Regulation of myogenesis and suppression of {\it mck\/} 5$\sp\prime$ enhancer elements by TGF$\beta$ and RAS

The regulation of muscle differentiation, like cell differentiation in general, is only now beginning to be understood. Here are described several key features to myogenesis: a beginning, some intermediary events, and an endpoint. Muscle differentiation proceeds spontaneously when myoblasts are cultured in serum-poor medium. Transforming growth factor type $\beta$ (TGF$\beta$), a component of fetal serum, was found to potently suppress muscle differentiation. Prolonged blockade of differentiation required replenishing TGF$\beta$. When TGF$\beta$ was removed, cells rapidly differentiated. Both TGF$\beta$ and RAS, which also blocks myogenesis, suppress the genes for a series of muscle-specific proteins. Regions that regulate transcription of one such gene, muscle creatine kinase (mck), were located by linking progressively smaller parts of the mck 5$\sp\prime$ region to the marker gene cat and testing the constructs for regulated expression of cat in myoblasts and muscle cells. The mck promoter is not muscle-specific but requires activation. Two enhancers were found: a weak, developmentally regulated enhancer within the first intron, and a strong, compact, and tightly developmentally regulated enhancer about 1.2 Kb upstream of the transcription start site. Activity of this enhancer is eliminated by activated ras. Suppression of activated N-RAS restores potency to the upstream enhancer. Further deletion shows the mck 5$\sp\prime$ enhancer to contain an enhancer core with low but significant muscle-specific activity, and at least one peripheral element that augments core activity. The core and this peripheral element were comprised almost entirely of factor-binding motifs. The peripheral element was inactive as a single copy, but was constitutively active in multiple copies. Regions flanking the peripheral element augmented its activity and conferred partial muscle-specificity. The enhancer core is also modulated by its 5$\sp\prime$ flanking region in a complex manner. Site-specific mutants covering most of the enhancer core and interesting flanking sequences have been made; all mutants tested diminish the activity of the 5$\sp\prime$ enhancer. Alteration of the site to which MyoD1 is reported to bind completely inactivates the enhancer. A theoretical analysis of cooperativity is presented, through which the binding of a constitutively expressed nuclear factor is shown to have weak positive cooperativity. In summary, TGF$\beta$, RAS, and enhancer-binding factors are found to be initial, intermediary, and final regulators, respectively, of muscle differentiation. ^

An extracytoplasmic function sigma factor operon regulates Myxococcus xanthus developmental gene expression

Myxococcus xanthus is a Gram-negative soil bacterium that undergoes multicellular development when high-density cells are starved on a solid surface. Expression of the 4445 gene, predicted to encode a periplasmic protein, commences 1.5 h after the initiation of development and requires starvation and high density conditions. Addition of crude or boiled supernatant from starving high-density cells restored 4445 expression to starving low-density cells. Addition of L-threonine or L-isoleucine to starving low-density cells also restored 4445 expression, indicating that the high-density signaling activity present in the supernatant might be composed of extracellular amino acids or small peptides. To investigate the circuitry integrating these starvation and high-density signals, the cis- and trans-acting elements controlling 4445 expression were identified. The 4445 transcription start site was determined by primer extension analysis to be 58 by upstream of the predicted translation start site. The promoter region contained a consensus sequence characteristic of e&barbelow;xtrac&barbelow;ytoplasmic f&barbelow;unction (ECF) sigma factor-dependent promoters, suggesting that 4445 expression might be regulated by an ECF sigma factor-dependent pathway, which are known to respond to envelope stresses. The small size of the minimum regulatory region, identified by 5′-end deletion analysis as being only 66 by upstream of the transcription start site, suggests that RNA polymerase could be the sole direct regulator of 4445 expression. To identify trans-acting negative regulators of 4445 expression, a strain containing a 4445-lacZ was mutagenized using the Himar1-tet transposon. The four transposon insertions characterized mapped to an operon encoding a putative ECF sigma factor, ecfA; an anti-sigma factor, reaA; and a negative regulator, reaB. The reaA and the reaB mutants expressed 4445 during growth and development at levels almost 100-fold higher than wild type, indicating that these genes encode negative regulators. The ecfA mutant expressed 4445-lacZ at basal levels, indicating that ecfA is a positive regulator. High Mg2+ concentrations over-stimulated this ecfA pathway possibly due to the depletion of exopolysaccharides and assembled type IV pili. These data indicate that the ecfA operon encodes a new regulatory stress pathway that integrates and transduces starvation and cell density cues during early development and is also responsive to cell-surface alterations.^