Functional and Adaptive Significance of Promoter Mutations That Affect Divergent Myocardial Expressions of TRIM72 in Primates

Abstract

Cis-regulatory elements play important roles in tissue-specific gene expression and in the evolution of various phenotypes, and mutations in promoters and enhancers may be responsible for adaptations of species to environments. TRIM72 is a highly conserved protein that is involved in energy metabolism. Its expression in the heart varies considerably in primates, with high levels of expression in Old World monkeys and near absence in hominids. Here, we combine phylogenetic hypothesis testing and experimentation to demonstrate that mutations in promoter are responsible for the differences among primate species in the heart-specific expression of TRIM72. Maximum likelihood estimates of lineage-specific substitution rates under local-clock models show that relative to the evolutionary rate of introns, the rate of promoter was accelerated by 78% in the common ancestor of Old World monkeys, suggesting a role for positive selection in the evolution of the TRIM72 promoter, possibly driven by selective pressure due to changes in cardiac physiology after species divergence. We demonstrate that mutations in the TRIM72 promoter account for the differential myocardial TRIM72 expression of the human and the rhesus macaque. Furthermore, changes in TRIM72 expression alter the expression of genes involved in oxidative phosphorylation, which in turn affects mitochondrial respiration and cardiac energy capacity. On a broader timescale, phylogenetic regression analyses of data from 29 mammalian species show that mammals with high cardiac expression of TRIM72 have high heart rate, suggesting that the expression changes of TRIM72 may be related to differences in the heart physiology of those species.

Keywords: TRIM72; heart metabolism; primate evolution; promoter mutations.

Fig. 1.

Different expression levels of TRIM72 in the heart of hominids and Old World monkeys. (A) The expression patterns of TRIM72 in hominids and Old World monkeys at the mRNA level (FPKM). Gorilla and orangutan data are from Brawand et al. (2011), and their skeletal muscle data are unavailable. Other data are from Pipes et al. (2013). (B) TRIM72 is rarely expressed in human heart, whereas highly expressed in skeletal muscle. Upper panel: representative western blots of TRIM72. Lower panel: mRNA expression data from GTEx(V6p) (GTEx Consortium 2013) (see also supplementary fig. S2, Supplementary Material online). (C) TRIM72 is highly expressed in the heart and skeletal muscle of rhesus macaque. Upper panel: representative western blots. Lower panel: expression levels of mRNA from RhesusBase (heart, n = 4, http://rhesusbase.cbi.pku.edu.cn/, last accessed March 24, 2021; Zhang et al. 2014). SkM, skeletal muscle.

Fig. 2.

Different activity of TRIM72 promoter between human and rhesus monkeys. (A) Comparison of TRIM72 promoter activity of human versus rhesus using luciferase reporter assays in H9c2 cells (n = 8). Left panel, promoter fragments are aligned to hg19 genome by BLAT. Hu-690, human full-length promoter (chr16: 31224983–31225673, hg19); Rh-690, homologous region of Hu-690 in rhesus (chr20: 28866360–28867045, rheMac8). (B) In vivo transgenic analysis of human and rhesus TRIM72 promoter activity in transgenic E12.5 mouse embryos (see also supplementary fig. S5, Supplementary Material online). (C) In vivo transgenic analysis of human and rhesus TRIM72 promoter activity in transgenic E13.5 mouse embryos (see also supplementary fig. S6, Supplementary Material online). In B and C, left and middle panels: representative transgenic E12.5 and E13.5 mouse embryos and slices of the heart (Red, Eosin. Blue, X-gal. Scale bar, 200 μm). Numbers of embryos with LacZ positive hearts over the total number of transgenic embryos screened are indicated. Right panel: LacZ activity (mean ± SE) in the heart of transgenic embryos. *P < 0.05, **P < 0.01 (Student’s t-test).

Fig. 3.

Ancestral reconstruction of the TRIM72 promoter and estimation of relative evolutionary rates in primates. (A) Ancestral TRIM72 promoter sequences at M3 and M9 reconstructed using maximum likelihood under the HKY+Γ₅ model using the BASEML program in the PAML package. Blue indicates the transcription factor binding sites and red nucleotides indicate mutations that change the binding affinity of corresponding trans-factors. The cardiac expression level of TRIM72 is given next to the sequence (data is missing for mouse lemur). (B and C) Species trees for TRIM72 promoter (B) and introns (C) showing branch lengths estimated under the HKY+Γ₅ model. The trees are unrooted but the root is placed on the mouse lemur branch for clarity. The branch ancestral to the Old World Monkeys (Cercopithecidae) is highlighted in blue, whereas the Hominid clade is in orange. The tree topology is used to fit the local-clock models which assign different rates for the colored branches. The node names of the trees are numbered for simplicity. #15: Primates; #16: Simiiformes; #17: Catarrhini; #18: Hominoide; #21: Cercopithecidae.

Fig. 4.

Mutations in the promoter contribute to the heart-specific expression change of TRIM72 in primates. (A) All human-specific mutations reduced the promoter activity of rhesus TRIM72 (n ≥ 6, mutation vs. rhesus as control). m1–m15, the Rhesus TRIM72 promoters (Rh-690) with one of the 15 rhesus-to-human mutations. The reporter activities of m1–m15 were compared with that of Rhesus TRIM72 promoter (Rh-690). Position of the mutations is shown in supplementary figure S8, Supplementary Material online. (B) Only two rhesus-specific mutations increased the promoter activity of human TRIM72 (n ≥ 6, mutation vs. human as control). M1–M15 indicate the human TRIM72 promoters (Hu-690) with one of the 15 human-to-rhesus mutations. The activities of M1–M15 were compared with human full-length promoter (Human Hu-690). Position of the mutations is shown in supplementary figure S8, Supplementary Material online. (C) Combination of M3 and M9 could restore the activity of human TRIM72 promoter to the rhesus level. M3, M9, and M3+M9, human full-length promoter with human-to-rhesus mutation 3, 9, and both 3 and 9, respectively. (D) Sanger sequencing of TRIM72 promoter region in edited hESC colonies. Sequence alignments of mutations 3 and 9 regions are shown. For each type of mutation, two clonal cell lines were expanded and tested. WT, promoter unmodified; Rh-F4 and Rh-C8, clones with TRIM72 promoter replaced with rhesus ortholog; M3-B2 and M3-B9, clones of M3; M9-H3 and M9-A3, clones of M9 (see also supplementary fig. S9, Supplementary Material online, for genome editing strategy). (E) Replacement in human TRIM72 promoter with rhesus-specific mutations upregulates TRIM72 mRNA expression in hESC-derived cardiomyocytes (n ≥ 6, mutation vs. wild type control). (F) EMSA result showing human-to-rhesus M3 decreases the binding affinity of NKX2-5 to TRIM72 promoter. The arrowhead indicates the protein-DNA complex of NKX2-5 and oligo probes. (G) EMSA result showing human-to-rhesus M9 increases the binding affinity of TBX5 to TRIM72 promoter. The arrowhead indicates the protein-DNA complex of TBX5 and oligo probes. In panels A–C and E, *P < 0.05, **P < 0.01, ***P< 0.005, ****P< 0.001 (Student’s t-test).

Fig. 5.

TRIM72 regulates mitochondrial respiratory capacity in cardiomyocytes. (A and B) KEGG pathway analysis showing that oxidative phosphorylation pathway is significantly altered in the Trim72 knockout (A, n = 3 for each group) or transgenic hearts (B, n = 4 for each group). (C and D) Knockdown (C) or overexpression (D) of TRIM72 affects mitochondrial respiration in NRVMs (n ≥ 6). Oligomycin is an antibiotic that inhibits ATP synthase by blocking its proton channel (F0 subunit). FCCP (carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone) is a potent uncoupler of oxidative phosphorylation. Rotenone is an inhibitor of complex I and antimycin A is an inhibitor of complex III in electron transport chain. NRVMs: neonatal rat ventricular myocytes. OCR, oxygen consumption rate; Ad-GFP, adenovirus expressing GFP; Ad-TRIM72, adenovirus expressing TRIM72. (E) Reduced enzyme activity of mitochondrial respiratory complex in cardiomyocytes from Trim72 knockout mice. (F) Increased enzyme activity of mitochondrial respiratory complex in cardiomyocytes from Trim72 transgenic mice. (G) Overexpression of TRIM72 upregulates mitochondrial respiration in hESC-derived cardiomyocytes (HCM, n = 6). (H) TRIM72 promoter mutations (Rhesus orthologue, M3 and M9) upregulate OCR in hESC-derived cardiomyocytes (n = 4–5). Edited HCM, TRIM72 promoter modified hESC-derived cardiomyocytes. (I–K) Area Under Curve (AUC) of panel H, showing that TRIM72 promoter mutations (Rhesus orthologue replacement, M3, and M9) upregulate total mitochondrial respiration (I), basal mitochondrial respiration (J), and maximal mitochondrial respiration (K) in hESC-derived cardiomyocytes (n = 4–5). In C–G and I–K, data are presented as mean ± SE. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way ANOVA with post hoc t-test).

Fig. 6.

TRIM72 regulates the expression of oxidative phosphorylation-related genes at transcriptional level. (A) Knockdown of TRIM72 downregulated the expression of oxidative phosphorylation-related genes at the mRNA level in NRVMs (n ≥ 3). (B) Overexpression of TRIM72 upregulated the expression of oxidative phosphorylation-related genes at the mRNA level in NRVMs (n ≥ 3). (C) ChIP-qPCR shows that TRIM72 binds to the promoter regions of genes related to oxidative phosphorylation. (D) Schematic shows how TRIM72 regulates mitochondrial metabolism. TRIM72 influences the mitochondrial efficiency by regulating the expression of respiration complex-related genes, and by affecting lipid absorption via regulating the expression of PPARα (Liu et al. 2015). Ndufa5 and Ndufv3 are subunits of NADH dehydrogenase (Complex I). Sdha is a subunit of succinate dehydrogenase (Complex II), Uqcrfs1 and Uqcrc1 are subunits of cytochrome bc1 complex (Complex III), and Cox6a1 is a subunit of cytochrome c oxidase (Complex IV). Ad-GFP, adenovirus expressing GFP; Ad-TRIM72, adenovirus expressing TRIM72.