Differentiation and fiber type-specific activity of a muscle creatine kinase intronic enhancer

Phillip Wl Tai,Charis L Himeda,Deri L Helterline,Barbara J Wold,John C Angello,Catherine L Smith,Katherine I Fisher-Aylor,Alexandra P Mackenzie,Stephen D Hauschka,Robert E Welikson

doi:10.1186/2044-5040-1-25

Phillip Wl Tai, Charis L Himeda + Show 8 more

Open Access

https://doi.org/10.1186/2044-5040-1-25

Copy DOI

Abstract

BackgroundHundreds of genes, including muscle creatine kinase (MCK), are differentially expressed in fast- and slow-twitch muscle fibers, but the fiber type-specific regulatory mechanisms are not well understood.ResultsModulatory region 1 (MR1) is a 1-kb regulatory region within MCK intron 1 that is highly active in terminally differentiating skeletal myocytes in vitro. A MCK small intronic enhancer (MCK-SIE) containing a paired E-box/myocyte enhancer factor 2 (MEF2) regulatory motif resides within MR1. The SIE's transcriptional activity equals that of the extensively characterized 206-bp MCK 5'-enhancer, but the MCK-SIE is flanked by regions that can repress its activity via the individual and combined effects of about 15 different but highly conserved 9- to 24-bp sequences. ChIP and ChIP-Seq analyses indicate that the SIE and the MCK 5'-enhancer are occupied by MyoD, myogenin and MEF2. Many other E-boxes located within or immediately adjacent to intron 1 are not occupied by MyoD or myogenin. Transgenic analysis of a 6.5-kb MCK genomic fragment containing the 5'-enhancer and proximal promoter plus the 3.2-kb intron 1, with and without MR1, indicates that MR1 is critical for MCK expression in slow- and intermediate-twitch muscle fibers (types I and IIa, respectively), but is not required for expression in fast-twitch muscle fibers (types IIb and IId).ConclusionsIn this study, we discovered that MR1 is critical for MCK expression in slow- and intermediate-twitch muscle fibers and that MR1's positive transcriptional activity depends on a paired E-box MEF2 site motif within a SIE. This is the first study to delineate the DNA controls for MCK expression in different skeletal muscle fiber types.

Highlights

Hundreds of genes, including muscle creatine kinase (MCK), are differentially expressed in fast- and slow-twitch muscle fibers, but the fiber type-specific regulatory mechanisms are not well understood
Sequence analysis of the intron 1 modulatory region modulatory region 1 (MR1) reveals multiple highly conserved sequence motifs To begin our characterization of mouse MR1 and its role in MCK gene expression, a 1,081-bp region (+740 to +1,721) was aligned to the MR1 regions of five other mammalian species to reveal the presence of potentially functional control elements (Figure 1 and Additional file 1, Figure S1)
The MCK small intronic enhancer (MCK-SIE) exhibits high sequence conservation and contains four motifs known to control the transcription of many muscle genes: two core E-boxes (CAnnTG) [41,42], a myocyte enhancer factor 2 (MEF2) site and an overlapping MAF half-site and alkaline phosphatase (AP)-1 site (Figure 1)

Summary

Introduction

Hundreds of genes, including muscle creatine kinase (MCK), are differentially expressed in fast- and slow-twitch muscle fibers, but the fiber type-specific regulatory mechanisms are not well understood. The 5’-enhancer (-1,256 to -1,050) is the best characterized of the known regions [18,20,23,24,25,26,27,28] It has the ability (1) to drive high-level transcription of reporter genes in skeletal and cardiac muscle in both transgenic mice and cell culture and (2) to function with heterologous promoters [29]. Deletion and mutation analyses within this region in cultured skeletal myocytes and in transgenic mice have defined seven control elements: muscle-specific (CArG) and serum response element promoters, activator protein 2 (AP-2), Six4/5, AT-rich, left and right E-boxes and myocyte enhancer factor 2 (MEF2) [23,24]. The MCK proximal promoter (-358 to +1) has been thoroughly studied It is active in skeletal and cardiac myocytes in culture and can function independently of the 5’-enhancer. The proximal promoter contains at least four active transcription factor binding sites: p53, E-box, CArG, and MPEX, a recently discovered sequence that recruits both Mycassociated zinc finger protein (MAZ) and Krupple-like factor 3 (KLF3) [30,31,32,33]

Methods

Results

Discussion

Conclusion