The murine adult IIB myosin heavy chain (IIB MyHC) gene is expressed only in certain skeletal muscle fibers. Within the proximal promoter are two A ؉ T-rich motifs, mAT1 and mAT2, which greatly enhance muscle-specific transcription; myogenic cells contain proteins that bind to these sequences. MEF-2 binds to both mAT1 and mAT2; a mutation abolishing its binding to mAT1 greatly diminishes the activity of the promoter. Both mAT motifs also form complexes with a protein requiring a target sequence typical of POU domain proteins, which migrate in electrophoretic mobility shift assays to the same position as a complex containing purified Oct-1 and which are supershifted by an antibody specific to Oct-1; this protein is therefore probably Oct-1. Footprinting experiments demonstrate that mAT1 is preferentially occupied by MEF-2 and mAT2 by Oct-1 and that these two proteins appear to bind cooperatively to their respective sites. Although the two mAT motifs have sequences that are very similar, they nonetheless exhibit distinct behaviors and perform differently in the activation of the promoter. The contribution of the IIB MyHC gene to specification of the myogenic phenotype is thus at least in part regulated by MEF-2 and Oct-1.

We have studied the effect of several myogenic regulatory factors on the activity of the promoter for a mouse gene encoding a skeletal myosin heavy chain (MyHC) expressed in adult (type IIB) muscle fibers. Co-transfection of myogenic factors is necessary for activity of the IIB promoter in mouse C2 myotubes in culture but not in quail myotubes in culture. Although this promoter contains one E-box within the first 192 base pairs upstream of the transcriptional start site, mutations in this motif demonstrate that it is not required for the transactivation effect of the myogenic factors. Analysis of other mutants suggests that the MEF2 and MHox DNA-binding factor binds to an evolutionarily conserved AT-rich motif. In addition, the IIB promoter appears to require the conserved TATA motif (CTATAAAAG) in order to be activated by the AT-rich sequences. The IIB promoter constructs produce RNA transcripts which begin at the natural site of transcriptional initiation in quail myotubes and in mouse C2 myotubes after co-transfection with myogenic factors; a second, minor, start site is also used in the co-transfected C2 myotubes. Results obtained after transfection of the mouse IIB promoter constructs in quail myotube cultures suggest that the overexpression of myogenic factors in C2 cultures does not result in an environment in which the control of IIB MyHC promoter activity is aberrant. Therefore, either the myogenic factors themselves, or other proteins induced by them, seem to interact directly with the basal transcription seem to interact directly with the basal transcription machinery to allow muscle-specific gene expression.

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ABSTRAm. Ontogenic changes in the mRNA levels of epidermal growth factor (EGF), transforming growth factor-a, EGF receptor, and thyroid hormone receptor (rerbA) were examined in developing rat kidneys. The mRNA levels of both EGF and thyroid hormone receptor rose dramatically during the postnatal period with the rise in thyroid hormone receptor message preceding the rise in EGF message. In addition, we examined renal mRNA levels in 1-wk-old rats treated with thyroxine (T4) from birth through d 6. Neonatal T4 treatment augmented the renal mRNA levels of EGF but decreased the levels of EGF-receptor and transforming growth factor-a. T4 treatment did not significantly affect the levels of renal mRNA for thyroid hormone receptor. Although the EGF and transforming growth factor-a peptides are similar and interact with the same receptor, our findings indicate that these homologous growth factors are regulated differently during development. In addition, hormones that influence growth and development, such as T4, may function both as positive and negative regulators of growth factor expression. (Pediatr Res 31: 330-334, 1992) born rodents (5, 6), and it has been proposed that EGF mediates these developmental effects of thyroid hormone (7).Thyroid hormones also have been shown to increase EGF as well as EGF mRNA levels in developing rodent kidneys (8). To more fully understand the interrelations among these polypeptide growth factors and thyroid hormones in developing rodent kidney, cDNA probes for EGF, TGF-a, EGF-R, and thyroid hormone receptor (r-erbA-P) were used to examine normal rat kidneys taken from animals ranging in age from 21 d postconception to adult. Northern blots were prepared and used to quantify changes in the relative amount of each message during development. The ontogenic changes in message levels were examined with respect to the normal postnatal increase in T4 secretion. In addition, newborn animals were injected with T4 for the first 6 d of life to prematurely increase circulating adult T4 levels. Northern blots prepared from the kidneys of treated and control animals were examined to determine the effect of T4 treatment on the message levels of EGF, TGF-a, EGF receptor, and thyroid hormone receptor. MATERIALS AND METHODS AbbreviationsSprague-Dawley rats were obtained from Simonson Laboratories. Date-bred rats from the same source provided the fetal EGF, epidermal growth factor and neonatal animals used in the study. The animals were TGF-a, transforming growth factor-a maintained in accordance with the NIH Guide for the Care and EGF-R, epidermal growth factor receptor Use of Laboratory Animals. For the T4 treatment studies, new-SSC, saline sodium citrate born rats were treated with T4 (0.4 yg/g body wt/d) from the day T3, triiodothronine of birth through d 6 of life; kidneys were collected on d 7. After T4, thyroxine decapitation, rat kidney tissues were removed, quickly frozen in poly A+, polyadenylated a bath of liquid nitrogen, and stored at -70°C. Before RNA extraction, the pooled kidney tissue...

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