Expression Profiles And Regulation Mechanism Of FIT1 Gene And Association Analyses Of Repetitive Elements With Muscle Disease (FSHD)

Section 1: Study on Porcine FIT1 Gene Expression Profiles and Regulation MechanismFat-induced transcript 1(FIT1/FITM1) gene is a member of the conserved gene family important for triglyceride-rich lipid droplet accumulation. FIT1 gene displays a similar muscle-specific expression across pig, mouse, and human. Triglyceride content in skeletal muscle plays a key role in meat quality and flavors. To gain better knowledge of the potential role of FIT1 gene in the growth and development of skeletal muscles as well as the correlations with pork meat quality traits, our attention is put on the expression profiling of FIT1 gene in muscles and the myogenic regulation mechanism of porcine FIT1 gene. The results were as follows.1. Using real time qRT-PCR, the expression of porcine FIT1 gene in 16 different tissues of Large White pigs at 4-month old was analyzed. Results showed that FIT1 gene exhibited a very restricted muscle-specific expression pattern in pig, with a high level in heart and skeletal muscle. Expression of FIT1 gene at various developmental stages in longissimus dorsi muscles and expression in C2C12 cells with various differentiation time showed that porcine FIT1 was expressed at the highest level at 4 months old with the lowest level at the embryonic stage; the expression of FIT1 in C2C12 cells was induced dramatically with differentiation.2. Approximately 1 kb of the FIT1 5′-flanking region(-1037 to +3 bp) was obtained. Alignment of the proximal sequence of this region among pig, mouse, rat and human indicated that two highly conserved E-box elements were present across these species. Various deletion fragments were introduced to a luciferase reporter vector and using dual luciferase activity assay, the region-397 to +3 bp was identified as the core promoter, which showed no activity in C3H10 T 1/2 fibroblast cells, and PK15 cells, lower in C2C12 myoblasts and stronger exclusively in C2C12 myotubes.3. Removal of the two conserved E-boxes from the region-397 to +3 bp showed that the region(-245 to +3 bp) comprising both E-box elements was necessary for the promoter activity of FIT1 in C2C12 myotubes. Site-directed mutagenesis to E-box1 and E-box2 showed that mutations in either E-box1 or E-box2 significantly reduced the activity of FIT1 promoter and to some extent E-box1 governs the promoter activity.4. Over-expression of MyoD1 significantly enhanced the promoter activity. siRNA against MyoD1 was used to further prove the role of MyoD1 in FIT1 promoter and results showed that inhibition of MyoD1 during differentiation dramatically decreased the FIT1 transcription activity.5. ChIP experiments were performed in C2C12 myotubes to confirm whether both E-box elements interacted with MyoD1. Using regular PCR and Ch IP-qPCR it was shown that MyoD could bind to either E-box site and the relative enrichment to E-box1 was larger than that to E-box2. This indicated that MyoD1 activated the promoter activity of FIT1 via the interaction with this E-box1 site.Section 2: Association Analyses of Repetitive Elements With Muscle Disease(FSHD)FSHD(Facioscapulohumeral muscular dystrophy) is an autosomal dominant myopathy resulting from the incompletely epigenetic derepression of D4Z4 macrosatellite repeats on the chromosome 4q during muscle development. The resulting toxic DUX4-fl mRNA and DUX4 protein were considered as the leading determinants of FSHD onset and progression whereas the DUX4-fl gene was not expressed in normal individuals. Based on the high incidence of FSHD in infants and adults, it is crucial to search for effective treatments on FSHD progression. Here, we aimed to investigate the potential role of DUX4-targeted repetitive elements in FSHD cells. The main results were shown as follows.1. 11 DUX4-targeted repetitive elements, including hsat2, L1MC4, L1P4 b, LTR12 D, LTR13, MER50 B, MER52 D, MER41 G, MER72, MLT2A1 and MLT2 D, were quantitatively analyzed in myogenic cells from FSHD(17Abic and 07Abic) and normal individuals(17Ubic and 07Ubic) at the transcriptional level. It was demonstrated that 9 repetitive RNAs out of 11(except MLT2A1 and MLT2D) were activated in differentiated 17 Abic myoblasts. In differentiated 07 Abic myoblasts all the other repetitive RNAs but MLT2A1, hsat2 and MER52D were activated as well.2. Conservative sequences of hsat2 and MLT2 D were amplified and cloned into the lentivirus vector pLKO-mUTL followed by the generation of the mature lentivirus particles with the packaging plasmid and the envelop plasmid in 293 T cells. Total RNAs used for RNA-seq were prepared from 17 Ubic myotubes transduced with individual lentivirus. FSHD-associated genes(ANO5, ATP2A1, CALCRL, CLYBL, CYP39A1, DAG1, FAM149 A, GAD1, IL32, PDLIM3, TLR3 and CCNA1) were selected from RNA-seq results and validated with qRT-PCR. The results showed that non-coding RNAs hsat2 and MLT2 D significantly reduced the levels of five FSHD-associated genes(ANO5, CLYBL, FAM149 A, PDLIM3 and IL32).3. Expression of FSHD-associated genes were investigated as well in 17 Ubic myotubes with or without lentiviruses using qRT-PCR. Results showed that lentiviruses without any insert sequence had no effect on FSHD-associated genes.