Abstract
BackgroundThe DGAT1 gene encodes an enzyme responsible for catalysing the terminal reaction in mammary triglyceride synthesis, and underpins a well-known pleiotropic quantitative trait locus (QTL) with a large influence on milk composition phenotypes. Since first described over 15 years ago, a protein-coding variant K232A has been assumed as the causative variant underlying these effects, following in-vitro studies that demonstrated differing levels of triglyceride synthesis between the two protein isoforms.ResultsWe used a large RNAseq dataset to re-examine the underlying mechanisms of this large milk production QTL, and hereby report novel expression-based functions of the chr14 g.1802265AA > GC variant that encodes the DGAT1 K232A substitution. Using expression QTL (eQTL) mapping, we demonstrate a highly-significant mammary eQTL for DGAT1, where the K232A mutation appears as one of the top associated variants for this effect. By conducting in vitro expression and splicing experiments in bovine mammary cell culture, we further show modulation of splicing efficiency by this mutation, likely through disruption of an exon splice enhancer as a consequence of the allele encoding the 232A variant.ConclusionsThe relative contributions of the enzymatic and transcription-based mechanisms now attributed to K232A remain unclear; however, these results suggest that transcriptional impacts contribute to the diversity of lactation effects observed at the DGAT1 locus.
Highlights
The diacylglyercol Oacyltransferase 1 (DGAT1) gene encodes an enzyme responsible for catalysing the terminal reaction in mammary triglyceride synthesis, and underpins a well-known pleiotropic quantitative trait locus (QTL) with a large influence on milk composition phenotypes
DGAT1 K232A is strongly associated with DGAT1 transcript abundance in the lactating mammary gland To test for regulatory effects on DGAT1 transcript levels, we performed an association analysis using a mammary RNAseq dataset representing 375 lactating cows (Methods)
DGAT1 K232A associates with splicing efficiency of DGAT1 introns Having observed differential splicing at exon 8 based on calculating percentage of reads spliced in [10] (PSI) of the alternative transcript structure, we investigated whether this variant associated with the efficiency of splicing at the neighbouring splicing junction
Summary
The DGAT1 gene encodes an enzyme responsible for catalysing the terminal reaction in mammary triglyceride synthesis, and underpins a well-known pleiotropic quantitative trait locus (QTL) with a large influence on milk composition phenotypes. Functional testing of the VNTR variant did not show any differences in DGAT1 expression between QTL genotypes in cell culture [6] This finding largely put the competing, gene expression-based hypothesis of the DGAT1 milk fat effect to rest at the time, with enzymatic differences deriving from the K232A mutation widely assumed as the underlying mechanism. The expression of DGAT1 transcripts was associated with K232A genotype (and milk fat percentage), and a strong correlation (r = 0.946) was observed between the DGAT1 eQTL and lactose yield (a trait with high genetic correlations with other milk yield phenotypes) Based on these observations, we have investigated mechanisms by which the K232A variant might mediate this effect. We present functional and statistical evidence suggesting splice-enhancement-based regulatory control of DGAT1 transcripts as an explanation for the eQTL, and by inference the milk fat and lactation effects attributed to this gene and mutation
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