Abstract
When environmental temperatures exceed a certain threshold, the upregulation of the ovine HSP90AA1 gene is produced to cope with cellular injuries caused by heat stress. It has been previously pointed out that several polymorphisms located at the promoter region of this gene seem to be the main responsible for the differences in the heat stress response observed among alternative genotypes in terms of gene expression rate. The present study, focused on the functional study of those candidate polymorphisms by electrophoretic mobility shift assay (EMSA) and in vitro luciferase expression assays, has revealed that the observed differences in the transcriptional activity of the HSP90AA1 gene as response to heat stress are caused by the presence of a cytosine insertion (rs397514115) and a C to G transversion (rs397514116) at the promoter region. Next, we discovered the presence of epigenetic marks at the promoter and along the gene body founding an allele-specific methylation of the rs397514116 mutation in DNA extrated from blood samples. This regulatory mechanism interacts synergistically to modulate gene expression depending on environmental circumstances. Taking into account the results obtained, it is suggested that the transcription of the HSP90AA1 ovine gene is regulated by a cooperative action of transcription factors (TFs) whose binding sites are polymorphic and where the influence of epigenetic events should be also taken into account.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Coulondre C, Miller JH, Farabaugh PJ, Gilbert W (1978) Molecular basis of base substitution hotspots in escherichia coli. Nature 274:775–780
Csermely P, Schnaider T, Soti C, Prohaszka Z, Nardai G (1998) The 90-kDa molecular chaperone family: structure, function, and clinical applications. A comprehensive review. Pharmacol Ther 79:129–168
Deaton AM, Bird A (2011) CpG islands and the regulation of transcription. Genes Dev 25:1010–1022. doi:10.1101/gad.2037511
Gagniuc P, Ionescu-Tirgoviste C (2013) Gene promoters show chromosome-specificity and reveal chromosome territories in humans. BMC Genomics 14:278. doi:10.1186/1471-2164-14-278
Gardiner-Garden M, Frommer M (1987) CpG islands in vertebrate genomes. J Mol Biol 196:261–282
Guisbert E, Czyz DM, Richter K, McMullen PD, Morimoto RI (2013) Identification of a tissue-selective heat shock response regulatory network. PLoS Genet 9:e1003466. doi:10.1371/journal.pgen.1003466
Hajkova P, el-Maarri O, Engemann S, Oswald J, Olek A, Walter J (2002) DNA-methylation analysis by the bisulfite-assisted genomic sequencing method. Methods Mol Biol 200:143–154. doi:10.1385/1-59259-182-5:143
Han G, Ma H, Chintala R, Fulton DJ, Barman SA, White RE (2009) Essential role of the 90-kilodalton heat shock protein in mediating nongenomic estrogen signaling in coronary artery smooth muscle. J Pharmacol Exp Ther 329:850–855. doi:10.1124/jpet.108.149112
Hu S et al (2013) DNA methylation presents distinct binding sites for human transcription factors. eLife 2:e00726. doi:10.7554/eLife.00726
Khrapunov S, Brenowitz M, Rice PA, Catalano CE (2006) Binding then bending: a mechanism for wrapping DNA. Proc Natl Acad Sci U S A 103:19217–19218. doi:10.1073/pnas.0609223103
Kim YJ, Kim JY, Ko AR, Kang TC (2013) Reduction in heat shock protein 90 correlates to neuronal vulnerability in the rat piriform cortex following status epilepticus. Neuroscience 255:265–277. doi:10.1016/j.neuroscience.2013.09.050
Klose RJ, Sarraf SA, Schmiedeberg L, McDermott SM, Stancheva I, Bird AP (2005) DNA binding selectivity of MeCP2 due to a requirement for A/T sequences adjacent to methyl-CpG. Mol Cell 19:667–678. doi:10.1016/j.molcel.2005.07.021
Korfanty J et al (2014) Crosstalk between HSF1 and HSF2 during the heat shock response in mouse testes. Int J Biochem Cell Biol 57C:76–83. doi:10.1016/j.biocel.2014.10.006
Landolin JM et al (2010) Sequence features that drive human promoter function and tissue specificity. Genome Res 20:890–898. doi:10.1101/gr.100370.109
LeBlanc S, Hoglund E, Gilmour KM, Currie S (2012) Hormonal modulation of the heat shock response: insights from fish with divergent cortisol stress responses. Am J Physiol Regul Integr Comp Physiol 302:R184–R192. doi:10.1152/ajpregu.00196.2011
Marcos-Carcavilla A et al (2008) Structural and functional analysis of the HSP90AA1 gene: distribution of polymorphisms among sheep with different responses to scrapie. Cell Stress Chaperones 13:19–29. doi:10.1007/s12192-007-0004-2
Marcos-Carcavilla A et al (2010) A SNP in the HSP90AA1 gene 5′ flanking region is associated with the adaptation to differential thermal conditions in the ovine species. Cell Stress Chaperones 15:67–81. doi:10.1007/s12192-009-0123-z
Medvedeva YA et al (2014) Effects of cytosine methylation on transcription factor binding sites. BMC Genomics 15:119. doi:10.1186/1471-2164-15-119
Miller SA, Dykes DD, Polesky HF (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16:1215
Morimoto RI (1998) Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators. Genes Dev 12:3788–3796
Nan X, Meehan RR, Bird A (1993) Dissection of the methyl-CpG binding domain from the chromosomal protein MeCP2. Nucleic Acids Res 21:4886–4892
Nan X, Campoy FJ, Bird A (1997) MeCP2 is a transcriptional repressor with abundant binding sites in genomic chromatin. Cell 88:471–481
Oner Y, Calvo JH, Serrano M, Elmaci C (2012) Polymorphisms at the 5 ′ flanking region of the HSP90AA1 gene in native Turkish sheep breeds. Livest Sci 150:381–385. doi:10.1016/j.livsci.2012.07.028
Pirkkala L, Nykanen P, Sistonen L (2001) Roles of the heat shock transcription factors in regulation of the heat shock response and beyond. FASEB J 15:1118–1131
Rieping M, Schoffl F (1992) Synergistic effect of upstream sequences, CCAAT box elements, and HSE sequences for enhanced expression of chimaeric heat shock genes in transgenic tobacco. Mol Gen Genet 231:226–232
Salces-Ortiz J, Gonzalez C, Moreno-Sanchez N, Calvo JH, Perez-Guzman MD, Serrano MM (2013) Ovine HSP90AA1 expression rate is affected by several SNPs at the promoter under both basal and heat stress conditions. PLoS ONE 8:e66641. doi:10.1371/journal.pone.0066641
Salces-Ortiz J, Gonzalez C, Martinez M, Mayoral T, Calvo JH, Serrano M (2015a) Looking for adaptive footprints in the HSP90AA1 ovine gene. BMC Evol Biol 15:7. doi:10.1186/s12862-015-0280-x
Salces-Ortiz J, Ramón M, González C, Pérez-Guzmán MD, Garde JJ, García-Álvarez O et al (2015b) Differences in the ovine HSP90AA1 gene expression rates caused by two linked polymorphisms at its promoter affect rams sperm DNA fragmentation under environmental heat stress conditions. PLoS ONE 10(21):e0116360. doi:10.1371/journal.pone.0116360
Salvatore P, Benvenuto G, Caporaso M, Bruni CB, Chiariotti L (1998) High resolution methylation analysis of the galectin-1 gene promoter region in expressing and nonexpressing tissues. FEBS Lett 421:152–158
Sud N et al (2007) Nitric oxide and superoxide generation from endothelial NOS: modulation by HSP90. Am J Physiol Lung Cell Mol Physiol 293:L1444–L1453. doi:10.1152/ajplung.00175.2007
Taipale M, Jarosz DF, Lindquist S (2010) HSP90 at the hub of protein homeostasis: emerging mechanistic insights. Nat Rev Mol Cell Biol 11:515–528. doi:10.1038/nrm2918
Tian WL et al (2014) High expression of heat shock protein 90 alpha and its significance in human acute leukemia cells. Gene 542:122–128. doi:10.1016/j.gene.2014.03.046
Verghese J, Abrams J, Wang Y, Morano KA (2012) Biology of the heat shock response and protein chaperones: budding yeast (Saccharomyces cerevisiae) as a model system. Microbiol Mol Biol Rev 76:115–158. doi:10.1128/MMBR.05018-11
Wade PA (2005) SWItching off methylated DNA. Nat Genet 37:212–213. doi:10.1038/ng0305-212
Warnecke PM, Stirzaker C, Song J, Grunau C, Melki JR, Clark SJ (2002) Identification and resolution of artifacts in bisulfite sequencing. Methods 27:101–107
Yang Z et al (2011) Novel insights into the role of HSP90 in cytoprotection of H2S against chemical hypoxia-induced injury in H9c2 cardiac myocytes. Int J Mol Med 28:397–403. doi:10.3892/ijmm.2011.682
Acknowledgments
This work was supported by the RTA2009-00098 INIA project. Judit Salces-Ortiz was supported by an INIA doctoral grant. We thank AGRAMA breeders association and CITA (Centro de Investigación y Tecnología Agroalimentaria de Aragón) for providing the biological samples and Facultad de Medicina, Universidad de Cantabria, for providing the technical support.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Additional supporting information associated with this article includes:
Supplementary Fig. 1
Electroforetic Motility Shift Assay (EMSA) using nuclear extracts from HepG2 cells under thermoneutral and heat stress conditions. Nuclear extracts from thermoneutral cultured cells were incubated with the D-668D-667-labelled oligonucleotide probe (lane 2) and nuclear extracts from heat shock cultured cells were incubated with the D-668D-667-labelled oligonucleotide probe (lane 3). The I-668D-667-labelled oligonucleotide probe was incubated with nuclear extracts from thermoneutral cultured cells (lane 5) and from heat shock cultured cells (lane 6). The I-668I-667-labelled oligonucleotide probe was incubated with nuclear extracts from thermoneutral cultured cells (lane 8) and from heat shock cultured cells (lane 9). In lanes 1, 4 and 7, nuclear extracts were not added. (DOCX 130 kb)
Supplementary Fig. 2
HSP90AA1 methylation analysis from bllos samples by bisulfite genomic sequencing (Primers are in light grey). Exons are dark grey shaded. CpGs of the CGI are highlighted in green and the CGI limits in blue: −745 to +1445 respect putative TSS(A+1). Uncertain genotyped methylated CpGs are in yellow. Some core promoter motifs are depicted: BRE is double underlined, TATA-box simple underlined and TSS (A+1) at the Inr highlighted in pink. g.660G > C and g.632_methyl-CpG are circled. (GIF 348 kb)
Supplementary Fig. 3
Electroforetic Motility Shift Assay (EMSA) comparing un-methylated CpG (CpG) vs methylated CpG (meCpG) associated with g.660G > C. Nuclear extracts from HepG2 cultured cells were incubated with the CpG-labelled oligonucleotide probe (lane 2), in the presence of increasing excess unlabelled CpG probe (lane 3-50x), in the presence of increasing excess unlabelled meCpG probe (lane 4-50x), meCpG-labelled (lane 6), meCpG-labelled with increasing excess unlabelled meCpG probe (lane 7-50x) and meCpG-labelled with increasing excess unlabelled CpG probe (lane 8-50x). In lanes 1 and 5 nuclear extracts were not added. (DOCX 142 kb)
Supplementary Table 1
EMSA oligonucleotides used in the present work. (XLSX 7 kb)
Supplementary Table 2
Primers designed for the different experiments performed in the work. (DOCX 19 kb)
Supplementary Table 3
Output of the RepeatMasker software. This software predicts the presence of two short interspersed elements in the HSP90AA1 ovine gene. (DOCX 13 kb)
Supplementary Table 4
Output obtained from using the RepeatMasker software. This software predicts: a fragment of Bov-tA2 SINE/tRNA-Core-RTE and a fragment of MIRc SINE/MIR. (DOCX 11 kb)
Rights and permissions
About this article
Cite this article
Salces-Ortiz, J., González, C., Bolado-Carrancio, A. et al. Ovine HSP90AA1 gene promoter: functional study and epigenetic modifications. Cell Stress and Chaperones 20, 1001–1012 (2015). https://doi.org/10.1007/s12192-015-0629-5
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12192-015-0629-5