The HMGB Protein Kl Ixr1, a DNA Binding Regulator of Kluyveromyces lactis Gene Expression Involved in Oxidative Metabolism, Growth, and dNTP Synthesis

Abstract

In the traditional fermentative model yeast Saccharomyces cerevisiae, ScIxr1 is an HMGB (High Mobility Group box B) protein that has been considered as an important regulator of gene transcription in response to external changes like oxygen, carbon source, or nutrient availability. Kluyveromyces lactis is also a useful eukaryotic model, more similar to many human cells due to its respiratory metabolism. We cloned and functionally characterized by different methodologies KlIXR1, which encodes a protein with only 34.4% amino acid sequence similarity to ScIxr1. Our data indicate that both proteins share common functions, including their involvement in the response to hypoxia or oxidative stress induced by hydrogen peroxide or metal treatments, as well as in the control of key regulators for maintenance of the dNTP (deoxyribonucleotide triphosphate) pool and ribosome synthesis. KlIxr1 is able to bind specific regulatory DNA sequences in the promoter of its target genes, which are well conserved between S. cerevisiae and K. lactis. Oppositely, we found important differences between ScIrx1 and KlIxr1 affecting cellular responses to cisplatin or cycloheximide in these yeasts, which could be dependent on specific and non-conserved domains present in these two proteins.

Keywords: IXR1; Kluyveromyces lactis; cisplatin sensitivity; dNTP pool; hypoxia.

Figure 1

(a) In silico comparison of Ixr1 amino acid sequence from S. cerevisiae and K. lactis.(upper) Scheme of protein domain distribution in Ixr1 protein from S. cerevisiae and K. lactis. (lower) Pairwise alignment of the Ixr1 double HMG-box domains arranged in tandem from S. cerevisiae (Uniprot code P33417) and K. lactis (Uniprot code Q6CMQ4). Conserved amino acids are highlighted in bold and conserved regions indicated by grey boxes. Small and hydrophobic amino acids (less tyrosine) are indicated in red, acidic amino acids in blue, basic amino acids (less histidine) in magenta and hydroxyl + sulfhydryl + amine (STYHCNGQ) in green. (b) Model superposition of HMG-box A (S. cerevisiae in dark green and K. lactis in dark blue) and HMG-box B (S. cerevisiae in light green and K. lactis in light blue). Enlarged frames show the conserved aromatic residues that form the cluster stabilizing the three-helix HMG folding.

Figure 2

Effect of cisplatin treatment on cell growth (in logarithmic and stationary phase) of strain W303 and derivatives. ixr1Δ null strain was transformed with the plasmid pAG426GAL-ccdB, either empty (Ø), or containing the IXR1 ORFs from S. cerevisiae or K. lactis under the control of the GAL1 promoter. Serial dilutions of the cells (1, 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵; from left to right) were made from an initial culture of OD₆₀₀ = 1.0 and were grown at 30 °C for three days on YPGal plates with different concentrations of cisplatin as specified.

Figure 3

Study of the cellular response of MW190-9b strain (black) and its derivative ixr1Δ (red) to cisplatin treatment. (a) Resistance to cisplatin in the wild type and klixr1Δ null strains. Cells from the K. lactis strain MW190-9B and its isogenic derivative MW190-9B-ixr1Δ were treated with several concentrations of cisplatin (0, 50, 100, 200, 400, and 600 µM). After 24 h of growth at 30 °C, optical density measurements at 600 nm were taken, and survival rates were calculated by normalization from untreated cultures. (b) rRNA cellular content determination by microfluidic-based automated electrophoresis. (c) mRNA levels of KlSFP1, KlCRF1, KlSCH9, KlTOR1, KlRAP1, KlIFH1, KlFHL1, and KlDOT6 were analyzed by RT-qPCR, before (solid) and after (streaked) the treatment with 600 µM of the chemotherapeutic agent cisplatin. Housekeeping gene KlTAF10 was used for gene normalization. * p < 0.05; ** p < 0.01.

Figure 4

Effect of KlIXR1 disruption on KlHEM13 expression under aerobic (solid) or hypoxic conditions (grid) in the MW190-9b strain from K. lactis were analyzed by (a) Northern blot and (b) RT-qPCR experiments. Blots for (a) were obtained in triplicate experiments and a representative picture is shown in upper left panel. KlSNR17A and KlTAF10 signal levels were used as references for normalization in (a,b), respectively. * p < 0.05; ** p < 0.01.

Figure 5

Analysis of KlIxr1 protein binding to the KlHEM13 promoter region. (a) EMSA assays showing sequence specificity to both binding sites found using the consensus sequences YYYATTGTTCTC and KTTSAAYKGTTYASA previously described [23,24]. Competition assays were conducted with increasing amounts of non-labeled ligands indicated. WT: ligand not mutated; M1: ligand mutated in first site; M2: ligand mutated in second site; NB: no retarded band (no protein added). (b) Klotz plots representing quantitative analysis of KlIxr1 binding to both KlHEM13 promoter deduced sites. DNA binding measured by fluorescence anisotropy changes of the 5′ fluorescein-labelled DNA (100 nM ligand) upon protein titration (see Section 2). The resulting semi-log binding isotherms were fitted to a 1:1 binding model with non-linear least squares regression. Data points are the average of 3 independent experiments, error bars representing standard deviations. HEM13A: first binding site deduced from KTTSAAYKGTTYASA [23] consensus sequence; HEM13B: second binding site deduced from YYYATTGTTCTC [24] consensus sequence; * indicates FAM labeled ligand. (c) Klotz plots representing competition analysis of KlIxr1 binding to both KlHEM13 promoter-assayed sites. DNA binding measured by fluorescence anisotropy changes of the 5′ fluorescein-labelled DNA (100 nM ligand) bound to KlIxr1 protein (200 nM) and upon non-labeled DNA competitor titration (see Section 2). The resulting semi-log binding isotherms were fitted to a logIC50 competitive model with non-linear least squares regression. * indicates FAM labeled ligand.

Figure 6

Analysis of the cellular response of MW190-9b strain and its derivative ixr1Δ to hydrogen peroxide and cadmium treatments. (a) Resistance to H₂O₂ and cadmium in the wild type and klixr1Δ null strains. Serial dilutions of the cells (1, 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵; from left to right) were made from an initial culture of OD₆₀₀ = 1.0 and were grown at 30 °C for three days on CM plates supplemented with the indicated concentrations of compound. (b) EMSA assays for the KlYCF1 promoter region contains two putative contiguous binding sites for KlIXR1 deduced by using the AYKGTT core consensus sequence [23] in silico searches. Competition assays were conducted with increasing amounts of non-labeled ligands indicated. WT: ligand not mutated; M1: ligand mutated in first site; M2: ligand mutated in second site. (c) Klotz plots representing quantitative analysis of KlIxr1 binding to KlYCF1 promoter-deduced site. DNA binding measured by fluorescence anisotropy changes of the 5′ fluorescein labelled DNA (100 nM ligand) upon protein titration (Section 2). The resulting semi-log binding isotherms were fitted to a 1:1 binding model with non-linear least squares regression. Data points are the average of 3 independent experiments. Competition analysis of KlIxr1 binding to both KlYCF1 promoter deduced sites were measured by fluorescence anisotropy changes of the 5′ fluorescein labelled DNA (100 nM ligand non mutated) bound to KlIxr1 protein (200 nM) and upon non-labeled DNA competitor titration (KlIxr1 binding sites individually mutated). The resulting semi-log binding isotherms were fitted to a logIC50 competitive model with non-linear least squares regression. * indicates FAM labeled ligand.

Figure 7

Analysis of petite phenotype in MW190-9b ixr1Δ null mutant. (a) (left) Colony size of MW190-9b strain and its derivative ixr1Δ in 2% w/v glucose, 2% w/v glycerol, or 2% w/v glucose supplemented with 50 µg/mL hemin. (right) Growth tracking of MW190-9b strain (black) and its derivative ixr1Δ (red) 2% w/v galactose or 2% w/v glycerol during 48h. Growth rates were calculated using respective 2% w/v glucose cultures as normalization elements. (b) RT-qPCR results of KlABF1, KlTEC1, KlSOK2, KlUME6, and KlDAL81 genes in wild-type (black) and ixr1Δ null strain (red), before (solid) and after (streaked) the treatment with 600 µM of the chemotherapeutic agent cisplatin. Housekeeping gene KlTAF10 was used for gene normalization. * p < 0.05; ** p <0.01. (c) Cellular response to cycloheximide treatment. Cell cultures of the K. lactis strain MW190-9B (black) and its isogenic derivative MW190-9B-ixr1Δ (red) were treated with several concentrations of cycloheximide (0, 25, 50, 100, 200, and 300 µg/mL). After 24 h of growth at 30 °C, optical density measurements at 600 nm were taken, and survival rates were calculated by normalization from untreated cultures.

Figure 8

Regulatory role of KlIxr1 in the KlMec1-KlRad53-KlDun1–dependent ribonucleotide reductase pathway of K. lactis. mRNA levels of KlMEC1, KlDUN1, KlRNR1, and KlRNR2 were analyzed by RT-qPCR in the MW190-9b strain (black) and its derivative ixr1Δ (red), before (solid) and after (streaked) the treatment with 600 µM of the chemotherapeutic agent cisplatin. Housekeeping gene KlTAF10 was used for gene normalization. * p < 0.05; ** p < 0.01.