Gene Expression
Section: Section of Gene Function and Regulation
Control of Myogenic Regulatory Factor Gene Expression
The key events in the development of the first skeletal muscles of the vertebrate embryo, the specification of myoblasts, their exit from the cell cycle and the activation of muscle-specific gene expression, are initiated and coordinated by the Myogenic Regulatory Factors (MRFs), Myf5, Myogenin, MyoD and Mrf4, members of the basic helix-loop-helix super-family of transcription factors. Myf5 is the first of these genes to be expressed and it is likely to be the determination gene for skeletal muscle. Using transgenic techniques we have defined the minimal DNA sequences required for the proper transcriptional control of Myogenin and have identified two of the regulatory pathways upstream of this gene. Having shown that Myogenin is controlled by Myf5, we have defined the minimal sequence by which Myf5 is regulated. This involved the adaptation of recent techniques whereby very large BAC based constructs (100+ kb of DNA) are required for full recapitulation of the expression pattern. Unlike other myogenic transcription factors Myf5 requires a large number of distinct enhancers, each driving an embryologically distinct group of skeletal muscle precursors, presumably because different signals provide the instructive signal in different anatomical locations. We have further shown that the requisite sequences are widely dispersed throughout 140 kb, including elements located within the adjacent Mrf4 gene. Extension of our interest to this latter gene has enabled us to demonstrate new developmental roles for Mrf4, and to explain that the unexpected phenotype following the knock-out of Mrf4 is most likely caused by deletion of an Myf5 enhancer element. We have also started the analysis of individual enhancer elements and have shown that the earliest functional enhancer is specific to a small population of cells in the dorsal part of the somite and is a required element if they are to express Myf5. Control of this Myf5 enhancer alone is at least as complex as control of the entire expression pattern of the downstream target, Myogenin.
External funding: The Medical Research Council and MYORES
The Regulation of Hox Gene Expression
In mammals there are four clusters of Hox genes, in each of which there is a direct relationship between the position of a gene within a cluster and both the timing of its expression and its final expression domain, a phenomenon known as colinearity which appears to have arisen very early in animal evolution. Genes at the 5' ends of clusters are activated late and expressed in posterior domains while genes at the 3' ends of clusters are activated early and expressed in anterior domains. The paralagous genes across the four clusters, eg. Hoxa4, Hoxb4, Hoxc4, and Hoxd4, have similar but not identical anterior boundaries of expression. Thus each pair of somites expresses a particular combination of Hox genes and it was therefore proposed that segmental identity was determined by the Hox code. Experiments in which Hox gene function is inactivated by targeted mutagenesis, or in which a given Hox gene is ectopically expressed in more anterior somites, indicate that the concept of the Hox code is generally valid. Given this, it follows that the establishment of the correct anterior boundary of expression is crucial. We have again used transgenic approaches to identify the sequences that control the expression of the Hoxb4 gene and have identified spatially specific enhancers that determine the correct anterior boundaries of expression. We have now identified cognate transcription factors which bind to these enhancers and have shown that imposition of an appropriate boundary also depends on interaction with the homologous promoter. We will continue to identify other transcription factors involved and to elucidate the mechanism of the interaction with the promoter.
In the Section of Gene Function and Regulation
