Data_Sheet_1_Regulation Between HSF1 Isoforms and HSPs Contributes to the Variation in Thermal Tolerance Between Two Oyster Congeners.docx

Heat shock transcription factor 1 (HSF1) plays an important role in regulating heat shock, which can activate heat shock proteins (HSPs). HSPs can protect organisms from thermal stress. Oysters in the intertidal zone can tolerate thermal stress. The Pacific oyster (Crassostrea gigas gigas) and Fujian oyster (C. gigas angulata)—allopatric subspecies with distinct thermal tolerances—make good study specimens for analyzing and comparing thermal stress regulation. We cloned and compared HSF1 isoforms, which is highly expressed under heat shock conditions in the two subspecies. The results revealed that two isoforms (HSF1a and HSF1d) respond to heat shock in both Pacific and Fujian oysters, and different heat shock conditions led to various combinations of isoforms. Subcellular localization showed that isoforms gathered in the nucleus when exposed to heat shock. The co-immunoprecipitation revealed that HSF1d can be a dimer. In addition, we selected HSPs that are expressed under the heat shock response, according to the RNA-seq and proteomic analyses. For the HSPs, we analyzed the coding part and the promoter sequences. The result showed that the domains of HSPs are conserved in two subspecies, but the promoters are significantly different. The Dual-Luciferase assay showed that the induced expression isoform HSF1d had the highest activity in C. gigas gigas, while the constitutively-expressed HSF1a was most active in C. gigas angulata. In addition, variation in the level of HSP promoters appeared to be correlated with gene expression. We argue that this gene is regulated based on the different expression levels between the two subspecies’ responses to heat shock. In summary, various stress conditions can yield different HSF1 isoforms and respond to heat shock in both oyster subspecies. Differences in how the isoforms and promoter are activated may contribute to their differential expressions. Overall, the results comparing C. gigas gigas and C. gigas angulata suggest that these isoforms have a regulatory relationship under heat shock, providing valuable information on the thermal tolerance mechanism in these commercially important oyster species.