Splice variants and seasonal expression of buffalo HSF genes

Abstract

In eukaryotes, the heat shock factors (HSFs) are recognized as the master regulator of the heat shock response. In this respect, the genes encoding the heat shock factors seem to be important for adaptation to thermal stress in organisms. Despite this, only few mammalian HSFs has been characterized. In this study, four major heat shock factor genes viz. HSF-1, 2, 4, and 5 were studied. The main objective of the present study was to characterize the cDNA encoding using conserved gene specific primers and to investigate the expression status of these buffalo HSF genes. Our RT-PCR analysis uncovered two distinct variants of buffalo HSF-1 and HSF-2 gene transcripts. In addition, we identified a variant of the HSF5 transcript in buffalo lacking a DNA-binding domain. In silico analysis of deduced amino acid sequences for buffalo HSF genes showed domain architecture similar to other mammalian species. Changes in the gene expression profile were noted by quantitative real-time PCR (qRT-PCR) analysis. We detected the transcript of buffalo HSF genes in different tissues. We also evaluated the seasonal changes in the expression of HSF genes. Interestingly, the transcript level of HSF-1 gene was found upregulated in months of high and low ambient temperatures. In contrast, the expression of the HSF-4 and 5 genes was found to be downregulated in months of high ambient temperature. This suggests that the intricate balance of different HSFs is adjusted to minimize the effect of seasonal changes in environmental conditions. These findings advance our understanding of the complex, context-dependent regulation of HSF gene expression under normal and stressful conditions.

Fig. 1

Detection of the buffalo HSF gene splice variant in tissues. a RT-PCR was performed for HSF-1 gene between exon 9 and 13, resulting in the amplification of a 273 bp fragment, corresponding to the original cloned product called “HSF-1Tv1” and a 357 bp fragment called “HSF-1Tv2”, containing extra 84 nucleotide. b RT-PCR was performed for HSF-2 gene between exon 10 and 12, resulting in the amplification of a 188 bp fragment, corresponding to the original cloned product called “HSF-2Tv1” and a 134 bp fragment called “HSF-2Tv2”, containing omission of 54 nucleotides. PCR products were electrophoresed on 2 % agarose gel. M indicates TrackIt ™ 50 bp DNA Ladder. The migration of size marker (bp) is shown to the left of gel

Fig. 2

Multiple sequence alignment of the identified heat shock factor sequences. Buffalo HSF-1, HSF-2, HSF-4 and HSF-5 protein sequences were aligned by using cluatalW2 program in BioEdit software. The secondary structure of HSF protein sequence is shown above the sequence. Arrow underneath the alignment indicates domain boundaries. Black and blue box depicts NLS and NES, respectively. Dot represents the conserved residues in the protein sequences

Fig. 3

Distinct tissue specific expression of buffalo HSF-1, 2, 4, and 5 genes (a–d). RT-qPCR analysis of HSF-1, 2, 4, and 5 transcripts in the indicated tissues was performed. The level of transcripts from the gene encoding ribosomal protein RPS 18 was used as an internal reference to normalize the HSF-1, 2, 4, and 5 mRNA, respectively. The normalized expression level was then compared to that of blood sample, which was arbitrarily given the value 1. Experiments were performed from tissues collected from n = 3 animals. Bar represents ± SEM. Within the graph, values labeled with different letters differ significantly from one another

Fig. 4

Seasonal differences in expression of buffalo HSF-1, 4, and 5 genes. (a–d) RT-qPCR analysis of HSF-1, 4, and 5 transcripts was performed in three different seasons as indicated. The level of transcripts from the gene encoding ribosomal protein RPS 18 was used as an internal reference to normalize the HSF-1, 4, and 5 mRNA, respectively. Summer season was chosen as calibrator arbitrarily and given the value 1. Blood samples collected from n = 3 Murrah buffalo heifers in 6 months) were used for the experiment. Bar represents ± SEM. Within the graph, values labeled with different letters differ significantly from one another