Jaime J. Carvajal

A BAC transgenic analysis of the Mrf4/Myf5 locus reveals interdigitated elements that control activation and maintenance of gene expression during muscle development

The muscle-specific transcription factors Myf5 and Mrf4 are two of the four myogenic regulatory factors involved in the transcriptional cascade responsible for skeletal myogenesis in the vertebrate embryo. Myf5 is the first of these four genes to be expressed in the mouse. We have previously described discrete enhancers that drive Myf5 expression in epaxial and hypaxial somites, branchial arches and central nervous system, and argued that additional elements are required for proper expression (Summerbell, D., Ashby, P. R., Coutelle, O., Cox, D., Yee, S. P. and Rigby, P. W. J. (2000) Development 127, 3745-3757). We have now investigated the transcriptional regulation of both Myf5 and Mrf4 using bacterial artificial chromosome transgenesis. We show that a clone containing Myf5 and 140 kb of upstream sequences is sufficient to recapitulate the known expression patterns of both genes. Our results confirm and reinforce the conclusion of our earlier studies, that Myf5 expression is regulated differently in each of a considerable number of populations of muscle progenitors, and they begin to illuminate the evolutionary origins of this complex regulation. We further show that separate elements are involved in the activation and maintenance of expression in the various precursor populations, reflecting the diversity of the signals that control myogenesis. Mrf4 expression requires at least four elements, one of which may be shared with Myf5, providing a possible explanation for the linkage of these genes throughout vertebrate phylogeny. Further complexity is revealed by the demonstration that elements which control Mrf4 and Myf5 are embedded in an unrelated neighbouring gene.

The Myogenic Factor Myf5 Supports Efficient Skeletal Muscle Regeneration by Enabling Transient Myoblast Amplification

The myogenic factor Myf5 defines the onset of myogenesis in mammals during development. Mice lacking both Myf5 and MyoD fail to form myoblasts and are characterized by a complete absence of skeletal muscle at birth. To investigate the function of Myf5 in adult skeletal muscle, we generated Myf5 and mdx compound mutants, which are characterized by constant regeneration. Double mutant mice show an increase of dystrophic changes in the musculature, although these mice were viable and the degree of myopathy was modest. Myf5 mutant muscles show a small decrease in the number of muscle satellite cells, which was within the range of physiological variations. We also observed a significant delay in the regeneration of Myf5 deficient skeletal muscles after injury. Interestingly, Myf5 deficient skeletal muscles were able to even out this flaw during the course of regeneration, generating intact muscles 4 weeks after injury. Although we did not detect a striking reduction of MyoD positive activated myoblasts or of Myf5‐LacZ positive cells in regenerating muscles, a clear decrease in the proliferation rate of satellite cell‐derived myoblasts was apparent in satellite cell‐derived cultures. The reduction of the proliferation rate of Myf5 mutant myoblasts was also reflected by a delayed transition from proliferation to differentiation, resulting in a reduced number of myotube nuclei after 6 and 7 days of culture. We reason that Myf5 supports efficient skeletal muscle regeneration by enabling transient myoblast amplification.

Analysis of a key regulatory region upstream of the Myf5 gene reveals multiple phases of myogenesis, orchestrated at each site by a combination of elements dispersed throughout the locus

Myf5 is the first myogenic regulatory factor to be expressed in the mouse embryo and it determines the entry of cells into the skeletal muscle programme. A region situated between -58 kb and -48 kb from the gene directs Myf5 transcription at sites where muscles will form. We now show that this region consists of a number of distinct regulatory elements that specifically target sites of myogenesis in the somite, limbs and hypoglossal cord, and also sites of Myf5 transcription in the central nervous system. Deletion of these sequences in the context of the locus shows that elements within the region are essential, and also reveals the combinatorial complexity of the transcriptional regulation of Myf5. Both within the -58 kb to -48 kb region and elsewhere in the locus, multiple sequences are present that direct transcription in subdomains of a single site during development, thus revealing distinct phases of myogenesis when subpopulations of progenitor cells enter the programme of skeletal muscle differentiation.

Transcriptional Dominance of Pax7 in Adult Myogenesis Is Due to High-Affinity Recognition of Homeodomain Motifs

Pax3 and Pax7 regulate stem cell function in skeletal myogenesis. However, molecular insight into their distinct roles has remained elusive. Using gene expression data combined with genome-wide binding-site analysis, we show that both Pax3 and Pax7 bind identical DNA motifs and jointly activate a large panel of genes involved in muscle stem cell function. Surprisingly, in adult myoblasts Pax3 binds a subset (6.4%) of Pax7 targets. Despite a significant overlap in their transcriptional network, Pax7 regulates distinct panels of genes involved in the promotion of proliferation and inhibition of myogenic differentiation. We show that Pax7 has a higher binding affinity to the homeodomain-binding motif relative to Pax3, suggesting that intrinsic differences in DNA binding contribute to the observed functional difference between Pax3 and Pax7 binding in myogenesis. Together, our data demonstrate distinct attributes of Pax7 function and provide mechanistic insight into the nonredundancy of Pax3 and Pax7 in muscle development.

Global transcriptional regulation of the locus encoding the skeletal muscle determination genes Mrf4 and Myf5

The linked Mrf4 and Myf5 genes encode two transcription factors essential for the determination and differentiation of skeletal muscle in the embryo. The locus is controlled by a multitude of interdigitated enhancers that activate gene expression at different times and in precisely defined progenitor cell populations. Manipulation of the enhancer-promoter composition of the locus reveals a novel mechanism for the regulation of such a gene cluster. Enhancers, promoters, and a new class of elements we call transcription balancing sequences, which can act as cryptic promoters, exist in a series of equilibria to ensure that enhancers and promoters together produce the highly dynamic and exquisitely specific expression patterns of the two genes. The proposed model depends upon nonproductive interactions between enhancers and both minimal and cryptic promoters, and is distinct from those developed for the beta-globin and Hox clusters. Moreover, it provides an explanation for the unexpected phenotypes of the three Mrf4 knockout alleles.

Identification of genetic variants that influence circulating IGF1 levels: a targeted search strategy

An important class of genetic variants that affect disease susceptibility may lie within regulatory elements that influence gene expression. Regulatory sequences are difficult to identify and may be distant from the genes they regulate, but many lie within evolutionarily conserved regions (ECRs). We used comparative genomics to identify 12 ECRs up to 75 kb 5 to and within introns of IGF1. These were screened by high-resolution melting curve analysis, and 18 single-nucleotide polymorphisms (SNPs) were identified, including five novel variants. We analysed two large population-based series of healthy women to test the nine SNPs with minor allele frequency (MAF) >1% within ECRs. Three of the nine SNPs within ECRs (rs35455143, rs35765817 and rs3839984) were significantly associated with circulating IGF1 levels in a multivariate analysis (P <or= 0.02 for each SNP, overall significance P < 0.001). All three are uncommon SNPs (MAF <or= 10%) that lie >70 kb 5 of IGF1. Two (rs35455143 and rs35765817) are in strong LD with each other and appear to have opposite effects on circulating IGF1. Our results on a subset of other SNPs in or near IGF1 were consistent with previously reported associations with IGF1 levels, although only one (rs35767: P = 0.05) was statistically significant. We believe that this is the first systematic study of an association between a phenotype and SNPs within ECRs extending over a large region adjacent to a gene. Targeting ECRs appears to be a useful strategy for identifying a subset of potentially functional non-coding regulatory SNPs.

Regulation of gene expression in vertebrate skeletal muscle.

During embryonic development the integration of numerous synergistic signalling pathways turns a single cell into a multicellular organism with specialized cell types and highly structured, organized tissues. To achieve this, cells must grow, proliferate, differentiate and die according to their spatiotemporal position. Unravelling the mechanisms by which a cell adopts the correct fate in response to its local environment remains one of the fundamental goals of biological research. In vertebrates skeletal myogenesis is coordinated by the activation of the myogenic regulatory factors (MRFs) in response to signals that are interpreted by their associated regulatory elements in different precursor cells during development. The MRFs trigger a cascade of transcription factors and downstream structural genes, ultimately resulting in the generation of one of the fundamental histotypes. In this review we discuss the regulation of the different MRFs in relation to their position in the myogenic cascade, the changes in the general transcriptional machinery during muscle differentiation and the emerging importance of miRNA regulation in skeletal myogenesis.

Members of the TEAD family of transcription factors regulate the expression of Myf5 in ventral somitic compartments

The transcriptional regulation of the Mrf4/Myf5 locus depends on a multitude of enhancers that, in equilibria with transcription balancing sequences and the promoters, regulate the expression of the two genes throughout embryonic development and in the adult. Transcription in a particular set of muscle progenitors can be driven by the combined outputs of several enhancers that are not able to recapitulate the entire expression pattern in isolation, or by the action of a single enhancer the activity of which in isolation is equivalent to that within the context of the locus. We identified a new enhancer element of this second class, ECR111, which is highly conserved in all vertebrate species and is necessary and sufficient to drive Myf5 expression in ventro-caudal and ventro-rostral somitic compartments in the mouse embryo. EMSA analyses and data obtained from binding-site mutations in transgenic embryos show that a binding site for a TEA Domain (TEAD) transcription factor is essential for the function of this new enhancer, while ChIP assays show that at least two members of the family of transcription factors bind to it in vivo.

Musculin and TCF21 coordinate the maintenance of myogenic regulatory factor expression levels during mouse craniofacial development

The specification of the skeletal muscle lineage during craniofacial development is dependent on the activity of MYF5 and MYOD, two members of the myogenic regulatory factor family. In the absence of MYF5 or MYOD there is not an overt muscle phenotype, whereas in the double Myf5;MyoD knockout branchiomeric myogenic precursors fail to be specified and skeletal muscle is not formed. The transcriptional regulation of Myf5 is controlled by a multitude of regulatory elements acting at different times and anatomical locations, with at least five operating in the branchial arches. By contrast, only two enhancers have been implicated in the regulation of MyoD. In this work, we characterize an enhancer element that drives Myf5 expression in the branchial arches from 9.5 days post-coitum and show that its activity in the context of the entire locus is dependent on two highly conserved E-boxes. These binding sites are required in a subset of Myf5-expressing cells including both progenitors and those which have entered the myogenic pathway. The correct levels of expression of Myf5 and MyoD result from activation by musculin and TCF21 through direct binding to specific enhancers. Consistent with this, we show that in the absence of musculin the timing of activation of Myf5 and MyoD is not affected but the expression levels are significantly reduced. Importantly, normal levels of Myf5 expression are restored at later stages, which might explain the absence of particular muscles in the Msc;Tcf21 double-knockout mice.

A Novel Gli3 Enhancer Controls the Gli3 Spatiotemporal Expression Pattern through a TALE Homeodomain Protein Binding Site

ABSTRACT The zinc finger transcription factor Gli3 is an essential mediator of hedgehog signaling. Gli3 has a dynamic expression pattern during embryonic development. In the neural tube, Gli3 transcripts are patterned along the anteroposterior and dorsoventral axes such that the initial broad expression in the posterior neural tube becomes dorsally restricted as neurogenesis takes place. Little is known about the molecular mechanisms that regulate this dynamic expression. Here, we report on a phylogenetic analysis of the Gli3 locus that uncovered a novel regulatory element, HCNE1. HCNE1 contains a compound Pbx/Meis binding site that binds Pbx and Meis/Prep proteins in vitro and in vivo. We show that HCNE1 recapitulates Gli3 expression in the developing neural tube and that mutations in the Pbx/Meis binding site affect the spatiotemporal control of HCNE1 transcriptional activity. Ectopic expression or loss of function of Pbx and Meis/Prep proteins in the chick and mouse embryo results in aberrant expression of endogenous Gli3 transcripts. We propose a novel role for TALE proteins in establishing the correct spatiotemporal expression pattern of Gli3 in the vertebrate spinal cord, thus implicating TALE transcription factors in early embryonic patterning events controlled by Sonic hedgehog signaling.