Jean N. Buskin

Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence

A DNA binding and dimerization motif, with apparent amphipathic helices (the HLH motif), has recently been identified in various proteins, including two that bind to immunoglobulin enhancers (E12 and E47). We show here that various HLH proteins can bind as apparent heterodimers to a single DNA motif and also, albeit usually more weakly, as apparent homodimers. The HLH domain can mediate heterodimer formation between either daughterless, E12, or E47 (Class A) and achaete-scute T3 or MyoD (Class B) to form proteins with high affinity for the kappa E2 site in the immunoglobulin kappa chain enhancer. The achaete-scute T3 and MyoD proteins do not form kappa E2-binding heterodimers together, and no active complex with N-myc was evident. The formation of a heterodimer between the daughterless and achaete-scute T3 products may explain the similar phenotypes of mutants at these two loci and the genetic interactions between them. A role of E12 and E47 in mammalian development, analogous to that of daughterless in Drosophila, is likely.

MYOD IS A SEQUENCE-SPECIFIC DNA BINDING PROTEIN REQUIRING A REGION OF MYC HOMOLOGY TO BIND TO THE MUSCLE CREATINE KINASE ENHANCER

MyoD is a skeletal muscle-specific protein that is able to induce myogenesis in a wide variety of cell types. In this report, we show that MyoD is a DNA binding protein capable of specific interaction with two regions of the mouse muscle creatine kinase gene upstream enhancer, both of which are required for full muscle-specific enhancer activity. MyoD shares antigenicity and DNA binding specificity with MEF1, a myocyte-specific DNA binding factor. The contiguous basic and myc homology regions of MyoD that are necessary and sufficient for specific DNA interaction are the same regions of the protein required to convert 10T1/2 fibroblasts into muscle. These findings suggest that the biological activity of MyoD is mediated via its capacity for specific DNA interaction.

The mouse muscle creatine kinase cDNA and deduced amino acid sequences: Comparison to evolutionarily related enzymes

SummaryThe nucleotide sequence of cloned DNA corresponding to full-length mouse muscle creatine kinase mRNA has been determined. This 1415 base pair DNA sequence and the deduced 381 amino acid sequence of the protein have been compared to creatine kinase sequences from other vertebrate species and to invertebrate guanidino kinase sequences. These comparisons show that the vertebrate muscle creatine kinases constitute a remarkably conserved protein family with a unit evolutionary period of 30. The creatine kinases also retain marked sequence similarity with the more distantly related invertebrate guanidino kinases. A portion of the sequence, presumably part of the ATP binding site, shows similarity to other nucleotide binding proteins with diverse functions. Comparisons of the untranslated regions of the creatine kinase cDNA sequences show that the 5′ untranslated regions are more highly conserved than are the 3′ untranslated regions; this may point to some regulatory function in the 5′ region.

Transgenic and tissue culture analyses of the muscle creatine kinase enhancer Trex control element in skeletal and cardiac muscle indicate differences in gene expression between muscle types

The muscle creatine kinase (MCK) gene is expressed at high levels only in differentiated skeletal and cardiac muscle. The activity of the cloned enhancer–promoter has previously been shown to be dependent on the Trex element which is specifically bound by a yet unidentified nuclear factor, TrexBF. We have further characterized the function of the Trex site by comparing wild-type and Trex-mutated MCK transgenes in five mouse skeletal muscles: quadriceps, extensor digitorum longus (EDL), soleus, diaphragm, and distal tongue, as well as in heart ventricular muscle. Several types of statistical analysis including analysis of variance (ANOVA) and rank sum tests were used to compare expression between muscle types and between constructs. Upon mutation of the Trex site, median transgene expression levels decreased 3- to 120-fold in the muscles examined, with statistically significant differences in all muscles except the EDL. Expression in the largely slow soleus muscle was more affected than in the EDL, and expression in the distal tongue and diaphragm muscles was affected more than in soleus. Median expression of the transgene in ventricle decreased about 18-fold upon Trex mutation. Transfections into neonatal rat myocardiocytes confirmed the importance of the Trex site for MCK enhancer activity in heart muscle, but the effect is larger in transgenic mice than in cultured cells.

BMP Induction of Cardiogenesis in P19 Cells Requires Prior Cell-Cell Interaction(s)

Mouse P19 embryonal carcinoma cells undergo cardiogenesis in response to high density and DMSO. We have derived a clonal subline that undergoes cardiogenesis in response to high density, but without requiring exposure to DMSO. The new subline retains the capacity to differentiate into skeletal muscle and neuronal cells in response to DMSO and retinoic acid. However, upon aggregation, these Oct 4‐positive cells, termed P19‐SI because they “self‐induce” cardiac muscle, exhibit increased mRNAs encoding the mesodermal factor Brachyury, cardiac transcription factors Nkx 2.5 and GATA 4, the transcriptional repressor Msx‐1, and cytokines Wnt 3a, Noggin, and BMP 4. Exposure of aggregated P19‐SI cells to BMP 4, a known inducer of cardiogenesis, accelerates cardiogenesis, as determined by rhythmic beating and myosin staining. However, cardiogenesis is severely inhibited when P19‐SI cells are aggregated in the presence of BMP 4. These results demonstrate that cell–cell interaction is required before P19‐SI cells can undergo a cardiogenic response to BMP 4. A concurrent increase in the expression of Msx‐1 suggests one possible process underlying the inhibition of cardiogenesis. The phenotype of P19‐SI cells offers an opportunity to explore new aspects of cardiac induction. Developmental Dynamics 235:2122–2133, 2006.

Zinc and the initiation of myoblast differentiation

Abstract Previous studies have indicated that a lack of available zinc inhibited myoblast differentiation as shown by a failure of the cells to fuse and low expression of creatine kinase mRNA and activity. However, the nature of the requirement for zinc and its relationship to the events leading to differentiation have been unclear. The current studies with C2C12 cells indicated that the muscle-specific enhancer present in the 5′-flanking region of the creatine kinase gene contributed to the zinc sensitivity of this enzyme. Because this enhancer can be activated by expression of the myogenic factors MyoD and myogenin, their sensitivity to zinc was investigated. The concentrations of both MyoD and, particularly, myogenin mRNA, were decreased by zinc deficiency. In vitro translation experiments suggested that these changes closely corresponded with alterations in their rates of synthesis. Further experiments failed to indicate a major effect of zinc on the stabilities of these mRNAs. Because an induction of myogenin mRNA is one of the earliest known events in myoblast differentiation, its particular sensitivity to lack of zinc suggests that zinc may be required before or during the initiation of myoblast differentiation.