ABSTRACT
The sarcomeric myosin gene, Myh7b, encodes an intronic microRNA, miR-499, which regulates cardiac and skeletal muscle biology, yet little is known about its transcriptional regulation. To identify the transcription factors involved in regulating Myh7b/miR-499 gene expression, we have mapped the transcriptional start sites and identified an upstream 6.2 kb region of the mouse Myh7b gene whose activity mimics the expression pattern of the endogenous Myh7b gene both in vitro and in vivo. Through promoter deletion analysis, we have mapped a distal E-box element and a proximal Ikaros site that are essential for Myh7b promoter activity in muscle cells. We show that the myogenic regulatory factors, MyoD, Myf5 and Myogenin, bind to the E-box, while a lymphoid transcription factor, Ikaros 4 (Eos), binds to the Ikaros motif. Further, we show that through physical interaction, MyoD and Eos form an active transcriptional complex on the chromatin to regulate the expression of the endogenous Myh7b/miR-499 gene in muscle cells. We also provide the first evidence that Eos can regulate expression of additional myosin genes (Myosin 1 and ß-Myosin) via the miR-499/Sox6 pathway. Therefore, our results indicate a novel role for Eos in the regulation of the myofiber gene program.
Subject(s)
Carrier Proteins/metabolism , Gene Expression Regulation , Ikaros Transcription Factor/metabolism , MicroRNAs/genetics , Myogenic Regulatory Factors/metabolism , Myosin Heavy Chains/genetics , Myosin Type II/genetics , Nerve Tissue Proteins/metabolism , Transcription, Genetic , Animals , Base Sequence , Carrier Proteins/chemistry , Carrier Proteins/physiology , Cells, Cultured , DNA-Binding Proteins , E-Box Elements , Humans , Ikaros Transcription Factor/physiology , Mice , MicroRNAs/biosynthesis , Molecular Sequence Data , Muscle, Skeletal/metabolism , MyoD Protein/metabolism , Myocardium/metabolism , Myogenic Regulatory Factors/physiology , Myosin Heavy Chains/biosynthesis , Myosin Type II/biosynthesis , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/physiology , Promoter Regions, Genetic , Protein Interaction Domains and Motifs , Transcription Initiation SiteABSTRACT
The ancient MYH7b gene, expressed in striated muscle and brain, encodes a sarcomeric myosin and the intronic microRNA miR-499. We find that skipping of an exon introduces a premature termination codon in the transcript that downregulates MYH7b protein production without affecting microRNA expression. Among other genes, endogenous miR-499 targets the 3' untranslated region of the transcription factor Sox6, which in turn acts as a repressor of MYH7b transcriptional activity. Thus, concerted transcription and alternative splicing uncouple the level of expression of MYH7b and miR-499 when their coexpression is not required.
Subject(s)
Alternative Splicing , Cardiac Myosins/genetics , Exons , Introns/genetics , MicroRNAs/metabolism , Myosin Heavy Chains/genetics , Protein Isoforms/genetics , Amino Acid Sequence , Animals , Cardiac Myosins/metabolism , Cell Line , Codon, Nonsense , Gene Expression Regulation , Humans , Mice , Mice, Inbred C57BL , MicroRNAs/genetics , Molecular Sequence Data , Myosin Heavy Chains/metabolism , Protein Isoforms/metabolism , SOXD Transcription Factors/genetics , SOXD Transcription Factors/metabolism , Sequence Alignment , Tissue Distribution , ZebrafishABSTRACT
Previous studies of the Sleeping Beauty (SB) transposon system, as an insertional mutagen in the germline of mice, have used reverse genetic approaches. These studies have led to its proposed use for regional saturation mutagenesis by taking a forward-genetic approach. Thus, we used the SB system to mutate a region of mouse Chromosome 11 in a forward-genetic screen for recessive lethal and viable phenotypes. This work represents the first reported use of an insertional mutagen in a phenotype-driven approach. The phenotype-driven approach was successful in both recovering visible and behavioral mutants, including dominant limb and recessive behavioral phenotypes, and allowing for the rapid identification of candidate gene disruptions. In addition, a high frequency of recessive lethal mutations arose as a result of genomic rearrangements near the site of transposition, resulting from transposon mobilization. The results suggest that the SB system could be used in a forward-genetic approach to recover interesting phenotypes, but that local chromosomal rearrangements should be anticipated in conjunction with single-copy, local transposon insertions in chromosomes. Additionally, these mice may serve as a model for chromosome rearrangements caused by transposable elements during the evolution of vertebrate genomes.
