Phosphorylation of Pax2 and Pax1 proteins by ERK1 associated complexes.

<p>(A) Diagrams of the pPERK1 and pPERK1m plasmids. The <i>pac</i> gene (open box) expression cassette is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030614#pone-0030614-g002" target="_blank">Figure 2C</a>. The <i>erk1</i> gene is under the control of its own 5′-flanking region (open boxes) and the 3′-flanking region of the <i>ran</i> gene (dotted box). ERK1m does not contain the predicted kinase domain (residues 26–202)(gray box). The filled box indicates the coding sequence of the HA epitope tag. (B) Similar levels of immunoprecipitated ERK1 protein from the vegetative and encysting pPERK1 cultures used in kinase assays. The pPERK1 stable transfectants were cultured in growth (Veg, vegetative growth) or encystation medium for 24 h (Enc, encystation) and then subjected to IP-kinase assays using anti-HA antibody. The addition of similar levels of the HA-tagged ERK1 protein from the vegetative and encysting pPERK1 cultures in each kinase reaction was confirmed by Western blot using an anti-HA antibody (left panel). The addition of similar levels of the HA-tagged ERK1 protein from the encysting pPERK1, and pPERK1m cultures in each kinase reaction was confirmed by Western blot using an anti-HA antibody (right panel). Equal amounts of protein loading were confirmed by SDS-PAGE and Coomassie blue staining. (C) Encystation-induced kinase activity of ERK1 for Pax2 substrate. The pPERK1 stable transfectants were cultured in growth (Veg, vegetative growth) or encystation medium for 24 h (Enc, encystation) and then subjected to IP-kinase assays using anti-HA antibody. Kinase activity was measured using purified recombinant Pax2 as a substrate. As a negative control, an IP-kinase assay was performed with the encysting 5′▵5N-Pac cultures which did not express the HA-tagged ERK1 protein (lane 5). Another IP-kinase assay was performed with the encysting pPERK1m cultures which expressed the ERK1m-HA protein without the predicted kinase domain (residues 26–202) (lane 7). To account for ERK1 autophosphorylation, an additional control without substrate but with immunoprecipitated ERK1-HA was also included (lane 1). (D) Encystation-induced kinase activity of ERK1 for Pax1 substrate. IP-kinase assays were performed as described above, except that Pax1 substrate was used.</p

Recruitment of Pax2 to the <i>cwp1-3</i> and <i>myb2</i> promoters.

<p>(A) Microarray analysis. Microarray data were obtained from the 5′▵5N-Pac and pPPax1 (or pPPax2) cell lines during vegetative growth. Fold changes are shown as the ratio of transcript levels in the pPPax1 (or pPPax2) cell line relative to the 5′▵5N-Pac cell line. Results are expressed as the means ± standard error of at least three separate experiments. (B) Pax2 and Pax1 overexpression generated similar gene expression patterns. The Venn diagrams illustrate the overlap of altered gene expression between the Pax2 and Pax1 overexpressing cells. Thirty eight and 185 genes were up-regulated (i.e. increased levels of gene expression relative to the control) in the Pax1 and Pax2 overexpressing cells, respectively. Among them, nineteen genes overlap. Fifty four and 172 genes were down-regulated in the Pax1 and Pax2 overexpressing cells, respectively. Among them, thirty genes overlap. (C) ChIP assays. The non-transfected WB cells were cultured in growth medium for 24 h and then subjected to ChIP assays. Anti-Pax2 antibody was used to assess binding of Pax2 to endogenous gene promoters. Preimmune serum was used as a negative control. Immunoprecipitated chromatin was analyzed by PCR using primers that amplify the 5′-flanking region of specific genes. At least three independent experiments were performed. Representative results are shown. Immunoprecipitated products of Pax2 yielded more PCR products of <i>pax2</i>, <i>cwp1</i>, <i>cwp2</i>, <i>cwp3</i>, <i>myb2</i>, and <i>ran</i> promoters, indicating that Pax2 was bound to these promoters. The <i>18S ribosomal RNA</i> gene promoter was used as a negative control for our ChIP analysis.</p

Domain architecture of Pax2 protein and alignment of the paired domains.

<p>(A) Schematic representation of the giardial Pax2 protein. The gray boxes indicate the paired domains. (B) Alignment of the paired domains. The paired domains from members of the human and <i>Drosophila</i> Pax family are analyzed by ClustalW 1.83 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030614#pone.0030614-Chenna1" target="_blank">[80]</a>. GenBank accession numbers for human Pax1 to 9 and <i>Drosophila</i> Prd are NM_006192, NM_000278, NM_181458, NM_006193, NM_016734, NM_000280, NM_001135254, NM_003466, NM_006194, and NM_164990, respectively. The open reading frame numbers (GenBank accession numbers) for the giardial Pax1 and Pax2 are 32686 (XM_001704983.1) and 16640 (XM_001709076.1) in the <i>G. lamblia</i> genome data base, respectively. Letters in black boxes, letters in gray boxes, and hyphens indicate identical amino acids, similar amino acids and gaps in the respective proteins, respectively. Gray boxes indicate the α helices in the paired domain of human Pax6 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030614#pone.0030614-Xu1" target="_blank">[58]</a>. The arrows indicate the key residues contacting the major groove in human Pax6 or <i>Drosophila</i> Prd <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030614#pone.0030614-Xu1" target="_blank">[58]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030614#pone.0030614-Xu2" target="_blank">[62]</a>. The arrowheads indicate the residues that make contact with the minor groove/phosphodiester backbone in human Pax6 or <i>Drosophila</i> Prd <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030614#pone.0030614-Xu1" target="_blank">[58]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030614#pone.0030614-Xu2" target="_blank">[62]</a>. Two regions (residues 185–205 and 226–248) rich in basic amino acid residues are underlined by dotted lines. (C) Alignment of N-terminal regions of giardial Pax2 and Pax1 ClustalW 1.83 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030614#pone.0030614-Chenna1" target="_blank">[80]</a>. Letters in black boxes, letters in gray boxes, and hyphens indicate identical amino acids, similar amino acids and gaps in the respective proteins, respectively.</p

Interaction between ERK1 and Pax2 (or Pax1).

<p>(A) Co-immunoprecipitation assay. The 5′Δ5N-Pac, pPERK1, and pPERK1m stable transfectants were cultured in encystation medium for 24 h. Proteins from cell lysates were immunoprecipitated using anti-HA antibody conjugated to beads. The precipitates were analyzed by Western blot with anti-HA, anti-Pax2, or anti-Pax1 antibody as indicated. (B) Expression of HA tagged ERK1, Pax2, and Pax1 proteins in whole cell extracts. The 5′Δ5N-Pac, pPERK1, and pPERK1m stable transfectants were cultured in encystation medium for 24 h (Enc, encystation) and then subjected to Western blot analysis. The blot was probed by anti-HA, anti-Pax2, anti-Pax1, and anti-RAN antibody. Equal amounts of protein loading were confirmed by SDS-PAGE and Coomassie blue staining. (C) RT-PCR analysis of gene expression in the ERK1- and ERK1m-overexpressing cell line. The 5′Δ5N-Pac, pPERK1, and pPERK1m stable transfectants were cultured in encystation medium for 24 h (Enc, encystation) and then subjected to RT-PCR analysis. PCR was performed using primers specific for <i>pax2</i>, <i>pax1</i>, <i>ran</i>, and <i>18S ribosomal RNA</i> genes.</p

Activation of <i>cwp1-3</i> and <i>myb2</i> gene expression in the Pax2 overexpressing cell line.

<p>(A) Overexpression of Pax2 increased the levels of CWP1 protein. The 5′▵5N-Pac, pPPax2, and pPPax2m1-3 stable transfectants were cultured in growth medium and then subjected to SDS-PAGE and Western blot. The blot was probed by anti-Pax2, anti-HA and anti-CWP1 antibody. Equal amounts of protein loading were confirmed by SDS-PAGE and Coomassie blue staining. Representative results are shown. (B) RT-PCR analysis of gene expression in the Pax2 and Pax2m1-3 overexpressing cell lines. The 5′▵5N-Pac, pPPax2, and pPPax2m1-3 stable transfectants were cultured in growth medium and then subjected to RT-PCR analysis using primers specific for <i>pax2-ha</i>, <i>pax2</i>, <i>cwp1</i>, <i>cwp2</i>, <i>cwp3</i>, <i>myb2</i>, <i>ran</i>, and <i>18S ribosomal RNA</i> genes. (C) Quantitative real-time PCR analysis of gene expression in the Pax2 and Pax2m1-3 overexpressing cell lines. Real-time PCR was performed using primers specific for <i>pax2-ha</i>, <i>pax2</i>, <i>cwp1</i>, <i>cwp2</i>, <i>cwp3</i>, <i>myb2</i>, <i>ran</i>, and <i>18S ribosomal RNA</i> genes. Similar mRNA levels of the <i>ran</i> and <i>18S ribosomal RNA</i> genes for these samples were detected. Transcript levels were normalized to 18S ribosomal RNA levels. Fold changes in mRNA expression are shown as the ratio of transcript levels in the pPPax2 or pPPax2m1-3 cell line relative to the 5′▵5N-Pac cell line. Results are expressed as the means ± standard error of at least three separate experiments. (D) Cyst count. The 5′▵5N-Pac, 5′▵5N-Pac, pPPax2, and pPPax2m1-3 stable transfectants were cultured in growth medium and then subjected to cyst count as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030614#s2" target="_blank"><b><u>Methods</u></b></a>”. The sum of total cysts is expressed as relative expression level over control. Values are shown as means ± standard error.</p

Localization of Pax2 mutants.

<p>(A) Diagrams of the Pax2 and Pax2m1-3 proteins. The gray box indicates the paired domain. Pax2m3 does not contain the C-terminal paired domain and C-terminal region (residues 172–302). Pax2m1 and Pax2m2 contain a mutation of two stretches of basic amino acids located inside of the paired domain (residues 185–205 for Pax2m1; residues 226–248 for Pax2m2). The <i>pax2</i> gene was mutated and subcloned to replace the wild type <i>pax2</i> gene in the backbone of pPPax2 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030614#pone-0030614-g002" target="_blank">Figure 2C</a>), and the resulting plasmids pPPax2m1-3 were transfected into <i>Giardia</i>. (B) Immunofluorescence analysis of Pax2m1-3 distribution. The pPPax2m1-3 stable transfectants were cultured in growth (Veg, vegetative growth) or encystation medium for 24 h (Enc, encystation) and then subjected to immunofluorescence analysis using anti-HA antibody for detection.</p

Analysis of <i>pax2</i> gene expression.

<p>(A) RT-PCR and quantitative real-time PCR analysis of <i>pax2</i> gene expression. RNA samples were prepared from <i>G. lamblia</i> wild-type non-transfected WB cells cultured in growth (Veg, vegetative growth) or encystation medium and harvested at 24 h (Enc, encystation). RT-PCR was performed using primers specific for <i>pax2</i>, <i>cwp1</i>, <i>ran</i>, and <i>18S ribosomal RNA</i> genes. Ribosomal RNA quality and loading controls are shown in the bottom panel. Representative results are shown on the left. Real-time PCR was performed using primers specific for <i>pax2</i>, <i>cwp1</i>, <i>ran</i> and <i>18S ribosomal RNA</i> genes. Transcript levels were normalized to 18S ribosomal RNA levels. Fold changes in mRNA expression are shown as the ratio of transcript levels in encysting cells relative to vegetative cells. Results are expressed as the means ± standard error of at least three separate experiments (right panel). (B) Pax2 protein levels in different stages. The wild-type non-transfected WB cells were cultured in growth (Veg, vegetative growth) or encystation medium for 24 h (Enc, encystation) and then subjected to SDS-PAGE and Western blot. The blot was probed by anti-Pax2, anti-RAN, or anti-α tubulin antibody. Representative results are shown. Equal amounts of protein loading were confirmed by SDS-PAGE and Coomassie blue staining. (C) Diagrams of the 5′▵5N-Pac and pPPax2 plasmid. The <i>pac</i> gene (open box) is under the control of the 5′- and 3′-flanking regions of the <i>gdh</i> gene (striated box). In construct pPPax2, the <i>pax2</i> gene is under the control of its own 5′-flanking region (open box) and the 3′-flanking region of the <i>ran</i> gene (dotted box). The filled black box indicates the coding sequence of the HA epitope tag. (D) Pax2 protein levels in pPPax2 stable transfectants. The pPPax2 stable transfectants were cultured in growth (Veg, vegetative growth) or encystation medium for 24 h (Enc, encystation) and then subjected to SDS-PAGE and Western blot. HA-tagged Pax2 protein was detected using an anti-HA antibody by Western blot analysis. The blot was also probed by anti-RAN or anti-α tubulin antibody. Equal amounts of protein loading were confirmed by SDS-PAGE and Coomassie blue staining. (E) Nuclear localization of Pax2. The pPPax2 stable transfectants were cultured in growth (Veg, left panels) or encystation medium for 24 h (Enc, right panels), and then subjected to immunofluorescence analysis using anti-HA antibody for detection. The product of pPPax2 localizes to the nuclei in both vegetative and encysting trophozoites (upper panels). The middle panels show the DAPI staining of cell nuclei. The bottom panels are the merged images of the DAPI staining and images of Pax2-HA.</p

Analysis of Pax2 binding ability.

<p>(A) Pax2 may bind to AT-rich sequence. Electrophoretic mobility shift assays were performed using purified Pax2 and various ³²P-end-lableled oligonucleotide probes as described. Components in the binding reaction mixtures are indicated above the lanes. The arrowhead indicates the shifted complex. “+”, “+/−”, and “−” represent moderate binding, weak binding, and no binding, respectively. “+++” and “++” represent strong binding. (B) Effect of distamycin A on the binding of Pax2 to DNA. ³²P end-labeled cwp1-45/−1 probe was incubated with Pax2 in the absence (lane 1) or presence of distamycin A (lanes 3–7). The arrowhead indicates the shifted complex. DistamycinA was dissolved in Me2SO. Adding Me2SO to the reaction mix did not decrease the Pax2 binding activity (lane 2).</p

Detection of Pax2 binding sites in multiple promoters.

<p>Electrophoretic mobility shift assays were performed using purified Pax2 and various ³²P-end-lableled oligonucleotide probes as described. Components in the binding reaction mixtures are indicated above the lanes. The arrowhead indicates the shifted complex. The transcription start sites of the <i>cwp2</i>, <i>cwp3</i>, and <i>myb2</i> genes determined from 24-h encysting cells are indicated by asterisks <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030614#pone.0030614-Lujan1" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030614#pone.0030614-Sun1" target="_blank">[14]</a>. The transcription start sites of the <i>ran</i> gene determined from vegetative cells are indicated by asterisks <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030614#pone.0030614-Sun2" target="_blank">[26]</a>. The AT-rich Inr elements spanning the transcription start sites are underlined. The translation start sites of the <i>cwp2</i> and <i>cwp3</i> genes are framed. “18S” represents 18S ribosomal RNA. “+”, “+/−”, and “−” represent moderate binding, weak binding, and no binding, respectively. “+++” and “++” represent strong binding.</p