MUNC, An eRNA Upstream From the MYOD Gene, Induces Myogenic Genes in trans, Independently of MyoD
Cichewicz, Magdalena, Biochemistry and Molecular Genetics - School of Medicine, University of Virginia
Dutta, Anindya, Department of Biochemistry and Molecular Genetics, University of Virginia
Myogenesis occurs during embryonic development while all tissues are being defined, and during regeneration of adult muscle tissue after injury and inflammation. Among myogenesis specific transcriptional factor, the two main players, MyoD and myogenin, are crucial for the process. In a healthy organism, all myogenic factors work in a strictly time-regulated manner to build new muscle fibers, and a feed-forward mechanism of the molecular cascade has been described. Although we know the main protein factors regulating myogenesis, still we are unable to explain the molecular pathology of various muscular dystrophies.
Since genome wide studies have become widely accessible, researchers now recognize novel transcripts which do not code for proteins but are functional as RNAs. It is established that the majority of transcripts produced by the mammalian genome does not express proteins, but is crucial for proper homeostasis of various biological systems. There are known examples of long noncoding RNAs (lncRNAs) which are tissue-specific, and some of them are cancer-specific. They function as scaffold molecules, chromatin modifiers, and post-transcriptional regulators of mRNAs.
The goal of my project was to describe novel pro-myogenic long noncoding RNAs induced during muscle differentiation, and to investigate their functions and mechanisms. In the beginning of the project, publicly available genome-wide datasets of differentiating murine myoblasts, C2C12 cells, were analyzed. After combining RNA-Seq, RNA PolII ChIP-Seq and H3K4me3 ChIP-Seq, a pool of potential long noncoding transcripts induced during differentiation was established. After confirmation that several of these lncRNAs are indeed induced during differentiation, we decided to focus on one specific transcript, MyoD Upstream NonCoding Element (MUNC), whose genomic locus partially overlaps with the DRR-enhancer for MyoD. We characterized the sequence of MUNC, and its two isoforms: unspliced and spliced. We found a functional human homolog of the transcript. In vitro and in vivo depletion experiments established that MUNC is pro-myogenic. By stable overexpression we learned that MUNC does not have one domain responsible for its action, rather at least two fragments of MUNC can independently induce myogenic transcripts. Partial overlap between the MUNC locus and an enhancer for MYOD sequence suggested that the only function of MUNC is activation of MYOD expression as an enhancer RNA (eRNA). To investigate this possibility, we generated a MYOD knockout cell line. By overexpressing MUNC in MYOD deleted cells we discovered that in the absence of MyoD, MUNC is still able to regulate specific genes, mostly skeletal muscle related. We characterized genes regulated by the coordinated action of MyoD and MUNC, and genes regulated by their separate pathways. Our study showed that MUNC does not only work as an enhancer inducing MYOD expression, but also has MyoD-independent functions during myogenesis. Thus it is both an eRNA and an lncRNA.
PHD (Doctor of Philosophy)
MUNC, MyoD, myogenesis, lncRNA, enhancer