Six homeoproteins and a linc-RNA cooperate at the fast MYH locus to lock terminal fast myofibre phenotype. Mus musculus

Thousands of long intergenic noncoding RNAs (lincRNAs) are encoded by the mammalian genome, which were reported to have multiple biological functions as transcriptional activators acting in cis 1 or trans 2, transcriptional repressors 3,4 or miRNAs decoys 5,6. However, the function of most lincRNAs has not yet been identified in vivo. Here, we demonstrate a role for linc-MYH, a novel long intergenic noncoding RNA, in adult fast-type myofibre specialization. Skeletal myofibre fast and slow phenotypes are established through differential expression of numerous fibre-specific genes7. We show linc-MYH and the fast MYH genes share a common enhancer located in the fast MYH genes locus and regulated by the Six1 homeoproteins. Muscle-specific Six1 mutant mice show increased expression of slow-type genes, and downregulation of linc-MYH and fast-type genes. linc-MYH function revealed by in vivo knockdown and wide transcriptomic analysis, is in fine to prevent expression of genes ensuring slow muscle contractile properties, and to increase fast-type muscle gene expression in fast-type myofibres. Thus, formation of efficient fast sarcomeric units and appropriate Ca++ cycling and excitation/contraction/relaxation coupling in fast- myofibres is achieved through the coordiante control of fast MYHs and linc-MYH expression by a Six bound enhancer. Overall design: Ten μg of shRNA-expressing vector were introduced into TA muscles of 8 week-old mice by electroporation. Two weeks following electroporation, TA myofibres expressing GFP were dissected under a Nikon SMZ1500 stereo microscope and frozen in liquid nitrogen before processing. The efficiency of each shRNA was established by determination of linc-MYH transcript levels in TA muscles transfected by each shlincMYH. The shRNA against 5'- TTCTGCTCACCACCTACAATT-3' sequence was selected for the knockdown experiment. After validation of RNA quality with the Bioanalyzer 2100 (using Agilent RNA6000 nano chip kit), 50 ng of total RNA were reverse transcribed following the Ovation PicoSL WTA System (Nugen). Briefly, the resulting double-strand cDNA was used for amplification based on SPIA technology. After purification according to Nugen protocol, 5 μg of single strand DNA was used for generation of Sens Target DNA using Ovation Exon Module kit (Nugen). 2.5 μg of Sens Target DNA were fragmented and labelled with biotin using Encore Biotin Module kit (Nugen). After control of fragmentation using Bioanalyzer 2100, the cDNA was then hybridized to GeneChip® Mouse Gene 1.0 ST (Affymetrix) at 45°C for 17 hours. After overnight hybridization, the ChIPs were washed using the fluidic station FS450 following specific protocols (Affymetrix) and scanned using the GCS3000 7G. The scanned images were then analyzed with Expression Console software (Affymetrix) to obtain raw data (cel files) and metrics for Quality Controls. The analysis of some of these metrics and the study of the distribution of raw data show no outlier experiment. Gastrocnemius muscles were collected from cSix1 KO and control mice. Total RNAs were extracted by Trizol Reagent (Invitrogen) according to manufacturer's instruction. After validation of RNA quality with the Bioanalyzer 2100 (using Agilent RNA6000 nano chip kit), 50 ng of total RNA were reverse transcribed following the Ovation PicoSL WTA System (Nugen). Briefly, the resulting double-strand cDNA was used for amplification based on SPIA technology. After purification according to Nugen protocol, 5 μg of single strand DNA was used for generation of Sens Target DNA using Ovation Exon Module kit (Nugen). 2.5 μg of Sens Target DNA were fragmented and labelled with biotin using Encore Biotin Module kit (Nugen). After control of fragmentation using Bioanalyzer 2100, the cDNA was then hybridized to GeneChip® Mouse Gene 1.0 ST (Affymetrix) at 45°C for 17 hours. After overnight hybridization, the ChIPs were washed using the fluidic station FS450 following specific protocols (Affymetrix) and scanned using the GCS3000 7G. The scanned images were then analyzed with Expression Console software (Affymetrix) to obtain raw data (cel files) and metrics for Quality Controls. The analysis of some of these metrics and the study of the distribution of raw data show no outlier experiment.

数千种长链基因间非编码RNA（long intergenic noncoding RNAs，lincRNAs）由哺乳动物基因组编码，既往研究表明其可作为顺式[1]或反式[2]转录激活因子、转录抑制因子[3,4]或miRNA诱饵[5,6]，发挥多种生物学功能。然而，大多数lincRNAs的体内功能仍未明确。
本研究揭示了一种新型长链基因间非编码RNA linc-MYH在成年快肌纤维特化中的作用。
骨骼肌快肌与慢肌表型的建立依赖于多种肌纤维特异性基因的差异表达[7]。本研究发现，linc-MYH与快肌MYH基因共享一个位于快肌MYH基因座内的增强子，该增强子受Six1同源域蛋白调控。肌肉特异性Six1突变小鼠的慢型基因表达上调，而linc-MYH与快型基因的表达则下调。
通过体内敲降实验与全转录组分析揭示的linc-MYH功能表明，其可精准抑制调控慢肌收缩特性的基因表达，并提升快肌纤维中快型肌肉基因的表达水平。
因此，快肌纤维中高效快肌肌节单位的形成、恰当的钙离子循环以及兴奋-收缩-松弛偶联，需通过Six结合增强子对快肌MYHs与linc-MYH表达的协同调控来实现。
实验设计：将10 μg表达短发夹RNA（short hairpin RNA，shRNA）的载体通过电穿孔法导入8周龄小鼠的胫前肌（TA）。电穿孔两周后，在尼康SMZ1500体式显微镜下解剖表达绿色荧光蛋白（green fluorescent protein，GFP）的TA肌纤维，置于液氮中速冻后备用。通过检测各shRNA转染的TA肌肉中linc-MYH的转录水平，验证各shRNA的敲降效率，最终选择靶向序列5'-TTCTGCTCACCACCTACAATT-3'的shRNA用于后续敲降实验。
使用Agilent RNA6000纳米芯片试剂盒通过2100生物分析仪验证RNA质量后，按照Ovation PicoSL WTA全转录组扩增试剂盒（Nugen）的步骤，对50 ng总RNA进行反转录。简言之，所得双链cDNA用于基于SPIA技术的扩增。按照Nugen试剂盒说明书纯化后，取5 μg单链DNA，使用Ovation Exon Module试剂盒（Nugen）制备靶标敏感DNA。取2.5 μg靶标敏感DNA，使用Encore Biotin Module试剂盒（Nugen）进行片段化与生物素标记。通过2100生物分析仪验证片段化效果后，将cDNA与GeneChip®小鼠基因1.0 ST芯片（Affymetrix）在45℃下杂交17小时。过夜杂交完成后，使用流体工作站FS450按照Affymetrix特定方案洗涤芯片，再通过GCS3000 7G扫描仪进行扫描。使用Expression Console软件（Affymetrix）分析扫描图像，以获取原始数据（cel文件）与质量控制指标。对这些指标及原始数据分布的分析显示，本实验无异常样本。
收集Six1条件性敲除（cSix1 KO）小鼠与对照小鼠的腓肠肌，按照Trizol试剂（Invitrogen）说明书提取总RNA。使用Agilent RNA6000纳米芯片试剂盒通过2100生物分析仪验证RNA质量后，按照Ovation PicoSL WTA全转录组扩增试剂盒（Nugen）的步骤，对50 ng总RNA进行反转录。简言之，所得双链cDNA用于基于SPIA技术的扩增。按照Nugen试剂盒说明书纯化后，取5 μg单链DNA，使用Ovation Exon Module试剂盒（Nugen）制备靶标敏感DNA。取2.5 μg靶标敏感DNA，使用Encore Biotin Module试剂盒（Nugen）进行片段化与生物素标记。通过2100生物分析仪验证片段化效果后，将cDNA与GeneChip®小鼠基因1.0 ST芯片（Affymetrix）在45℃下杂交17小时。过夜杂交完成后，使用流体工作站FS450按照Affymetrix特定方案洗涤芯片，再通过GCS3000 7G扫描仪进行扫描。使用Expression Console软件（Affymetrix）分析扫描图像，以获取原始数据（cel文件）与质量控制指标。对这些指标及原始数据分布的分析显示，本实验无异常样本。