Project description:The 9p21.3 cardiovascular disease locus is the most influential common genetic risk factor for coronary artery disease, accounting for ~10-15% of disease among non-African populations. The ~60kb risk haplotype is human-specific and lacks coding genes, hindering efforts to decipher its function. Genetic studies implicate the 9p21.3 locus and other risk genes to effects in the vascular wall. Here, we use genome editing to delete the entire risk on non-risk haplotype from the genomes of human iPSCs and perform genomewide transcriptional profiling along the timecourse of their differentiation into vascular smooth muscle cells (VSMCs). These studies identify a network of ~3000 genes governed by the risk haplotype in VSMCs that predict deficits in cell division, adhesion and contraction, which we confirmufunctionally. Remarkably, deleting the risk haplotype reverts VSMCs to resemble the non-risk VSMCs, suggesting that the risk region drives a cell state transition. transcriptionally and functionally. . Deleting the risk haplotype reverts these cells to reverted to the non-risk of iPSCs we show that the non-risk haplotype has little effect on locus we produce iPSCs from risk and non-risk individuals, delete each haplotype using genome editing and generate vascular smooth muscle cells (VSMCs). We show that risk VSMCs exhibit aberrant adhesion and contraction, concomitant with dramatically altered global transcriptional changes that are enriched in previously identified cardiovascular disease genes and pathways. Unexpectedly, deleting the risk haplotype rescues VSMC transcriptional identity and function, while expressing the 9p21.3-associated long non-coding RNA ANRIL induces risk phenotypes in non-risk VSMCs. This studies shows that the risk haplotype dominantly predisposes VSMCs to adopt perturbed phenotypes associated with cardiovascular disease and establishes haplotype-edited iPSCs as powerful tools for functionally annotating human-specific variation in non-coding genomic regions.

Project description:Background : The Blonde Aquitaine (BA) is a French beef breed with enhanced muscularity of unknown genetic origin. Targeted sequencing has shown that the BA is not a carrier of any of the bovine MSTN mutations. At the protein level the BA shows a transition to a faster muscle isoform phenotype with a greater proportion of type IIX and a smaller proportion of type I fibres than comparison Charolais (CH). Associated modifications in the activity of oxidative and glycolytic enzymes were also previously detected. Collectively, these changes are often observed in breeds of production species selected for enhanced growth and efficiency, but the underlying molecular mechanisms are unknown. Method : We examined the transcriptome of the Longissimus thoracis muscle at 4 gestational stages (110, 180, 210, and 260 dpc) in BA and CH foetuses. These time points coincide with primary and secondary myogenesis and functional differentiation. Total RNA was hybridised to the Agilent one-colour microarray platform containing 45,220 probes mapped to 17646 protein coding bovine genes. Following normalisation, we used a combination of differential expression (limma method, Smyth & al 2005) and a network theory method based on differential co-expression analysis (Hudson & al 2009) to compare and contrast the two muscle transcriptomes. Conclusions : In both breeds the most developmentally-regulated genes included: embryonic muscle subunit isoforms (e.g. MYL4) whose expression dropped dramatically prior to birth when they are replaced by the adult isoforms; metabolic enzymes (e.g. the mitochondrially-localised sarcomeric creatine kinase CKMT2) that prepare the foetus for functional independence postpartum, and 1 probe out of 4 representing a gene of unknown function (C13H20ORF194) also previously published as highly developmentally regulated in Wagyu by Hereford longissimus muscle (Hudson & al 2013). In contrasting the breeds, MSTN was the 2nd most differentially expressed muscle gene between the breeds, being approximately 4-fold down-regulated in CH at each of the time points. Given MSTN is a growth inhibitor, this result may reflect either divergent muscle mass or fibre composition in foetuses and could account for the low muscle development of BA calves at birth. Among the most differentially connected transcriptional regulators we prioritised expected molecules like MSTN, but also unexpected ones like PROX1, NMI, MBD1, NRIP2 and TAF12. Although not differentially expressed at the transcript level these molecules are transcriptionally rewired between the breeds and may form attractive new candidates for the development of differential muscle mass and fibre composition in cattle. 2 beef breeds: Blond Aquitaine (BA) and Charolais (CH) ; 4 gestational stages per breed (110, 180, 210, and 260 dpc); 3 animals (biological replicats) per stage and per breed, except for BA 180 dpc, BA 260 dpc and CH 110 dpc (only 2 animals due to technical problem during hybridization).

Project description:Background : The Blonde Aquitaine (BA) is a French beef breed with enhanced muscularity of unknown genetic origin. Targeted sequencing has shown that the BA is not a carrier of any of the bovine MSTN mutations. At the protein level the BA shows a transition to a faster muscle isoform phenotype with a greater proportion of type IIX and a smaller proportion of type I fibres than comparison Charolais (CH). Associated modifications in the activity of oxidative and glycolytic enzymes were also previously detected. Collectively, these changes are often observed in breeds of production species selected for enhanced growth and efficiency, but the underlying molecular mechanisms are unknown. Method : We examined the transcriptome of the Longissimus thoracis muscle at 4 gestational stages (110, 180, 210, and 260 dpc) in BA and CH foetuses. These time points coincide with primary and secondary myogenesis and functional differentiation. Total RNA was hybridised to the Agilent one-colour microarray platform containing 45,220 probes mapped to 17646 protein coding bovine genes. Following normalisation, we used a combination of differential expression (limma method, Smyth & al 2005) and a network theory method based on differential co-expression analysis (Hudson & al 2009) to compare and contrast the two muscle transcriptomes. Conclusions : In both breeds the most developmentally-regulated genes included: embryonic muscle subunit isoforms (e.g. MYL4) whose expression dropped dramatically prior to birth when they are replaced by the adult isoforms; metabolic enzymes (e.g. the mitochondrially-localised sarcomeric creatine kinase CKMT2) that prepare the foetus for functional independence postpartum, and 1 probe out of 4 representing a gene of unknown function (C13H20ORF194) also previously published as highly developmentally regulated in Wagyu by Hereford longissimus muscle (Hudson & al 2013). In contrasting the breeds, MSTN was the 2nd most differentially expressed muscle gene between the breeds, being approximately 4-fold down-regulated in CH at each of the time points. Given MSTN is a growth inhibitor, this result may reflect either divergent muscle mass or fibre composition in foetuses and could account for the low muscle development of BA calves at birth. Among the most differentially connected transcriptional regulators we prioritised expected molecules like MSTN, but also unexpected ones like PROX1, NMI, MBD1, NRIP2 and TAF12. Although not differentially expressed at the transcript level these molecules are transcriptionally rewired between the breeds and may form attractive new candidates for the development of differential muscle mass and fibre composition in cattle.

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TRANSCRIPTIONAL ANALYSIS OF MUSCLE FROM CATTLE WITH ALTERNATIVE NCAPG/LCORL GENOTYPES

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