ChIP analysis of Yna1p and Yna2p occupying the <i>YNT1</i> gene promoter in different physiological conditions and genetic backgrounds.

<p><b>A</b>. GFP-tagged Yna1p expressed from its own nitrate-inducible promoter in the wild type (Yna1::GFP) and <i>YNA2Δ</i> (Yna1::GFP-<i>YNA2Δ</i>) background was analysed by ChIP in cells grown under nitrate-inducing (I) or glutamine-repressing (R) conditions. <b>B</b>. Experimental set-up for the GFP-tagged Yna2p in the wild ytpe (Yna2^OE::GFP) and <i>YNA1Δ</i> background (Yna2^OE::GFP-<i>YNA1Δ</i>) was identical to the one described for Yna1p with the exception that the Yna2p-GFP fusion protein was expressed from the strong MOX promoter in order to be able to analyse Yna2p occupancy also in a <i>YNA1Δ</i> background in which <i>YNA2</i> is normally not expressed. All values shown in this figure represent the amount of <i>YNT1</i> promoter DNA relative to the amount of input DNA in the same sample. Experiments were done in two biological repetitions and in each experiment ChIP analysis was duplicated. Error bars thus represent the standard deviations of four measurements in each condition and strain.</p

Yna1p and Yna2p interact <i>in vivo</i>.

<p>Fluorescence of the YFP protein is only visible in the reconstituted protein in the AL-YFP18 strain (wild type background) when both proteins Yna1p-YFP^Nterm and Yna2p-YFP^Cterm are simultaneously expressed (under nitrate-inducing, I, and proline non-inducing, NI, conditions) but is not detected under repressing conditions on glutamine (R). DAPI-staining detects the nuclei. Scale bars refer to 2 μm.</p

The <i>H</i>. <i>polymorpha</i> nitrate utilization cluster (GenBank #AJ223294) and its regulation.

<p><b>A.</b> The complete sequence (nt 1–13684) consists of five nitrate assimilation genes (grey shaded) flanked by a predicted glutathione S-transferase (<i>ORF-1)</i> on the one side and by a predicted <i>RAD3</i> homolog (<i>ORF-3</i>) on the other. Numbers (in bp) above the coding regions correspond to the size of the ORFs and numbers between the genes indicate the length of the intergenic regions. <i>YNT1</i>, nitrate transporter; <i>YNI1</i>, nitrite reductase; <i>YNA1</i>, Zn(II)₂Cys₆ transcriptional activator; <i>YNR1</i>, nitrate reductase; <i>YNA2</i>, Zn(II)₂Cys₆ transcriptional activator. <b>B. Northern blots showing the effect of different nitrogen sources on the expression of the nitrate assimilation genes in the <i>H</i>. <i>polymorpha</i> wild type strain (L1).</b> The actin gene mRNA signal was used as loading control. Nitrogen sources were 50mM alone or in combinations as indicated above the autoradiographs. N, nitrate; A, ammonium; Q, glutamine; E, glutamate; P, proline. Mean densitometric values (± standard error) of separate experiments, normalised to the loading control signal, are reported (expressed as percentage of maximum induction value i.e., growth on nitrate). nd, not-detectable. <b>C. Northern blot analysis investigating the role of <i>YNA1</i> and <i>YNA2</i> in the expression of the nitrate cluster genes.</b> Mutant strains are derived from the wild type (L1) and carry either a marker insertion at the <i>YNA1</i> locus (L1-yna1^I6632) or maker insertion and internal deletion at the <i>YNA2</i> locus (L1-yna2^{Δ12274–12595}). The actin gene signal was used as loading control.</p

Yna1p and Yna2p bind <i>in vitro</i> and <i>in vivo</i> to a region containing a conserved DNA motif present in all nitrate-responsive promoters.

<p><b>A.</b> Alignment of the promoter regions of genes belonging to the nitrate cluster detects a conserved motif (5′CGGAGA) indicated by six consecutive asterisks. This motif could represent a putative upstream activating sequence (UAS) bound by Yna1p and Ynap2. Probes used in the band shift assays are indicated as YNT1 probe 1 and YNT1 probe 2; the two different derivatives of YNT1 probe 2 containing the indicated changes in conserved nucleotides are shown as m1 and m2. <b>B.</b><i>In vitro</i> interaction experiments using electric mobility shift assays (EMSA). <b>FP*, labelled probe (double-stranded oligonucleotide as indicated in A) was incubated without recombinant proteins; P*, labelled probe incubated with the indicated recombinant protein but no unlabelled, specific competitor DNA has been added.; +1P, same conditions as in P* but an equimolar amount of the unlabelled double-stranded oligonucleotide was present in the incubation mixture as specific competitor for complex formation; +2P, same conditions as in +1P but double molar amount of the unlabelled double-stranded oligonucleotide was present in the incubation mixture as specific competitor.</b> Radiolabelled probes described in panel A were incubated with recombinant <i>HIS</i>-Yna1p_(1–185) and <i>HIS</i>-Yna2p_(1–180) proteins expressed in <i>E</i>. <i>coli</i> and purified (as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135416#pone.0135416.s006" target="_blank">S6 Fig</a>). <b>While Yna1p</b>_{<b>(1–185)</b>}<b>binds both</b><i>YNT1</i> probes 1 and 2, Yna2p_(1–180) doesn’t interact with the <i>YNT1</i> probe 2. Exchanging 3 nucleotides in the conserved 5′-CGGAGA-3′strech of YNT1 probe 2 disrupts the <b>Yna1p</b>_{<b>(1–185)</b>}<b>binding capability (m2). Likewise, exchange in an upstream conserved residue potentially forming a partial inverted repeat with the CGGAGA motif also disrupts complex formation (m1). This indicates that both motifs are involved in binding Yna1p. C.</b><i>In vivo footprinting</i> methylation protection profile of the <i>YNT1</i> gene promoter. Cells from the wild type strain (wt) as well as from the <i>yna</i> mutant strains (y1, corresponding to <b>L1-<i>yna1</i></b>^{<b><i>I6632</i></b>}<b>, and y2, corresponding to L1-<i>yna2</i></b>^{<b><i>Δ12274–12595</i></b>}<b>) have been incubated at the standard physiological conditions</b><i>(I</i>, <i>nitrate; NI</i>, <i>proline</i>) and subjected to ligation-mediated methylation protection footprinting as detailed in Materials and Methods. The sequence pattern of a control reaction containing <i>in vitro</i> methylated DNA is shown in lane V and the corresponding sequence derived from the <i>YNT1</i> promoter is aligned to the left of the autoradiograph. The sequence corresponding to the banding pattern in the autoradiograph lies between the diagonal dotted lines. In some cases guanines are not properly resolved in the gel but appear as compressed regions. Adenines can become hypersensitive and eventually appear as strong as guanines, mainly in the <i>in vivo</i> lanes. For a better orientation, vertical dotted lines next to the V lane (<i>in vitro</i> control reaction) and next to the sequence correspond to each other. Protected guanines are marked with a triangle and hypersensitive adenines are marked with circles. Asterisks on the displayed vertical sequence correspond to the asterisks marking the conserved residues in the <i>YNT1</i> promoter region used for designing <i>YNT1</i> probes 1 and 2. Note that the sequence displayed next to the autoradiograph is complementary to the sequence in panel A. Below the figure, the scheme shows the location of protected regions (white rectangles) in the <i>YNT1</i> promoter. The conserved 5′-CGGAGA-3′ sequence is indicated by the grey rectangle and asterisks.</p

Nuclear accumulation of Yna1p is Yna2p-dependent.

<p><b>A, B, C.</b> Yna1p expressed as a GFP fusion protein from its own promoter in the L1 wild type strain and the <b>L1-<i>yna1</i></b>^{<b><i>I6632</i></b>}<b>and L1-<i>yna2</i></b>^{<b><i>Δ12274–12595</i></b>}<b>null mutants is shown. Pictures of this figure were taken from strains incubated on nitrate but identical results were obtained on all media, i.e. also on non-inducing (proline), inducing-repressing (nitrate and glutamine) or repressing (glutamine) conditions</b>. Photographs of the cultures incubated on proline, nitrate and glutamine or only glutamine are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135416#pone.0135416.s004" target="_blank">S4 Fig</a>. In the inlet of each photograph a higher magnification shows details of one representative cell of the experiment. <b>D, E, F.</b> Yna2p-GFP localization in a strain expressing the fusion gene from the MOX promoter (to be able to detect the protein also in a <i>YNA1</i> mutant). A detail for each strain is shown as inlet in the photographs. Scale bars refer to 2 μm.</p