter entry was inhibited under all test conditions in cells overexpressing Myb2. In contrast, concomitant promoter entries by Myb2 and Myb3 diminished in cells overexpressing Myb1, except that Myb3 promoter entry was slightly affected under prolonged iron depletion. Together, these results suggest that Myb2 and Myb3 may coactivate basal and iron-inducible ap65-1 transcription against Myb1 through conditional and competitive promoter entries.

Activation of multifarious transcription of an adhesion protein ap65-1 gene by a novel Myb2 protein in the protozoan parasite Trichomonas vaginalis.

Multifarious transcription of the adhesion protein ap65-1 gene in the human pathogen, Trichomonas vaginalis, is critically regulated by the coordination of two similar but opposite oriented DNA regulatory regions, MRE-1/MRE-2r and MRE-2f, both of which are binding sites for multiple Myb-like proteins. In the present study, MRE-1/MRE-2r was demonstrated to be composed of multiple overlapping promoter elements, among which the entire region is required for growth-related ap65-1 transcription, and the 5'-MRE-1 antagonizes the suppressive activity of the 3'-MRE-2r in iron-inducible transcription. The recombinant Myb2 protein derived from a previously identified myb2 gene was demonstrated to recognize distinct sequence contexts in MRE-2r and MRE-2f, whereas Myb2 in the nuclear lysate preferentially binds to MRE-2f to MRE-2r. Iron repletion resulted in persistent repression of the myb2 gene, and temporal activation/deactivation of Myb2 promoter entry, which was also activated by prolonged iron depletion. The hemagglutinintagged Myb2 when overexpressed during iron-depleted conditions facilitated basal and growth-related ap65-1 transcription to a level that was achieved in iron-replete cells, whereas ironinducible ap65-1 transcription was abolished with knockdown of Myb2. These findings demonstrated that Myb2 is involved in activation of growth-related and iron-inducible transcription of the ap65-1 gene, possibly through differential promoter selection in competition with other Myb proteins.

Multifarious transcriptional regulation of adhesion protein gene ap65-1 by a novel Myb1 protein in the protozoan parasite Trichomonas vaginalis.

The transcription efficiency of an adhesion protein gene, ap65-1, in Trichomonas vaginalis varies with changes in the iron supply and with the growth stage. In the present study, two Myb recognition elements, MRE-1/MRE-2r and MRE-2f, were found to play antagonistic roles in regulating the iron-inducible activity of an ap65-1 reporter gene. Intriguingly, either of these elements was shown to be sufficient to repress basal activity, but together they were also shown to activate growth-related activity of the reporter gene in iron-depleted cells. A myb1 gene which encodes a 24-kDa protein containing a Myb-like R2R3 DNA binding domain was identified from Southwestern screening of MRE-2f-binding proteins. The Myb1 protein was detected as a major 35-kDa protein which exhibited variations in nuclear concentration with changes in the iron supply. A recombinant Myb1 protein was shown to differentially interact with MRE-1/MRE-2r and MRE-2f in vitro. Overexpression of hemagglutinin-tagged Myb1 in T. vaginalis resulted in repression or activation of ap65-1 transcription in iron-depleted cells at an early and a late stage of cell growth, respectively, while iron-inducible ap65-1 transcription was constitutively repressed. The hemagglutinin-tagged Myb1 protein was found to constantly occupy the chromosomal ap65-1 promoter at a proximal site, but it also selected two more distal sites only at the late growth stage. Together, these observations suggest that Myb1 critically regulates multifarious ap65-1 transcription, possibly via differential selection of multiple promoter sites upon environmental changes.

Involvement of multiple DNA elements in iron-inducible transcription of the ap65-1 gene in the protozoan parasite Trichomonas vaginalis.

A reputed iron-responsive region, which contains multiple nuclear protein-binding DNA sequences, was shown previously to regulate iron-inducible transcription of the ap65-1 gene in the protozoan pathogen, Trichomonas vaginalis. These DNA sequences include two overlapping MYB recognition elements (MRE-1/MRE-2r) and three abutted T-tract elements. Additional nuclear protein-binding DNA sequences flanking the 5' (AGTGAAGTGA) and 3' (MRE-2f) of the iron-responsive region were identified in the present study. A stable promoter assay and primer extension revealed that transcriptional activity of the ap65-1 promoter is iron inducible as well as growth related, being lowest in the early logarithmic phase and highest in the mid-logarithmic phase. Subsequent mutational analysis of individual DNA elements of the ap65-1 promoter suggests that closely spaced T-tract elements together with an intervening GAAGGAAG sequence within the iron-responsive region are most critical for regulation of overall transcriptional activity, whereas an additional AGTGAAGTGA and MRE-2f together with an upstream T-rich region are required for optimal iron-inducible activity, and the MRE-1/MRE-2r overlap is only involved in growth-related activity. These observations suggest that expression of the ap65-1 gene is dynamically regulated under various growth conditions via interactions among multiple DNA regulatory elements.

Characterization of a bi-directional promoter for divergent transcription of a PHD-zinc finger protein gene and a ran gene in the protozoan pathogen Giardia lamblia.

We showed previously that transcription of the ran gene in Giardia lamblia is regulated by an AT-rich initiator. In the present study, the ran initiator was found to regulate transcription of a neighbouring PHD zinc-finger protein gene. Deletion and scanning mutagenesis of the phd promoter in a firefly luciferase reporter system showed that the promoter activity is determined by multiple single-stranded T-tract DNA elements distributed into a distal domain spanning the ran initiator (-134/-103) and a proximal domain (-88/-48) spanning phd messenger RNA (mRNA) start sites (-74, -55 and -53 relative to the first ATG). The promoter activity is repressed by the single T-tract element on a non-template strand of the ran initiator, and is activated by closely spaced T-tract elements on the opposite strand. The T-tract elements in the phd and ran initiators compete for similar ssDNA binding proteins. Mutation of -47/-42 resulted in dramatic reduction of luciferase activity without changing luciferase mRNA levels, indicating the potential involvement of a regulatory mechanism in PHD protein translation. These findings suggest that G. lamblia uses multiple copies of a T-tract element as both core and distal elements in regulating transcription initiation, and that expression of the phd gene is regulated at multiple levels.