title: Dissection of the elements of osmotic stress response transcription factor Hot1 involved in the interaction with MAPK Hog1 and in the activation of transcription.
abstract: The response to hyperosmotic stress is mediated by the HOG pathway. The MAP kinase Hog1 activates several transcription factors, regulates chromatin-modifying enzymes and, through its interaction with RNA polymerase II, it directs this enzyme to osmotic stress-controlled genes. For such targeting, this kinase requires the interaction with transcription factors Hot1 and Sko1. However, phosphorylation of these proteins by Hog1 is not required for their functionality. In this study, we aim to identify the Hot1 elements involved in Hog1-binding and in the activation of transcription. Two-hybrid experiments demonstrated that the Hot1 sequence between amino acids 340 and 534 and the CD element of Hog1 are required for the interaction between the two proteins and the Hot1-dependent transcription regulation. Inside this Hot1 region, short sequence KRRRR (KR4, amino acids 381-385) is essential for the kinase binding. Our data show that another element, sequence EDDDDD (ED5, amino acids 541-546), is essential for Hot1 binding to chromatin. Under osmotic stress conditions, both Hot1 elements, Hog1-interaction KR4 and DNA-binding ED5, are involved in the appropriate recruitment of Hog1 and RNA polymerase II to genes controlled by this transcription factor. Moreover, both sequences are required for osmotolerance and KR4 is necessary for the functionality of the HOG pathway. According to several experiments described in this study, the Hot1 protein is capable of forming homodimers.
authors.affiliation: Departament de Bioquímica i Biologia Molecular, Facultat de Ciències Biològiques, Universitat de València, Burjassot, Valencia, Spain.
authors.forename: Mercè
authors.initials: M
authors.lastname: Gomar-Alba
authors.forename: Paula
authors.initials: P
authors.lastname: Alepuz
authors.forename: Marcel lí
authors.initials: Ml
authors.lastname: del Olmo
mesh_terms: D002843:Chromatin; D047369:Chromatin Immunoprecipitation; D015966:Gene Expression Regulation, Fungal; D047468:Immunoprecipitation; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D064587:Osmoregulation / Q000235:genetics*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020218:Response Elements / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics*
chemical_list: D002843:Chromatin; C120535:HOT1 protein, S cerevisiae; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C088589:HOG1 protein, S cerevisiae; D020928:Mitogen-Activated Protein Kinases
keywords: Activation of transcription; HOG pathway; Hot1; Osmotic stress; Saccharomyces cerevisiae