A Novel YY1-miR-1 Regulatory Circuit in Skeletal Myogenesis Revealed by Genome-Wide Prediction of YY1-miRNA Network

Leina Lu,Peiyong Jiang,Eric Z Chen,Huating Wang,Kun Sun,Lijun Wang,Liang Zhou,Hao Sun,Xiaoxi Su,Andre Van Wijnen

doi:10.1371/journal.pone.0027596

Abstract

microRNAs (miRNAs) are non-coding RNAs that regulate gene expression post-transcriptionally, and mounting evidence supports the prevalence and functional significance of their interplay with transcription factors (TFs). Here we describe the identification of a regulatory circuit between muscle miRNAs (miR-1, miR-133 and miR-206) and Yin Yang 1 (YY1), an epigenetic repressor of skeletal myogenesis in mouse. Genome-wide identification of potential down-stream targets of YY1 by combining computational prediction with expression profiling data reveals a large number of putative miRNA targets of YY1 during skeletal myoblasts differentiation into myotubes with muscle miRs ranking on top of the list. The subsequent experimental results demonstrate that YY1 indeed represses muscle miRs expression in myoblasts and the repression is mediated through multiple enhancers and recruitment of Polycomb complex to several YY1 binding sites. YY1 regulating miR-1 is functionally important for both C2C12 myogenic differentiation and injury-induced muscle regeneration. Furthermore, we demonstrate that miR-1 in turn targets YY1, thus forming a negative feedback loop. Together, these results identify a novel regulatory circuit required for skeletal myogenesis and reinforce the idea that regulatory circuitries involving miRNAs and TFs are prevalent mechanisms.

Highlights

MicroRNAs are non-coding single-stranded RNAs of 21 to 25 nucleotides and constitute a novel class of gene regulators that are found in many eukaryotic organisms. miRNAs negatively regulate their targets at the post-transcriptional level through binding to their 39 UTR regions leading to either mRNA cleavage or translational repression depending on the degree of complementarities between the miRNA and the target [1]
We reasoned that functional Yin Yang 1 (YY1) regulation on miRNA genes should lead to silencing of their expression in myoblasts and upregulation of miRNA expression levels upon myoblasts differentiation as YY1 is known as a repressor of muscle genes and YY1 itself is gradually down-regulated in myogenic differentiation [19]
Total RNAs were isolated from undifferentiated C2C12 myoblasts in growth medium (GM) or myotubes formed in differentiation medium (DM) for 1 or 3 days and subjected to miRNA array analyses using microRNA CHIPv4 [26]

Summary

Introduction

MicroRNAs (miRNAs) are non-coding single-stranded RNAs of 21 to 25 nucleotides and constitute a novel class of gene regulators that are found in many eukaryotic organisms. miRNAs negatively regulate their targets at the post-transcriptional level through binding to their 39 UTR regions leading to either mRNA cleavage or translational repression depending on the degree of complementarities between the miRNA and the target [1]. Skeletal muscle growth and regeneration are attributed to satellite cells-muscle stems cells, which are characterized by the expression of paired-box transcription factor 7 (Pax7) and when activated, become immature muscle cells or myoblasts which will proliferate and differentiate. A major portion of our understanding of myogenic differentiation is focused at the level of transcription, orchestrated by a complex network of transcription factors (TFs) including MyoD, Myogenic Factor 5 (Myf5), Myogenin, Myogenic Regulatory Factor 4 (MRF4), and myocyte enhancer factor 2 (Mef). A major portion of our understanding of myogenic differentiation is focused at the level of transcription, orchestrated by a complex network of transcription factors (TFs) including MyoD, Myogenic Factor 5 (Myf5), Myogenin, Myogenic Regulatory Factor 4 (MRF4), and myocyte enhancer factor 2 (Mef2) These factors activate muscle specific genes to coordinate myoblasts to terminally withdraw from the cell cycle and subsequently fuse into multinucleated myotubes [8]. On the other hand, is required for satellite cell proliferation and sustained expression of Pax prevents myogenic induction and muscle terminal differentiation [9]

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Conclusion