Cis-regulation of MyoD:a systems analysis of a fate master regulator.
Doctoral thesis, UCL (University College London).
Myogenesis is highly regulated and its activation in the embryo is controlled by a series of complex transcriptional regulatory networks that ultimately result in the expression of myogenic regulatory factors (MRFs). The MRFS, particularly MyoD and Myf5, are responsible, in concert with a vast range of cofactors, for directing the expression of genes responsible for muscle formation and activity. Several candidate proteins have emerged as being responsible for MRF expression, as well as numerous downstream effectors involved in muscle formation in vivo. Several cis-regulatory elements have been identified for MyoD, but only a handful of factors have been identified that bind these elements. In addition, knockout experiments of these regions do not result in a complete loss of MyoD expression, suggesting a certain level of redundancy and the existence of other yet unidentified cis-regulatory modules. In this study, novel potential regulatory regions within the MyoD upstream genomic locus were identified by comparative genomics. These regions, named ReMos 9, 10 and 11, were conserved in mammals, chick and fish. Reporter assays in C2C12 cells using these regions cloned upstream of the MyoD promoter revealed that they positively enhanced the promoter activity. A synergy was uncovered between ReMo 9 and 10, which have a strong positive effect on promoter activity, but none individually; ReMo 11 seemed to disrupt this synergy. In addition, ReMos 9+10 and the CER enhancer were shown, by double fluorescent RNA in situ hybridisations, to be transcribed and possess cryptic promoter activity. This suggested that these elements acted as alternative promoters and encoded RNAs that regulated MyoD gene expression. Furthermore, the use of a newly engineered database generated predictions of DNA-binding factors interacting with the cis-regulatory regions, as well as protein interaction networks involved in MyoD regulation. These predictions were refined and constrained with biological input data derived by microarrays of single-cells transiently expressing relevant constructs. A list of candidate muscle-specific binding factors was then tested in vitro by siRNA knockdown experiments, and showed that MyoD disrupts the positive synergistic effect of ReMos 9 and 10 on the PRR. In conclusion, this study identified a number of regions that seem to be involved in MyoD regulation, and candidate factors binding to the MyoD cis-regulatory regions. Further in vivo validation will identify their function in MyoD spatio-temporal gene expression.
|Title:||Cis-regulation of MyoD:a systems analysis of a fate master regulator|
|Additional information:||Authorisation for digitisation not received|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Medical Sciences > Medicine (Division of) > Wolfson Inst for Biomedical Research|
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