CF2 transcription factor is involved in the regulation of Mef2 RNA levels, nuclei number and muscle fiber size.

CF2 and Mef2 influence a variety of developmental muscle processes at distinct stages of development. Nevertheless, the exact nature of the CF2-Mef2 relationship and its effects on muscle building remain yet to be resolved. Here, we explored the regulatory role of CF2 in the Drosophila embryo muscle formation. To address this question and not having proper null CF2 mutants we exploited loss or gain of function strategies to study the contribution of CF2 to Mef2 transcription regulation and to muscle formation. Our data point to CF2 as a factor involved in the regulation of muscle final size and/or the number of nuclei present in each muscle. This function is independent of its role as a Mef2 collaborative factor in the transcriptional regulation of muscle-structural genes. Although Mef2 expression patterns do not change, reductions or increases in parallel in CF2 and Mef2 transcript abundance were observed in interfered and overexpressed CF2 embryos. Since CF2 expression variations yield altered Mef2 expression levels but with correct spatio-temporal Mef2 expression patterns, it can be concluded that only the mechanism controlling expression levels is de-regulated. Here, it is proposed that CF2 regulates Mef2 expression through a Feedforward Loop circuit.

Co-operation between enhancers modulates quantitative expression from the Drosophila Paramyosin/miniparamyosin gene in different muscle types.

The distinct muscles of an organism accumulate different quantities of structural proteins, but always maintaining their stoichiometry. However, the mechanisms that control the levels of these proteins and that co-ordinate muscle gene expression remain to be defined. The paramyosin/miniparamyosin gene encodes two thick filament proteins transcribed from two different promoters. We have analysed the regulatory regions that control expression of this gene and that are situated in the two promoters, the 5' and the internal promoters, both in vivo and in silico. A distal muscle enhancer containing three conserved MEF2 motifs is essential to drive high levels of paramyosin expression in all the major embryonic, larval and adult muscles. This enhancer shares sequence motifs, as well as its structure and organisation, with at least four co-regulated muscle enhancers that direct similar patterns of expression. However, other elements located downstream of the enhancer are also required for correct gene expression. Other muscle genes with different patterns of expression, such as miniparamyosin, are regulated by other basic mechanisms. The expression of miniparamyosin is controlled by two enhancers, AB and TX, but a BF modulator is required to ensure the correct levels of expression in each particular muscle. We propose a mechanism of transcriptional regulation in which similar enhancers are responsible for the spatio-temporal expression of co-regulated genes. However, it is the interaction between enhancers which ensures that the correct amounts of protein are expressed at any particular time in a cell, adapting these levels to their specific needs. These mechanisms may not be exclusive to neural or muscle tissue and might represent a general mechanism for genes that are spatially and temporally co-regulated.

CF2 activity and enhancer integration are required for proper muscle gene expression in Drosophila.

The creation of the contractile apparatus in muscle involves the co-activation of a group of genes encoding muscle-specific proteins and the production of high levels of protein in a short period of time. We have studied the transcriptional control of six Drosophila muscle genes that have similar expression profiles and we have compared these mechanisms with those employed to control the distinct expression profiles of other Drosophila genes. The regulatory elements controlling the transcription of co-expressed muscle genes share an Upstream Regulatory Element and an Intronic Regulatory Element. Moreover, similar clusters of MEF2 and CF2 binding sites are present in these elements. Here, we demonstrate that CF2 depletion alters the relative expression of thin and thick filament components. We propose that the appropriate rapid gene expression responses during muscle formation and the maintenance of each muscle type is guaranteed in Drosophila by equivalent duplicate enhancer-like elements. This mechanism may be exceptional and restricted to muscle genes, reflecting the specific requirement to mediate rapid muscle responses. However, it may also be a more general mechanism to control the correct levels of gene expression during development in each cell type.