An intragenic MEF2-dependent enhancer directs muscle-specific expression of microRNAs 1 and 133

Abstract

The muscle-specific microRNAs, miR-1 and miR-133, play important roles in muscle growth and differentiation. Here, we show that the MEF2 transcription factor, an essential regulator of muscle development, directly activates transcription of a bicistronic primary transcript encoding miR-1-2 and 133a-1 via an intragenic muscle-specific enhancer located between the miR-1-2 and 133a-1 coding regions. This MEF2-dependent enhancer is activated in the linear heart tube during mouse embryogenesis and thereafter controls transcription throughout the atrial and ventricular chambers of the heart. MEF2 together with MyoD also regulates the miR-1-2/-133a-1 intragenic enhancer in the somite myotomes and in all skeletal muscle fibers during embryogenesis and adulthood. A similar muscle-specific intragenic enhancer controls transcription of the miR-1-1/-133a-2 locus. These findings reveal a common architecture of regulatory elements associated with the miR-1/-133 genes and underscore the central role of MEF2 as a regulator of the transcriptional and posttranscriptional pathways that control cardiac and skeletal muscle development.

Fig. 1.

Down-regulation of miR-1 and 133 in hearts of MEF2 mutant mice. Mef2c and Mef2d were deleted in the heart by breeding Mef2c^loxP/−; Mef2d^loxP/loxP mice to Nkx2.5-Cre transgenic mice (KO). RNA was isolated from hearts of these mutant mice, which are viable, and WT littermates, and expression of the indicated miRNAs was detected by real-time PCR.

Fig. 2.

The miR-1-2/133a-1 gene and primary transcripts encoding miR-1-2 and miR-133a-1. (A) The genomic structure of the mouse miR-1-2/133a-1 gene is shown. The premiR-1-2 stem–loop is 2.5 kb upstream of the premiR-133a-1. Locations of enhancer 1 (E1) and the 330-bp intragenic enhancer (E2) are shown. Primers used in the RACE and RT-PCR are also shown. The proposed structure of the primary transcript containing stem–loop sequences of both miRs is shown. This transcript was not detected in RT-PCR. (B) Structures of primary transcripts of miR-1-2 and miR-133a-1 mapped by 5′- and 3′-RACE. (Upper) Three transcripts were identified for pri-miR-1-2, and two of them were spliced internally. (Lower) Two transcripts were identified for pri-miR-133a-1. Both transcripts were spliced and did not contain premiR-1-2 sequences.

Fig. 3.

Muscle-specific expression of the intragenic miR-1-2/133a-1 enhancer. (A) β-Galactosidase staining was performed on embryos from staged mating of a stable transgenic line bearing the 2.5-kb hsp68-lacZ construct. LacZ expression in the heart is observed as early as E8.5. LacZ is also expressed in somites starting from E9.5. At E11.5, lacZ expression is observed in body wall musculature. (B) Transverse sections revealed lacZ expression in the outflow tract and four chambers of the heart as well as in somites. Embryos from the stable transgenic line were stained for β-galactosidase activity, sectioned, and counterstained with Nuclear Fast red. h, heart; oft, outflow tract; ra, right atrium; la, left atrium; rv, right ventricle; lv, left ventricle; m, somite myotomes.

Fig. 4.

Delineation of a miR-1-2/133a-1 muscle-specific enhancer. Summary of transgenic constructs used to delineate the intragenic miR-1-2/133a-1 enhancer. Fractions of F₀ transgenic embryos showing cardiac and skeletal muscle (SKM) expression at E12.5 are indicated in the right column. Construct 1 is identical to the construct used in Fig. 3. Evolutionary conservation of the 2.5-kb fragment is shown at the bottom.

Fig. 5.

Analysis of the MEF2 site and E-box in the miR-1-2/133a-1 enhancer. (A) Binding of MEF2C to the MEF2-binding site in the minimal enhancer by gel mobility shift assay. A ³²P-labeled oligonucleotide probe containing the MEF2-binding site and total-cell extract from COS-1 cells transfected with a MEF2C expression plasmid formed a DNA–protein complex in the assay. A ³²P-labeled oligonucleotide probe containing a mutated MEF2-binding site did not form a DNA–protein complex with the MEF2C protein. The complex was supershifted by using a MEF2C-specific antibody, and unlabeled WT oligonucleotide containing the MEF2-binding site competed for binding. (B) Mutation of the MEF2-binding site in the 2.5-kb enhancer abolished expression in somites at E12.5. (C) Heart and transverse sections showed loss of ventricular chamber expression in the MEF2 mutant embryos. Expression in the atrial chambers was not affected. ra, right atrium; la, left atrium; rv, right ventricle; lv, left ventricle. (D) Binding of MyoD and E12 complex to the E-box binding site in the minimal enhancer element by gel mobility shift assay. A ³²P-labeled oligonucleotide containing the E-box-binding site and total-cell extract from COS-1 cells transfected with myc-tagged MyoD and E12 expression plasmids formed a DNA–protein complex in the assay. Mutant E-box probe did not form a DNA–protein complex with MyoD and E12. The complex was supershifted by using a Myc-specific antibody and unlabeled WT oligonucleotide containing the E-box binding site competed for binding. The asterisk represents nonspecific binding. (E) Mutation of the E-box-binding site in the 2.5-kb enhancer abolished expression in somites and ventral myoblasts at E12.5. Expression in the heart was not affected by the E-box mutation.

Fig. 6.

Delineation of an intragenic miR-1-1/133a-2 muscle-specific enhancer. (A) Summary of transgenic constructs used to delineate the intragenic miR-1-1/133a-2 enhancer. Fractions of F₀ transgenic embryos showing cardiac and skeletal muscle (SKM) expression at E12.5 are indicated in the right column. (B) Representative transgenic embryo showing lacZ expression from construct 1 and transverse section revealing lacZ expression in the outflow tract and four chambers of the heart as well as in somites. ra, right atrium; la, left atrium; rv, right ventricle; lv, left ventricle; m, somite myotomes.