Genome-wide H3K4me3 analysis in Angus cattle with divergent tenderness. Bos taurus

Tenderness is one of the most important properties of meat quality, which is influenced by genetic and environmental factors. As an intensively studied epigenetic marker, histone methylation, occurring on arginine and lysine residues, has pivotal regulatory functions on gene expression. To examine whether histone methylation involves in beef tenderness variation, we analyzed the transcriptome and H3K4me3 enrichment profiles of muscle strips obtained from the longissimus dorsi (LD) of Angus steers previously classify to the tender or tough group. We first plotted a global bovine H3K4me3 map on chromosomes and called peak-enriched regions and genes. We found that majorities of H3K4me3 on genes were occupying the first intron and intergenic regions and its maps displayed similar patterns in tender and tough groups, with high H3K4me3 enrichment surrounding the transcription start site (TSS). We also explored the relationship of H3K4me3 and gene expression. The results showed that H3K4me3 enrichment is highly positively correlated with gene expression across the whole genome. Cluster analysis results confirmed the relationship of H3K4me3 enrichment and gene expression. By using a pathway-based approach in genes with H3K4me3 enrichment in promoter regions from the tender cluster, we revealed that those genes involved in the development of different tissues–connective tissue, skeletal and muscular system and functional tissues–; while in tough group those genes engaged in cell death, lipid metabolism and small molecule biochemistry. The results from this study provide a deep insight into understanding of the mechanisms of epigenetic regulations in meat quality and beef tenderness. Overall design: Nineteen purebred Angus steers were obtained from the Wye Farm. At approximately 12 months of age, the animals were serially harvested. Immediately after harvest, samples of longissimus dorsi (LD) from the right side of the carcass were obtained and placed in RNAlater solution at -80°C. The carcass were stored at 4°C for a total of 14 days. After this period, steaks were obtained from the LD at the level of the 12th intercostal space and then frozen. For measurement of the WBSF, steaks were thawed at room temperature to an internal temperature of 4°C. Then, the steaks were cooked to a core temperature of 70°C using a George Foreman Lean Mean Fat Grilling Machine. The cooked steaks were then cooled down to room temperature. Using a sharp cylinder, especially designed for muscle, six cores (1.27 cm in diameter) were sampled parallel to the muscle fiber orientation. The Warner-Bratzler shear forces (WBSF) of the cores were obtained. The average WBSF of the six cores was calculated and used as the WBSF for the samples. From these 19 steers, 4 with the lowest WBSF values (6.77±0.56 kg) were identified as tender and 5 samples with the largest WBSF values (19.93±0.39 kg) labeled as tough. Then both groups underwent further analysis..

嫩度是肉品质最重要的属性之一，其受遗传与环境因素共同调控。作为被广泛研究的表观遗传标记（epigenetic marker），发生于精氨酸（arginine）与赖氨酸（lysine）残基的组蛋白甲基化（histone methylation）对基因表达具有关键调控作用。为探究组蛋白甲基化是否参与牛肉嫩度的变异调控，本研究对先前被划分为嫩度组与硬度组的安格斯公牛背最长肌（longissimus dorsi, LD）肌条的转录组（transcriptome）及H3K4me3富集谱进行了分析。本研究首先绘制了全牛染色体水平的H3K4me3图谱，并对峰富集区域及基因进行了识别。研究发现，基因区域内的H3K4me3多富集于第一内含子及基因间区，且嫩度组与硬度组的H3K4me3图谱模式相似，在转录起始位点（Transcription Start Site, TSS）周围呈现高富集状态。本研究还探究了H3K4me3与基因表达之间的关联，结果显示全基因组范围内H3K4me3富集水平与基因表达量呈显著正相关。聚类分析结果进一步验证了H3K4me3富集与基因表达之间的关联。本研究针对嫩度聚类中启动子区存在H3K4me3富集的基因采用通路富集分析方法，发现这些基因参与不同组织的发育过程——包括结缔组织、骨骼与肌肉系统及功能组织；而硬度组中的相关基因则主要参与细胞死亡、脂质代谢及小分子生物化学过程。本研究结果为解析肉品质及牛肉嫩度的表观遗传调控机制提供了深入的理论依据。

实验设计：本研究从Wye Farm获取了19头纯种安格斯公牛。待其生长至约12月龄时，对所有公牛进行分批屠宰。屠宰后即刻采集胴体右侧背最长肌（LD）样本，置于RNAlater溶液中并保存于-80℃。将胴体置于4℃条件下储存14天，随后在第12肋间隙水平采集背最长肌肌块并冷冻保存。为测定沃纳-布拉茨勒剪切力（Warner-Bratzler Shear Force, WBSF），将肌块置于室温解冻至内部温度为4℃，随后使用George Foreman Lean Mean Fat Grilling Machine将其加热至核心温度70℃，之后将熟制后的肌块冷却至室温。采用专为肌肉组织设计的锋利取样器，沿肌纤维方向平行采集6个直径为1.27cm的肌芯，测定每个肌芯的WBSF值，以6个肌芯的平均剪切力作为样本的WBSF值。基于19头公牛的WBSF测定结果，选取WBSF值最低的4头（6.77±0.56 kg）划分为嫩度组，选取WBSF值最高的5头（19.93±0.39 kg）划分为硬度组，随后对两组样本开展后续分析。