HMGB proteins involved in TOR signaling as general regulators of cell growth by controlling ribosome biogenesis.

The number of ribosomes and their activity need to be highly regulated because their function is crucial for the cell. Ribosome biogenesis is necessary for cell growth and proliferation in accordance with nutrient availability and other external and intracellular signals. High-mobility group B (HMGB) proteins are conserved from yeasts to human and are decisive in cellular fate. These proteins play critical functions, from the maintenance of chromatin structure, DNA repair, or transcriptional regulation, to facilitation of ribosome biogenesis. They are also involved in cancer and other pathologies. In this review, we summarize evidence of how HMGB proteins contribute to ribosome-biogenesis control, with special emphasis on a common nexus to the target of rapamycin (TOR) pathway, a signaling cascade essential for cell growth and proliferation from yeast to human. Perspectives in this field are also discussed.

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.

Comparative transcriptome analysis of yeast strains carrying slt2, rlm1, and pop2 deletions.

The function of the genes SLT2 (encoding the Mpk1 protein), RLM1, and POP2 have previously been related to several stress responses in yeasts. DNA arrays have been used to identify differences among the transcriptomes of a Saccharomyces cerevisiae wild type strain and its derivative Δslt2, Δrlm1, and Δpop2 mutants. Correspondence analyses indicate that the vast majority of genes that show lower expression in Δrlm1 also show lower expression in Δslt2. In contrast, there is little overlap between the results of the transcriptome analyses of the Δpop2 strain and the Δslt2 or Δrlm1 strains. The DNA array data were validated by reverse Northern blotting and chromatin immunoprecipitation (ChIp). ChIp assays demonstrate Rlm1p binding to specific regions of the promoters of two genes that show expression differences between the Δrlm1 mutant and wild type strains. Interestingly, RLM1 deletion decreases the transcription of SLT2, encoding the Mpk1p kinase that phosphorylates Rlm1p, suggesting a feedback control in the signal transduction pathway. Also, deletion of RLM1 causes a decrease in the mRNA level of KDX1, which is paralogous to SLT2. In contrast, deletion of POP2 is accompanied by an increase of both SLT2 and KDX1 levels. We show that SLT2 mRNA increase in the Δpop2 strain is due to a decrease in RNA turnover, consistent with the expected loss of RNA-deadenylase activity in this strain.

Characterization of a gene similar to BIK1 in the yeast Kluyveromyces lactis.

In Saccharomyces cerevisiae, Bik1p is a microtubule plus-end-tracking protein that plays several roles in mitosis and ploidy. KlBik1p (from Kluyveromyces lactis) maintains the same structural-domain organization as does S. cerevisiae Bik1p. As part of its characterization, we constructed a stable klbik1 mutant which is sensitive to benomyl only at 14 degrees C and has a higher frequency of crescent-shaped nuclei than S. cerevisiae bik1 mutants. This phenotype is partially rescued by S. cerevisiae BIK1. Other phenotypes associated with bik1 are not present in the K. lactis mutant. By fusion to GFP we were able to show the functionality of the KlBik1p CAP-Gly domain and found that the fusion protein changes its cellular location during the cell cycle.

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.