Differential expression of mitochondrial protein genes
Main Cows: Adult
In total, 16,166 genes including were available for analysis after filtering (out of 24,616 annotated Ensembl genes). A gene was considered as differentially expressed (DE) in a tissue if the expression was different from the average expression across all other tissues (LFC >|0.6|, FDR <0.01). Across all genes, 13 to 40% of genes in total were DE in one or more tissues and as high as 50% each of the DE genes were over-expressed or under-expressed (Figure 1). Table 1 provides a summary of the number of DE genes by category across tissues. The highest overall numbers of DE genes among the tissues were in blood leukocytes(N= 9,218), loin muscle (N= 7,560), brain caudal lobe (N= 7,504), and brain cerebellum (N= 7,161), and the lowest in the ovary (N= 3,003), omental fad pad (N= 3,008), and mediastinal lymph node (N= 3,428). The DE genes in heart, skeletal muscles and tongue were significantly enriched for OXPHOS, metabolic pathways and neurodegenerative diseases pathways, and enriched for metabolic pathways in liver and kidney cortex (Table 2).
More than 99% of the MP genes originate from the nuclear genome (NuMP). The proportion of DE NuMP genes across the tissues varied from 12 to 60% with higher proportions (>50%) in heart and skeletal muscles. The proportion of under or over-expressed DE NuMP genes within tissues varied considerably. A relatively greater proportion of NuMP genes were over-expressed in heart, kidney cortex, skeletal muscles, and tongue, and under-expressed in blood leukocytes, lymph nodes, placenta, lungs, mammary, and thymus (Figure 2). The expression of NuMP genes was similar between animals in the Main Cows as indicated by the clustering together of same tissues, with the exception of five tissues (Figure 2; adipose, ovary, kidney cortex, and leukocytes).
In contrast to NuMP, differential expression of MtMP genes were observed in less than 50% of tissues (14 out of 29 tissues). The proportion of DE MtMP genes within tissues ranged widely from 0 (no genes) to 100% (all 13 MtMP genes). Specifically, MtMP genes were 100% DE in heart, leg muscle, latissimus dorsi muscle, loin muscle, and tongue, and ranged between 50-75% in other tissues (leukocytes, placenta, thymus, rib muscle, and lymph node). Unlike NuMP genes, all DE MtMP genes were expressed in a single direction (i.e. either all over-expressed or all under-expressed) meaning every DE MtMP gene was over-expressed in heart, tongue, muscles and kidney cortex, and under-expressed in blood leukocytes, placenta, lymph node, pituitary, thymus, and thyroid (Figure 3). Further, there were similarities between the expression of DE MtMP and NuMP genes within a tissue. For instance, every tissue showing over-expression of DE MtMP genes invariably showed predominant over-expression of NuMP genes and similarly for under-expression. In addition to MtMP genes, some of the non-protein coding genes from the mitochondrial genome were also DE in several tissues (16s rRNA, 12s rRNA, tRNA-Pro and tRNA-Ser).
Within groups of tissues with either over-expression of MP genes (heart, skeletal muscles, liver and kidney cortex) or under-expression (leukocytes, thymus, placenta and lymph node), we examined all overlapping genes and their functional enrichment. In heart and skeletal muscles, there were 1,088 over-expressed genes in common including 320 NuMP and seven MtMP genes. Altogether across these 1,088 genes, there was significant enrichment for OXPHOS, metabolic pathways and neurodegenerative disease pathways as in these individual tissues. Similarly, liver and kidney cortex had 1,249 over-expressed genes in common including 223 NuMP genes (0 MtMP genes) and these were significantly enriched for metabolic pathways and peroxisome, valine, leucine and isoleucine degradation. In contrast, the DE genes in common for tissues in the under-expression group was low (63) with only 20 NuMP genes (0 MtMP genes). Across all 63 genes, there was enrichment for adrenergic signalling in cardiomyocytes, dilated cardiomyopathy, cardiac muscle contraction and hypertrophic cardiomyopathy. Altogether, these results indicated a significant role of the over-expressed MP genes contributing to the enriched pathways in the over-expression tissue group, while this pattern was not observed in the under-expression group.
Main Cows: Foetuses
The analysis for functional enrichment of overall DE genes in six foetal tissues showed significant enrichment of OXPHOS and metabolic pathways only in heart and lungs but not in leg muscle (Additional file 1). The NuMP genes were over-expressed in the heart and under-expressed in the remaining tissues, including leg muscles (Additional file 2). Similarly, the MtMP genes were prominently over-expressed in heart, under-expressed in the lungs and not significant in the remaining tissues (Additional file 3). Higher expression of NuMP genes was observed in liver of the male foetus and it did not cluster with liver of female foetus.
Co-expression network analysis of mitochondrial protein genes
The gene co-expression network constructed based on the affinity matrix from genes correlated in expression > |0.95| in adult cows had altogether 3,643 genes clustered into four major network clusters I-IV (Figure 4). The NuMP genes were concentrated in two main clusters (I and IV) indicating co-expression among NuMP genes and the remaining NuMP genes were sparsely scattered across all other clusters. Similarly, MtMP genes were all grouped in cluster I. Clusters I and IV containing subgroups of highly co-expressed NuMP and MtMP and NuMP genes are referred to as NuMP-MtMP and NuMP clusters respectively. Within the NuMP-MtMP cluster, the MP genes from the respective genomes were highly co-expressed. The NuMP-MtMP cluster was significantly enriched for OXPHOS, metabolic pathways and mitochondrial diseases’ pathways. Similarly, the NuMP cluster (cluster IV) was over-represented for signalling pathways, contraction, metabolic pathways and myopathies related to the heart (Table 3). The gene functions of the non-mitochondrial protein genes (Non-MP) in the NuMP-MtMP cluster were associated with heart and muscle functioning, signalling, and contraction (Additional file 4 and5).
We tested if the co-expression of NuMP genes in the NuMP-MtMP cluster was due to random chance using a Chi-square goodness of fit test. The frequency of NuMP genes in the cluster was significantly higher than random (ꭓ2 = 307.6, p<0.01), supporting that the cluster was enriched with co-expressed MP.
Further, we investigated the effect of TAD on the co-expression by comparing the number of 651 TAD mapped genes in the NuMP-MtMP cluster with the mean from 100 randomly generated samples of 651 genes from 3,022 TAD mapping genes across the clusters. It showed involvement of NuMP-MtMP genes in a similar number of TADs (472 ±10 vs 484), but NuMP-MtMP were more likely to be present in groups of two or more within TADs. The total number of genes occurring in a two or more in a TAD was 282 and 116 (±6) in NuMP-MtMP cluster and random samples respectively. This indicated that co-expression in the NuMP-MtMP cluster was enriched within TADs.
Validation of patterns of mitochondrial protein gene expression
The key findings on the expression of MP genes from the Main Cows dataset were validated using two independent datasets (Validation Cow and Validation Sheep). Both validation sets confirmed the general trends of MP gene expression and coexpression in tissues.
Firstly, both validation datasets confirmed the over-expression of MP genes in heart and skeletal muscles, and under-expression in blood leukocytes as in the adult tissues of the Main Cows dataset (Additional file 6-13). Further, expression of MP genes within tissues, as indicated by LFC values between Main Cows and Validation Cow (Figure 5), were highly correlated (R2 0.67-0.96) except for thyroid (R2 0.01). Similarly, the correlation of LFC values between Main Cows and Validation Sheep was high (R2 0.6-0.87), except for mammary and lungs (R2 0.36, 0.34) (Additional file 14). We investigated the poor correlation of gene expression in thyroid between the Main Cows and the Validation Cow. At least 35 DE NuMP genes in common between the datasets were expressed in opposite directions. These genes were mainly enriched for metabolic pathways, pyruvate metabolism and synthesis of antibiotics. Interestingly, the expression of NuMP genes between Validation Cow and Validation Sheep were moderately correlated including thyroid (R2 0.59) except for lung and mammary tissues (Additional file 15).
Secondly, the ‘either all over-expression or all under-expression’ of DE MtMP genes within tissues was supported by findings from both validation datasets. Further, the expression of MtMP genes in the direction of the dominant DE NuMP genes also remained evident across datasets.
Thirdly, the co-expression of MtMP and NuMP genes in a cluster were reproduced in the Validation Cow (Additional file 16), and to some extent in Validation Sheep (Additional file 17). The co-expression of MP genes in the NuMP-MtMP cluster in the Validation Cow was more than expected by random chance (ꭓ2=207.847, p<0.01) indicating that this cluster was enriched for co-expression of MP genes.
Finally, the overlap of genes in NuMP-MtMP clusters across the Main Cow and validation datasets was higher than would be expected if genes were randomly allocated to clusters. In particular, the occurrence of MtMP genes were almost coincidental (13/13) between cow datasets and 12/13 genes in common between cow and sheep datasets. Similarly, a considerable proportion of NuMP genes and also non-mitochondrial protein genes, were in common across datasets (Figure 6).