Deep Sequencing the Transcriptome Reveals Seasonal Adaptive Mechanisms in a Hibernating Mammal

Abstract

Mammalian hibernation is a complex phenotype involving metabolic rate reduction, bradycardia, profound hypothermia, and a reliance on stored fat that allows the animal to survive for months without food in a state of suspended animation. To determine the genes responsible for this phenotype in the thirteen-lined ground squirrel (Ictidomys tridecemlineatus) we used the Roche 454 platform to sequence mRNA isolated at six points throughout the year from three key tissues: heart, skeletal muscle, and white adipose tissue (WAT). Deep sequencing generated approximately 3.7 million cDNA reads from 18 samples (6 time points ×3 tissues) with a mean read length of 335 bases. Of these, 3,125,337 reads were assembled into 140,703 contigs. Approximately 90% of all sequences were matched to proteins in the human UniProt database. The total number of distinct human proteins matched by ground squirrel transcripts was 13,637 for heart, 12,496 for skeletal muscle, and 14,351 for WAT. Extensive mitochondrial RNA sequences enabled a novel approach of using the transcriptome to construct the complete mitochondrial genome for I. tridecemlineatus. Seasonal and activity-specific changes in mRNA levels that met our stringent false discovery rate cutoff (1.0×10−11) were used to identify patterns of gene expression involving various aspects of the hibernation phenotype. Among these patterns are differentially expressed genes encoding heart proteins AT1A1, NAC1 and RYR2 controlling ion transport required for contraction and relaxation at low body temperatures. Abundant RNAs in skeletal muscle coding ubiquitin pathway proteins ASB2, UBC and DDB1 peak in October, suggesting an increase in muscle proteolysis. Finally, genes in WAT that encode proteins involved in lipogenesis (ACOD, FABP4) are highly expressed in August, but gradually decline in expression during the seasonal transition to lipolysis.

Gene expression changes controlling distinct adaptations in the heart and skeletal muscle of a hibernating mammal

“…We used tissues from the same animals for both muscle types, combined with Illumina HiSeq sequencing technologies, to make the most direct comparison between the gene programs of the heart and skeletal muscle during hibernation. This comparative analysis improves on previous transcriptome analyses performed in our lab that relied on subtractive hybridization (7), EST identification (17), or 454 sequencing technologies that used pooled samples and therefore lacked statistical validation of expression levels at any given time point (38).…”

“…Any contig containing a predicted coding domain was selected and trimmed to include only this domain plus up to 100 bases on both ends of the domain. Before reads were matched to the contigs, mitochondrially encoded genes were screened out by the use of the thirteen-lined ground squirrel mitochondrial genome sequence (38) and the National Center for Biotechnology Information's (NCBI's) megablast program (2) because of the high density of mitochondrial genomes in heart and skeletal muscle. The selected contigs were then compared with the NCBI RefSeq human mRNA sequences using NCBI Blastn (2).…”

“…Maximum fold change for each gene was calculated as the collection point with the highest average read counts by the collection point with the lowest average read counts. Tissue specificity for heart and skeletal muscle was calculated for each gene by dividing the percentage of read counts in that tissue divided by the total number of read counts in all other transcriptomic samples, including heart, skeletal muscle, along with cortex, hypothalamus (88), BAT (37), and WAT (38), which were obtained from other transcriptomic experiments.…”

“…Figure 2 shows the number of transcripts that were identified with Ͼ75% specificity for each tissue type, relative to the other five ground squirrel tissues on which we have previously performed transcriptome analysis (brown and white adipose, hypothalamus, cortex, heart, and skeletal muscle) (37,38,88). Of those transcripts, the skeletal muscle has twice as many mRNAs with Ͼ95% specificity compared with the heart.…”

Dynamic changes in global and gene specific DNA methylation during hibernation in adult thirteen-lined ground squirrels,Ictidomys tridecemlineatus

“…Transcriptional differences occurring during hibernation are also expressed in a tissue-specific manner (Fujii et al, 2006). However, the exact regulatory mechanisms that mediate transcriptional activation and repression in these altered metabolic states are largely unknown, particularly at the level of global transcriptional controls (Hampton et al, 2011;Yan et al, 2008).…”