Mónica Lamas-Maceiras

Kluyveromyces lactis HIS4 transcriptional regulation: similarities and differences to Saccharomyces cerevisiae HIS4 gene

Sequence analysis of the Kluyveromyces lactis HIS4 (KlHIS4) gene promoter reveals relevant differences in comparison to the Saccharomyces cerevisiae HIS4 homologous gene. Among them are the absence of a Rap1 binding site and the presence of only three putative Gcn4 binding consensus sites instead of the five described in the S. cerevisiae promoter. Since these factors are implicated in the general control, we investigated the transcriptional regulation of the KlHIS4 gene under conditions of amino acid starvation and discovered that the mechanisms previously described for S. cerevisiae HIS4 regulation and related to general control are not functional in K. lactis. The expression analysis of the KlHIS4 gene under phosphate starvation or high adenine supply shows that factors, such as Bas1 or Bas2, involved in the basal control may also operate in a different way in K. lactis. Interestingly, and also in contrast to the HIS4 regulation in S. cerevisiae, we found domains for Nit2‐like and yeast‐Ap1‐like binding sequences. Northern analyses showed transcriptional activation under ammonia starvation and oxidative stress. The EMBL accession number for the KlHIS4 promoter is AJ238494.

Involvement of Pta1, Pcf11 and a KlCYC1 AU-rich element in alternative RNA 3'-end processing selection in yeast.

This work reports the involvement of yeast RNA processing factors Pta1 and Pcf11 in alternative 3′‐end RNA processing. The pta1‐1 and pcf11‐2 mutations changed the predominance of KlCYC1 1.14 and 1.5 kb transcript isoforms. Mutation of the KlCYC1 3′‐UTR AU‐rich sequence at positions 679–690 (mutant M1) altered transcript predominance. Moreover, expression of M1 in the yeast mutants partially suppressed their effects in the predominance pattern. The combination of the M1 and M2 (694–698 deletion) mutations abolished the alternative processing. Pta1 involvement in this selection was confirmed using the Pta1‐td degron strain.

High Mobility Group B Proteins, Their Partners, and Other Redox Sensors in Ovarian and Prostate Cancer

Cancer cells try to avoid the overproduction of reactive oxygen species by metabolic rearrangements. These cells also develop specific strategies to increase ROS resistance and to express the enzymatic activities necessary for ROS detoxification. Oxidative stress produces DNA damage and also induces responses, which could help the cell to restore the initial equilibrium. But if this is not possible, oxidative stress finally activates signals that will lead to cell death. High mobility group B (HMGB) proteins have been previously related to the onset and progressions of cancers of different origins. The protein HMGB1 behaves as a redox sensor and its structural changes, which are conditioned by the oxidative environment, are associated with different functions of the protein. This review describes recent advances in the role of human HMGB proteins and other proteins interacting with them, in cancerous processes related to oxidative stress, with special reference to ovarian and prostate cancer. Their participation in the molecular mechanisms of resistance to cisplatin, a drug commonly used in chemotherapy, is also revised.

Transcriptome analysis of the thermotolerant yeast Kluyveromyces marxianus CCT 7735 under ethanol stress

The thermotolerant yeast Kluyveromyces marxianus displays a potential to be used for ethanol production from both whey and lignocellulosic biomass at elevated temperatures, which is highly alluring to reduce the cost of the bioprocess. Nevertheless, contrary to Saccharomyces cerevisiae, K. marxianus cannot tolerate high ethanol concentrations. We report the transcriptional profile alterations in K. marxianus under ethanol stress in order to gain insights about mechanisms involved with ethanol response. Time-dependent changes have been characterized under the exposure of 6% ethanol and compared with the unstressed cells prior to the ethanol addition. Our results reveal that the metabolic flow through the central metabolic pathways is impaired under the applied ethanol stress. Consistent with these results, we also observe that genes involved with ribosome biogenesis are downregulated and gene-encoding heat shock proteins are upregulated. Remarkably, the expression of some gene-encoding enzymes related to unsaturated fatty acid and ergosterol biosynthesis decreases upon ethanol exposure, and free fatty acid and ergosterol measurements demonstrate that their content in K. marxianus does not change under this stress. These results are in contrast to the increase previously reported with S. cerevisiae subjected to ethanol stress and suggest that the restructuration of K. marxianus membrane composition differs in the two yeasts which gives important clues to understand the low ethanol tolerance of K. marxianus compared to S. cerevisiae.

Promoter-Terminator Gene Loops Affect Alternative 3'-End Processing in Yeast.

Many eukaryotic genes undergo alternative 3′-end poly(A)-site selection producing transcript isoforms with 3′-UTRs of different lengths and post-transcriptional fates. Gene loops are dynamic structures that juxtapose the 3′-ends of genes with their promoters. Several functions have been attributed to looping, including memory of recent transcriptional activity and polarity of transcription initiation. In this study, we investigated the relationship between gene loops and alternative poly(A)-site. Using the KlCYC1 gene of the yeast Kluyveromyces lactis, which includes a single promoter and two poly(A) sites separated by 394 nucleotides, we demonstrate in two yeast species the formation of alternative gene loops (L1 and L2) that juxtapose the KlCYC1 promoter with either proximal or distal 3′-end processing sites, resulting in the synthesis of short and long forms of KlCYC1 mRNA. Furthermore, synthesis of short and long mRNAs and formation of the L1 and L2 loops are growth phase-dependent. Chromatin immunoprecipitation experiments revealed that the Ssu72 RNA polymerase II carboxyl-terminal domain phosphatase, a critical determinant of looping, peaks in early log phase at the proximal poly(A) site, but as growth phase advances, it extends to the distal site. These results define a cause-and-effect relationship between gene loops and alternative poly(A) site selection that responds to different physiological signals manifested by RNA polymerase II carboxyl-terminal domain phosphorylation status.

Dual function of Ixr1 in transcriptional regulation and recognition of cisplatin-DNA adducts is caused by differential binding through its two HMG-boxes

Ixr1 is a transcriptional factor involved in the response to hypoxia, which is also related to DNA repair. It binds to DNA through its two in-tandem high mobility group box (HMG-box) domains. Each function depends on recognition of different DNA structures, B-form DNA at specific consensus sequences for transcriptional regulation, or distorted DNA, like cisplatin-DNA adducts, for DNA repair. However, the contribution of the HMG-box domains in the Ixr1 protein to the formation of different protein-DNA complexes is poorly understood. We have biophysically and biochemically characterized these interactions with specific DNA sequences from the promoters regulated by Ixr1, or with cisplatin-DNA adducts. Both HMG-boxes are necessary for transcriptional regulation, and they are not functionally interchangeable. The in-tandem arrangement of their HMG-boxes is necessary for functional folding and causes sequential cooperative binding to specific DNA sequences, with HMG-box A showing a higher contribution to DNA binding and bending than the HMG-box B. Binding of Ixr1 HMG boxes to specific DNA sequences is entropy driven, whereas binding to platinated DNA is enthalpy driven for HMG-box A and entropy driven for HMG-box B. This is the first proof that HMG-box binding to different DNA structures is associated with predictable thermodynamic differences. Based on our study, we present a model to explain the dual function of Ixr1 in the regulation of gene expression and recognition of distorted DNA structures caused by cisplatin treatment.

KlGcr1 controls glucose-6-phosphate dehydrogenase activity and responses to H2O2, cadmium and arsenate in Kluyveromyces lactis

It has been previously reported that Gcr1 differentially controls growth and sugar utilization in Saccharomyces cerevisiae and Kluyveromyces lactis, although the regulatory mechanisms causing activation of glycolytic genes are conserved (Neil et al., 2004). We have found that KlGCR1 deletion diminishes glucose consumption and ethanol production, but increases resistance to oxidative stress caused by H2O2, cadmium and arsenate, glucose 6P dehydrogenase activity, and the NADPH/NADP(+) and GSH/GSSG ratios in K. lactis. The gene KlZWF1 that encodes for glucose 6P dehydrogenase, the first enzyme in the pentose phosphate pathway, is transcriptionally regulated by KlGcr1. The high resistance to oxidative stress observed in the ΔKlgcr1 mutant strain, could be explained as a consequence of an increased flux of glucose through the pentose phosphate pathway. Since mitochondrial respiration decreases in the ΔKlgcr1 mutant (García-Leiro et al., 2010), the reoxidation of the NADPH, produced through the pentose phosphate pathway, has to be achieved by the reduction of other molecules implied in the defense against oxidative stress, like GSSG. The higher GSH/GSSG ratio in the mutant would explain its phenotype of increased resistance to oxidative stress.

Ixr1 Regulates Ribosomal Gene Transcription and Yeast Response to Cisplatin

Ixr1 is a Saccharomyces cerevisiae HMGB protein that regulates the hypoxic regulon and also controls the expression of other genes involved in the oxidative stress response or re-adaptation of catabolic and anabolic fluxes when oxygen is limiting. Ixr1 also binds with high affinity to cisplatin-DNA adducts and modulates DNA repair. The influence of Ixr1 on transcription in the absence or presence of cisplatin has been analyzed in this work. Ixr1 regulates other transcriptional factors that respond to nutrient availability or extracellular and intracellular stress stimuli, some controlled by the TOR pathway and PKA signaling. Ixr1 controls transcription of ribosomal RNAs and genes encoding ribosomal proteins or involved in ribosome assembly. qPCR, ChIP, and 18S and 25S rRNAs measurement have confirmed this function. Ixr1 binds directly to several promoters of genes related to rRNA transcription and ribosome biogenesis. Cisplatin treatment mimics the effect of IXR1 deletion on rRNA and ribosomal gene transcription, and prevents Ixr1 binding to specific promoters related to these processes.

Delineating the HMGB1 and HMGB2 interactome in prostate and ovary epithelial cells and its relationship with cancer

High Mobility Group B (HMGB) proteins are involved in cancer progression and in cellular responses to platinum compounds used in the chemotherapy of prostate and ovary cancer. Here we use affinity purification coupled to mass spectrometry (MS) and yeast two-hybrid (Y2H) screening to carry out an exhaustive study of HMGB1 and HMGB2 protein interactions in the context of prostate and ovary epithelia. We present a proteomic study of HMGB1 partners based on immunoprecipitation of HMGB1 from a non-cancerous prostate epithelial cell line. In addition, HMGB1 and HMGB2 were used as baits in yeast two-hybrid screening of libraries from prostate and ovary epithelial cell lines as well as from healthy ovary tissue. HMGB1 interacts with many nuclear proteins that control gene expression, but also with proteins that form part of the cytoskeleton, cell-adhesion structures and others involved in intracellular protein translocation, cellular migration, secretion, apoptosis and cell survival. HMGB2 interacts with proteins involved in apoptosis, cell motility and cellular proliferation. High confidence interactors, based on repeated identification in different cell types or in both MS and Y2H approaches, are discussed in relation to cancer. This study represents a useful resource for detailed investigation of the role of HMGB1 in cancer of epithelial origins, as well as potential alternative avenues of therapeutic intervention.

The HMGB protein Ixr1 interacts with Ssn8 and Tdh3 involved in transcriptional regulation

Ixr1 is a Saccharomyces cerevisiae transcriptional factor that extensively regulates the response to hypoxia and controls other important cellular functions and DNA repair. During aerobic growth, the Ixr1 repressor function is predominant on regulated promoters of hypoxic genes, although activator effects are also observed on other genes. During hypoxia, Ixr1 expression increases and the number of genes activated by Ixr1 also increase. In this work we demonstrate that the NH2-terminal region of Ixr1 is involved in transcriptional activation. We also present the first analysis about Ixr1 interactions with three factors that have been previously identified as important players in the yeast hypoxic response, Cyc8, Tup1 and Ssn8; results demonstrate that only Ssn8 binds to Ixr1. We have also looked for other Ixr1-binding proteins associated with transcriptional regulation, by co-purification and mass spectrometry identification. Tdh3, a protein involved in transcriptional silencing, is among the new identified Ixr1-binding proteins. Differential phosphorylation of Ixr1 is found when comparing aerobic and hypoxic yeast growth. Implication of these results in transcriptional regulation mediated by Ixr1 is discussed.