Molecular insights into meat quality determination: role of calcineurin in oxidative fibre conversion and in regulation of myosin heavy chain genes

活体动物肌肉的纤维类型组成对其死后肉的数量和质量有很大的影响。有利的肉质性状，特别是猪肉品质，如嫩度和颜色，与大多数氧化纤维类型的丰度有关，这是由肌球蛋白重链（MyHC）慢速和2a亚型的存在所定义的。负责将快速糖酵解纤维转化为氧化纤维的最重要的细胞途径是钙调磷酸酶信号传导。钙调神经磷酸酶是一种依赖钙的丝氨酸-苏氨酸磷酸酶，对其下游蛋白因子或基因的控制知之甚少。该项目是关于使用集成基因组学方法来识别钙调神经磷酸酶的新分子靶标（蛋白质因子和DNA调控元件），这将极大地促进我们对钙调神经磷酸酶如何差异和协调地将快速糖酵解转化为氧化纤维的理解。这项提议背后的科学动力基于最近的两项重要发现。首先，我们发现calcalineurin对出生后MyHC基因（2a， 2x和2b）有差异调节，这揭示了calcalineurin介导氧化纤维型结果的一个新的机制过程（图1）。我们发现钙调磷酸酶的早期反应是诱导快速氧化的MyHC2a基因，以及更快和更容易糖酵解的MyHC2x和MyHC2b基因的下调。相比之下，钙调磷酸酶仅在确定了产后快速MyHC基因的差异调控后，才检测到氧化性最强的MyHCslow被上调。因此，钙调磷酸酶似乎在快速糖酵解纤维表型（MyHC2x和2b）的早期下调中发挥关键作用，以促进MyHC2a的氧化结果和随后的MyHCslow表达。其次，我们证明了钙调神经磷酸酶复合物能够结合MyHC染色质DNA（图2），这表明钙调神经磷酸酶-染色质相互作用可能是钙调神经磷酸酶信号传导的重要机制。我们假设染色质结合对于钙调神经磷酸酶介导氧化表型的协调同种异构体特异性程序具有重要的功能。我们提出的策略是将ChIP-on-chip分析与我们最近从钙调神经磷酸酶过表达研究中获得的表达微阵列数据整合起来，用于全基因组检测蛋白质因子结合（如钙调神经磷酸酶）与启动子的结合。这种联合方法将极大地增强对MyHC和基因组中其他促进氧化纤维转化的启动子上的新型反式调节因子和DNA调节顺式作用元件的鉴定。该项目将为钙调磷酸酶信号传导的分子机制提供急需的基础知识，钙调磷酸酶信号传导负责促进有利的肉质纤维。一个可能具有商业重要性的项目结果是确定钙调磷酸酶的候选靶基因，这些基因可以开发成DNA标记，用于标记辅助选择以改善肉质。这些标记的开发需要考虑目标物种SNP开发（如猪）和关联研究的成本、潜在的商业利益和选择可能产生的额外影响。另一个可能的商业结果是确定候选效应基因作为药理学靶点。精确靶向下游基因有可能最大限度地减少不必要的副作用，并安全地改善特定的肌肉参数。在农场动物生产中，直接对靶基因或蛋白质活性进行药理学修饰以改善肉质不一定是唯一可行的方法。根据目标的不同，战略性地使用免疫方法，例如GnRH疫苗（Improvac）对公猪进行绝育，可能是提高动物产量的有效策略。

Fibre type composition of a muscle in a live animal has a strong bearing on its subsequent meat quantity and quality at post mortem. Favourable meat quality traits, in particular pork quality, like tenderness and colour, are associated with the abundance of the most oxidative fibre types, as defined by the presence of myosin heavy chain (MyHC) slow and 2a isoforms. The most important cellular pathway that is responsible for converting fast-glycolytic fibres to oxidative fibres is calcineurin signalling. Little is known about the downstream protein factors or genes that are under the control of calcineurin, a calcium-dependent serine-threonine phosphatase. This project is about the use of an integrated genomics approach to identify novel molecular targets (protein factors and DNA regulatory elements) of calcineurin, which will greatly advance our understanding of how calcineurin differentially and co-ordinately converts fast-glycolytic to oxidative fibres. The scientific impetus behind the proposal is based on 2 recent key findings. Firstly, we discovered that calcineurin differentially regulates the post-natal MyHC genes (2a, 2x and 2b), which reveals a novel mechanistic process in which calcineurin mediates an oxidative fibre type outcome (Fig.1). We found that an early response to calcineurin is the induction of the fast-oxidative MyHC2a, and the down-regulation of the faster and more glycolytic MyHC2x and MyHC2b genes. By contrast, the up-regulation of the most oxidative MyHCslow by calcineurin is only detected after the differential regulation of the post-natal fast MyHC genes has been well established. Hence calcineurin appears to play a crucial role in the early down-regulation of the fast-glycolytic fibre phenotype (MyHC2x and 2b) to facilitate the oxidative outcome of MyHC2a and subsequent MyHCslow expression. Secondly, we demonstrated that calcineurin complex is able to bind MyHC chromatin DNA (Fig.2), which suggests that calcineurin-chromatin interaction could be an important mechanism of calcineurin signalling. We hypothesise that chromatin binding is functionally important for calcineurin to mediate the coordinated isoform-specific programme of an oxidative phenotype. The strategy proposed is to integrate the use of ChIP-on-chip analysis for the genome-wide detection of protein-factor binding (such as calcineurin) to promoters, with our recently acquired expression microarray data from calcineurin over-expression studies. This combined approach would greatly enhance the identification of novel trans-regulatory factors and DNA regulatory cis-acting elements at MyHC and other promoters in the genome that contribute to oxidative fibre conversion. This project will provide much needed underpinning knowledge of the molecular mechanisms of calcineurin signalling that are responsible for the promotion of favourable meat quality fibres. A possible project outcome that is of commercial importance is the identification of candidate target genes of calcineurin that could be developed into DNA markers for marker-assisted selection to improved meat quality. The exploitation of such markers needs to take into account the costs of target-species SNP development (e.g. in pigs) and association studies, potential commercial benefit and possible additional effects of selection. Another possible commercial outcome is the identification of candidate effector genes as pharmacological targets. The precise targeting of downstream genes has the potential to minimise unwanted side effects, and to safely improve specific muscle parameters. In farm animal production, the direct pharmacological modification of a target gene or protein activity to improve meat quality is not necessarily the only approach available. Depending on the target, the strategic use of an immunisation approach, as exemplified by the GnRH vaccine (Improvac) for sterilisation of boars, could be an effective strategy in improving animal production.