The C-terminal region of the Hot1 transcription factor binds GGGACAAA-related sequences in the promoter of its target genes Mercè Gomar-Alba a , Catarina Amaral b , Alejandro Artacho c , Giuseppe D'Auria c,d , Catarina Pimentel b , Claudina Rodrigues-Pousada b , Marcel·lí del Olmo a, ⁎ a Departament de Bioquímica i Biologia Molecular, Facultat de Ciències Biològiques, Universitat de València, Burjassot, Valencia, Spain b Genomics and Stress Laboratory, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal c Joint Unit of Research in Genomics and Health, Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunidad Valenciana (FISABIO)-Salud Pública, Valencia, Spain d Centro de Investigación en Red en Epidemiología y Salud Pública (CIBEResp), Madrid, Spain abstract article info Article history: Received 30 July 2015 Received in revised form 7 October 2015 Accepted 8 October 2015 Available online xxxx Keywords: Osmotic stress Saccharomyces cerevisiae Hot1 DNA binding domain Sequence recognition Response to hyperosmotic stress in the yeast Saccharomyces cerevisiae involves the participation of the general stress response mediated by Msn2/4 transcription factors and the HOG pathway. One of the transcription factors activated through this pathway is Hot1, which contributes to the control of the expression of several genes in- volved in glycerol synthesis and flux, or in other functions related to adaptation to adverse conditions. This work provides new data about the interaction mechanism of this transcription factor with DNA. By means of one-hybrid and electrophoretic mobility assays, we demonstrate that the C-terminal region, which corresponds to amino acids 610–719, is the DNA-binding domain of Hot1. We also describe how this domain recognizes se- quence 5’-GGGACAAA-3’ located in the promoter of gene STL1. The bioinformatics analysis carried out in this work allowed the identification of identical or similar sequences (with up to two mismatches) in the promoter of other Hot1 targets, where central element GGACA was quite conserved among them. Finally, we found that small variations in the sequence recognized by Hot1 may influence its ability to recognize its targets in vivo. © 2015 Elsevier B.V. All rights reserved. 1. Introduction The yeast Saccharomyces cerevisiae can be affected by several ad- verse conditions while it grows. Adaptation and cell survival under these conditions require the activation of signal transduction pathways, which results in major transcriptional changes (reviewed in [1]). In dif- ferent stress situations, S. cerevisiae displays a common mechanism, the Environmental Stress Response (ESR), which requires the activity of transcription factors Msn2 and Msn4 and affects the expression of ap- proximately 900 genes [2,3]. One well-studied stress condition is hyperosmolarity due to high concentrations of salt, sorbitol or glucose [1,4,5,6]. The specific response to this adverse condition is mediated by the High Osmolarity Glycerol (HOG) pathway. Two osmo-sensing mechanisms (branches SLN1 and SHO1) provoke the activation of MAPK Hog1 by phosphorylation [7– 15], which results in its nuclear translocation. Hog1 regulates several unrelated transcription factors (Hot1, Msn1, Smp1, Gcn4, Skn7, Sko1, Msn2/4 and Rtg1/3), each of which is responsible for controlling the ex- pression of a subset of osmo-responsive genes [16–21]. Hog1 also regulates chromatin-modifying enzymes [22,23] and interacts with RNA polymerase II [24–26]. Hot1 has been identified in a two-hybrid screen that used HOG1 cod- ing sequences as a bait [17]. This transcription factor has at least three well-known features [27]: (1) Hog1 binding; (2) phosphorylation by Hog1; and (3) DNA binding at the promoter of its target genes. It has been recently described that the Hot1 basic sequence between amino acids 381–385 (KRRRR, KR4) is the docking site for Hog1 MAPK and is required for HOG pathway functionality [28]. This region does not con- tain Hog1 phosphorylation sites, and this post-translational modifica- tion is not actually necessary for the activation of Hot1 target genes [24]. Together all these data suggest that the interaction itself between Hot1 and MAPK is needed for the transcription of Hot1-regulated genes [28]. Once activated by Hog1, the recruitment of this transcription factor in the regulatory regions of stress responsive genes targets the Hog1-RNA PolII complex to them [25,26]. Hot1 binds to the promoter of several genes, including STL1, GPD1, GPP2 and HGI1[17,18,29,30], and is involved in their transcriptional in- duction under osmotic stress. In HGI1, which is partially controlled by this factor, binding occurs only under stress conditions, while for STL1 gene Hot1 interacts with its promoter also under non stress conditions [28,29]. Studies carried out by Capaldi et al. [31] have suggested that due to the inhibition of Msn2/4 at high glucose concentrations, Hot1 together Biochimica et Biophysica Acta 1849 (2015) 1385–1397 ⁎ Corresponding author at: Departament de Bioquímica i Biologia Molecular, Facultat de Ciències Biològiques, Universitat de València, Dr. Moliner, 50, E-46100, Burjassot,Valencia, Spain. E-mail address: m.del.olmo@uv.es (M. del Olmo). http://dx.doi.org/10.1016/j.bbagrm.2015.10.007 1874-9399/© 2015 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagrm