Peter W. J. Rigby

Know Your Neighbors: Three Phenotypes in Null Mutants of the Myogenic bHLH Gene MRF4

The data from the MRF4, Hox, and globin studies suggest that many other knockout experiments may be subject to similar effects. The examples we have discussed involve loci that contain two or more related genes, and these may be especially prone to the evolution of joint locus control elements and other interrelated cis-regulatory interactions. It is equally plausible that our current picture is skewed toward these cases by investigator knowledge of the identity of neighboring genes and that similar long-range effects will be seen for unrelated adjacent genes. We therefore feel that until substantially more is understood about cis-regulation over substantial regions of the genome it is prudent to consider these effects in design and in interpretation. One might think that direct complementation testing would clarify which knockouts are subject to neighborhood complications, but in the mouse this solution is not straightforward. It has proved frustratingly difficult to capture and reintroduce into mice pieces of DNA that faithfully recapitulate the full developmental pattern and correct level of expression of the wildtype gene. The culprits appear to be the very same aspects of gene structure that are at work in generating the neighborhood problem: the dispersal of pertinent regulatory elements over very large stretches of DNA that may also include additional genes. Moreover, minigenes that might be induced to express at will complementing protein coding sequences, in the manner of heat shock regulated constructions in Drosophila, are unreliable in current mouse transgenic formats. For these reasons the application of improved, if somewhat more complex, knockout strategies provides a more immediate solution to neighborhood uncertainties. To avoid deleting sequences that may have regulatory effects on adjacent genes, one can instead introduce an effectively positioned stop codon. This can be coupled with removal of the selection cassette by site-specific recombinases such as yeast Flp or phage P1 Cre. Alternatively, the so-called “hit-and-run” strategy, which provides for simultaneous introduction of subtle nonsense or missense mutations together with the elimination of all selection cassette residue (Ramirez-Solis et al. 1993xRamirez-Solis, R, Zheng, II, Whiting, J, Krumlauf, R, and Bradley, A. Cell. 1993; 73: 279–294Abstract | Full Text PDF | PubMed | Scopus (227)See all ReferencesRamirez-Solis et al. 1993), can be used. This design achieves the cleanest introduction of small and specific mutations, but calls for somewhat greater effort in establishing and identifying the desired ES cell line. Finally, for many existing knockouts, an immediate challenge is to consider their phenotypes with attention to the identities and activities of neighboring genes.

Gene regulatory networks and transcriptional mechanisms that control myogenesis.

We discuss the upstream regulators of myogenesis that lead to the activation of myogenic determination genes and subsequent differentiation, focusing on the mouse model. Key upstream genes, such as Pax3 and Pax7, Six1 and Six4, or Pitx2, participate in gene regulatory networks at different sites of skeletal muscle formation. MicroRNAs also intervene, with emerging evidence for the role of other noncoding RNAs. Myogenic determination and subsequent differentiation depend on members of the MyoD family. We discuss new insights into mechanisms underlying the transcriptional activity of these factors.

Negative regulation of viral enhancers in undifferentiated embryonic stem cells

Many viral genomes, including those of SV40 and MuLV, are not efficiently expressed in undifferentiated embryonal carcinoma (EC) cells but are expressed in differentiated derivatives. This regulation appears to be at the level of transcription. We have used DNA-mediated gene transfer to analyze the function of several viral promoters in EC cells. We show that the SV40 early promoter works efficiently in an enhancer-independent fashion following transfection into undifferentiated cells. Strikingly, the promoter in the LTR of MSV does not function in such cells; but when upstream sequences, including the enhancer, are deleted expression ensues. Replacement of the SV40 enhancer by that of MSV results in inactivation of the SV40 early promoter in these cells. We propose that the undifferentiated cells contain a trans-acting regulatory factor (or factors) that reduces transcription by interacting with viral enhancers.

Activation of mouse genes in transformed cells

We have used molecular hybridization and cDNA cloning techniques to isolate mouse cellular genes activated in SV40-transformed cells and we show that many of the clones belong to one of four sets. We characterize the cytoplasmic transcripts and genomic sequences homologous to two of these sets. The Set 1 transcription unit(s) is activated in all SV40-transformed cell lines analyzed, and experiments with tsA-mutant-transformed lines show that activation appears to require functional large T-antigen. This transcription unit(s) is also activated in mouse fibroblasts transformed by other agents, including retroviruses and chemical carcinogens. Activation of the Set 2 transcription unit(s) is more restricted, being confined to cell lines transformed by SV40 and retroviruses with distinctive biological properties.

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.

Mox2 is a component of the genetic hierarchy controlling limb muscle development.

The skeletal muscles of the limbs develop from myogenic progenitors that originate in the paraxial mesoderm and migrate intothe limb-bud mesenchyme. Among the genes known to be important for muscle development in mammalian embryos are those encoding the basic helix-loop-helix (bHLH) myogenic regulatory factors (MRFs; MyoD, Myf5, myogenin and MRF4),, and Pax3, a paired-type homeobox gene that is critical for the development of limb musculature,,. Mox1 and Mox2 are closely related homeobox genes that are expressed in overlapping patterns in the paraxial mesoderm and its derivatives,. Here we show that mice homozygous for a null mutation of Mox2 have a developmental defect of the limb musculature, characterized by an overall reduction in muscle mass and elimination of specific muscles. Mox2 is not needed for the migration of myogenic precursors into the limb bud, but it is essential for normal appendicular muscle formation and for the normal regulation of myogenic genes, as demonstrated by the downregulation of Pax3 and Myf5 but not MyoD in Mox2-deficient limb buds. Our findings show that the MOX2 homeoprotein is an important regulator of vertebrate limb myogenesis.

An adenovirus E1A-like transcription factor is regulated during the differentiation of murine embryonal carcinoma stem cells

The expression of the E2A transcription unit of the adenovirus E1A deletion mutant dl312 in murine embryonal carcinoma (EC) stem cells suggests that they contain an activity that will complement viral E1A. We have prepared from these cells in vitro transcription extracts that use E1A-inducible promoters more efficiently than do extracts from a differentiated cell line. Mixing experiments demonstrate that the EC phenotype is dominant. Gel retardation assays using the E2A promoter detect a binding activity present in F9 and PCC4 EC cells but not in differentiated cells. Our data indicate that EC stem cells contain a transcription factor that is analogous to viral E1A and is likely to be involved in the control of cellular gene expression during differentiation.

Transcripts regulated during normal embryonic development and oncogenic transformation share a repetitive element

We have previously isolated cDNA clones homologous to mRNAs present at elevated levels in transformed mouse fibroblasts. Clones of Set 1 contain a dispersed repetitive element present thousands of times in the mouse genome. This repeat identifies in mouse embryos a large number of transcripts that are quantitatively regulated during development. At maximal expression these RNAs constitute between 1% and 3% of polyadenylated RNA. A pattern of Set 1-related transcripts very similar to that observed in midgestation embryos is found in pluripotential EC and EK cell lines, and the abundance of these RNAs decreases upon differentiation in vitro. However, the F9 line of EC cells, which has a more restricted developmental capacity, exhibits a much simpler pattern of transcripts containing the Set 1 repeat.

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.

Regulation of RNA polymerase III transcription in response to F9 embryonal carcinoma stem cell differentiation

B2 genes are rodent-specific middle repetitive elements transcribed by RNA polymerase III. They are expressed in the ectoderm and mesoderm but not in the embryonic or extraembryonic endoderm of early mouse embryos. This tissue specificity is mimicked in vitro by embryonal carcinoma and embryonic stem cell lines. Nuclear run-on experiments show that the down-regulation of B2 genes during F9 embryonal carcinoma cell differentiation into endoderm occurs at the transcriptional level and that other class III genes, including those encoding tRNA, show a similar response. We have used cell-free extracts to investigate the molecular mechanisms responsible. The specific down-regulation of transcription by RNA polymerase III during F9 cell differentiation is due to a reduction in the activity of the general class III transcription factor TFIIIB.