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

ABSTRACT: 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. 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.

ORGANISM(S): Mus musculus

SUBMITTER: iori sakakibara 

PROVIDER: E-GEOD-46151 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress