Ovine HSP90AA1 gene promoter: functional study and epigenetic modifications

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 extracted 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.

Keywords: Allele-specific methylation; EMSA; Epigenetic marks; HSP90AA1; Luciferase; Polymorphisms; Sheep.

Fig. 1

Electrophoretic mobility shift assay (EMSA) using nuclear extracts from HepG2 cells under thermoneutral conditions. Nuclear extracts from cultured cells were incubated with C₋₆₆₀-labelled oligonucleotide probe alone (lane 2), in the presence of increasing excess unlabelled C₋₆₆₀ probe (lanes 3-10×, 4-25×, 5-50×), G₋₆₆₀-labelled (lane 7), G₋₆₆₀-labelled with increasing excess unlabelled C-660 probe (lanes 8-10×, 9-25×, 10-50×). In lanes 1 and 6, nuclear extracts were not added

Fig. 2

Electrophoretic mobility shift assay (EMSA) using nuclear extracts from HepG2 cells under thermoneutral conditions. Nuclear extracts from cultured cells were incubated with the D₋₇₀₄-labelled oligonucleotide probe alone (lane 2), in the presence of excess unlabelled D₋₇₀₄ probe (lane 3), in the presence of excess unlabelled I₋₇₀₄ probe (lane 4), the I₋₇₀₄-labelled oligonucleotide probe alone (lane 6), in the presence of excess unlabelled I₋₇₀₄ probe (lane 7), in the presence of excess unlabelled I₋₇₀₄ probe (lane 8) and D₋₇₀₄ in the presence of excess unlabelled canonical GR sequence binding oligonucleotide (lane 9). In lanes 1 and 5, nuclear extracts were not added

Fig. 3

Electrophoretic mobility shift assay (EMSA) using nuclear extracts from HepG2 cells under thermoneutral conditions. Nuclear extracts from cultured cells were incubated with D₋₆₆₈D₋₆₆₇-labelled oligonucleotide probe alone (lane 2), in the presence of excess unlabelled I₋₆₆₈D₋₆₆₇ probe (lane 3), in the presence of excess unlabelled I₋₆₆₈I₋₆₆₇ probe (lane 4), the I₋₆₆₈D₋₆₆₇-labelled oligonucleotide probe alone (lane 6), in the presence of excess unlabelled D₋₆₆₈D₋₆₆₇ probe (lane 7), in the presence of excess unlabelled I₋₆₆₈I₋₆₆₇ probe (lane 8), the I₋₆₆₈I₋₆₆₇-labelled oligonucleotide probe alone (lane 10), in the presence of excess unlabelled D₋₆₆₈D₋₆₆₇ probe (lane 11) and in the presence of excess unlabelled I₋₆₆₈D₋₆₆₇ probe (lane 12). In lanes 1, 5 and 9, nuclear extracts were not added. The grey arrow indicates the binding of a complex of proteins with more efficiency to D₋₆₆₈D₋₆₆₇ and I₋₆₆₈I₋₆₆₇ whereas the protein indicated with the purple arrow seems to have more efficiency to I₋₆₆₈D₋₆₆₇. The blue arrow indicates a protein that binds in all three possible genotypes, even though it seems to have more preference to I₋₆₆₈D₋₆₆₇, as during competition, it does not disappear

Fig. 4

Luciferase assays for several polymorphisms at the HSP90AA1 promoter. Each alternative allele was transiently expressed in HepG2 cells for luciferase assays. Firefly luciferase activity was normalized with Renilla luciferase activity. Data are represented compared to pGL3-Basic and the means SD are for three replicates. a g.660G>C alternative genotypes under thermoneutral conditions. b g.703_704ins(2)A alternative genotypes under thermoneutral conditions. Ns non-significant c g.667_668insC and g.666_667insC alternative genotypes under thermoneutral conditions. d g.667_668insC and g.666_667insC alternative genotypes under heat stress conditions

Fig. 5

Diagram of the short interspersed elements (SINEs) predicted in the HSP90AA1 gene by RepeatMasker software. The two SINEs predicted are highlighted in pink. One SINE (35 bp long) is located in the intergenic region (1090 bp upstream of the Inr) and the second one (50 bp long) located in the third intron of the HSP90AA1. Exons 1, 2 and 3 are marked in grey

Fig. 6

Description of the HSP90AA1 promoter CpG island motifs. HSE (purple), TATA-box (pink) and TSS(A₊₁) (grey and underlined) at the Inr (yellow). Furthermore, HSP90AA1 promoter polymorphisms (g.703_704ins(2)A, g.667_668insC, g.666_667insC, g.660G>C, g.601A>C, g.528A>G, g.524G>T, g.522A>G, g.516_517insG, g.468G>T, g.444A>G, g.304A>G, g.296A>G, g.295C>T, g.252C>G) are positioned in the figure. Polymorphism’s names are based on their distances to TSS. Polymorphism susceptible of allele-specific methylation are underlined In addition, g.660G>C is highlighted in orange and g.632_methyl-CpG in green

Fig. 7

A schematic drawing of the effect that g.667_668insC, g.666_667insC and g.660G>C could have in the HSP90AA1 expression regulation. They are located in a putative distal enhancer binding sequence which allows activators to bind depending on their genotype. Those activators recruit different mediator transcription factors which form a hairpin that allows an approach with the core promoter and promotes transcription. Adapted from original drawing from Pearson Education, Inc. (publishing as Pearson Benjamin Cummings, Copyright © 2008)

Fig. 8

Possible mechanisms of transcriptional repression by MeCP2 (adapted from (Wade 2005). Interaction of MeCP2 with two methylated DNA sites results in local recruitment of chromatin-remodeling machine. This factor alters histone-DNA contacts. Furthermore, histone deacetylation by HDAC (histone deacetylases) and histone methylation by HMT (histone methyltransferases) facilitate formation of repressive chromatin conformation and therefore contribute to transcriptional repression