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的肉的数量和质量有很大的影响。有利的肉质性状，特别是猪肉质量，如嫩度和颜色，与最氧化的纤维类型的丰度有关，如肌球蛋白重链（MyHC）慢和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神经磷酸酶信号传导。对钙调神经磷酸酶（一种钙依赖性丝氨酸-苏氨酸磷酸酶）控制下的下游蛋白因子或基因知之甚少。该项目是关于使用一个集成的基因组学方法来确定新的分子靶点（蛋白质因子和DNA调控元件）的钙调磷酸酶，这将大大推进我们的钙调磷酸酶如何差异和协调转换快速糖酵解氧化纤维的理解。该提案背后的科学动力基于最近的两项关键发现。首先，我们发现钙调神经磷酸酶差异调节出生后MyHC基因（2a，2x和2b），这揭示了一种新的机制过程，其中钙调神经磷酸酶介导氧化纤维型结果（图1）。我们发现，对钙调神经磷酸酶的早期反应是诱导快速氧化的MyHC 2a，以及下调更快和更多糖酵解的MyHC 2x和MyHC 2b基因。相比之下，最氧化的MyHC慢钙调磷酸酶的上调后，才检测到出生后的快速MyHC基因的差异调节已经很好地建立。因此，钙调神经磷酸酶似乎在快速糖酵解纤维表型（MyHC 2x和2b）的早期下调中起着至关重要的作用，以促进MyHC 2a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，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，染色质结合是钙调神经磷酸酶介导的协调异构体特异性的氧化表型程序的功能重要。提出的策略是整合使用ChIP芯片分析的全基因组检测的蛋白质因子结合（如钙调神经磷酸酶）的启动子，与我们最近获得的表达微阵列数据从钙调神经磷酸酶过表达的研究。这种结合的方法将大大提高新的反式调节因子和DNA调节顺式作用元件在基因组中的MyHC和其他启动子，有助于氧化纤维转换的鉴定。该项目将提供急需的钙调神经磷酸酶信号分子机制的基础知识，负责促进有利的肉质纤维。一个可能的项目成果是具有商业重要性的是钙调磷酸酶的候选靶基因的鉴定，可以开发成DNA标记，用于标记辅助选择，以改善肉质。利用这些标记需要考虑目标物种SNP开发（例如在猪中）和关联研究的成本、潜在的商业利益和可能的额外选择效应。另一个可能的商业成果是鉴定作为药理学靶点的候选效应基因。下游基因的精确靶向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，靶基因或蛋白质活性的直接药理学修饰以改善肉质不一定是唯一可用的方法。根据目标，免疫方法的战略性使用，例如用于公猪绝育的GnRH疫苗（GnRHac），可能是提高动物生产的有效策略。

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.