Identification of two E-boxes that negatively modulate the activity of MyoD on the alpha-sarcoglycan core promoter

The alpha-SG promoter is composed of a plethora of cis-regulatory elements, whose individual contribution to alpha-SG gene expression modulation remains unknown. We have identified a negative regulatory element in the alpha-SG distal promoter including two conserved E-boxes (E1 and E2), which interact with MyoD. We found that E1 and E2 negatively modulate the transactivation potential of MyoD on the alpha-SG core promoter. Moreover, such negative effect is mainly mediated by E2, which is surrounded by conserved nucleotides conferring MyoD binding capacity. Our results suggest that modulation of MyoD activity by E1, and particularly E2, contributes to the negative regulation of alpha-SG gene expression during myogenic differentiation.     

Positive autoregulation of the myogenic determination gene MyoD1

Transfection of cDNA expression vectors encoding either MyoD1 or myogenin into 10T1/2 cells converts them to myogenic cells. We show that transfection of 10T1/2 cells with the MyoD1 cDNA activates expression of endogenous MyoD1 mRNA, indicating that MyoD1 is subject to positive autoregulation. This activation of endogenous MyoD1 mRNA was also observed in Swiss 3T6 cells, but not in several other fibroblast or adipoblast cell lines transfected with the MyoD1 cDNA. In addition, transfection of the MyoD1 cDNA leads to activation of myogenin expression, and transfection of the myogenin cDNA leads to activation of MyoD1 expression. Thus, MyoD1 and myogenin appear to function in a positive autoregulatory loop that could either: account for or contribute to the stability of myogenic commitment; or amplify the level of expression of both MyoD1 and myogenin above a critical threshold that is required for activation of the myogenic program.     

Dynamic cerebral autoregulation is preserved in neurally mediated syncope.

To test whether cerebral autoregulation is impaired in patients with neurally mediated syncope (NMS), we evaluated 15 normal subjects and 37 patients with recurrent NMS. Blood pressure (BP), heart rate, and cerebral blood velocity (CBV) (transcranial Doppler) were recorded at rest and during 80 degrees head-up tilt (HUT). Static cerebral autoregulation as assessed from the change in cerebrovascular resistance during HUT was the same in NMS and controls. Properties of dynamic cerebral autoregulation were inferred from transfer gain, coherence, and phase of the relationship between BP and CBV estimated from filtered data segments (0.02-0.8 Hz). During the 3 min preceding syncope, dynamic cerebral autoregulation of subjects with NMS did not differ from that of controls nor did it change over the course of HUT in patients with NMS or in control subjects. Dynamic cerebral autoregulation was also unaffected by the degree of orthostatic intolerance as inferred from latency to onset of syncope. We conclude that cerebral autoregulation in patients with recurrent syncope does not differ from that of normal control subjects.     

12-O-tetradecanoylphorbol-13-acetate activation of the MDR1 promoter is mediated by EGR1.

P-glycoprotein, the product of the MDR1 gene (multidrug resistance gene 1), is an energy-dependent efflux pump associated with treatment failure in some hematopoietic malignancies. Its expression is regulated during normal hematopoietic differentiation, although its function in normal hematopoietic cells is unknown. To identify cellular factors that regulate the expression of MDR1 in hematopoietic cells, we characterized the cis- and trans-acting factors mediating 12-O-tetradecanoylphorbol-13-acetate (TPA) activation of the MDR1 promoter in K562 cells. Transient-transfection assays demonstrated that an MDR1 promoter construct containing nucleotides -69 to +20 conferred a TPA response equal to that of a construct containing nucleotides -434 to +105. TPA induced EGR1 binding to the -69/+20 promoter sequences over a time course which correlated with increased MDR1 promoter activity and increased steady-state MDR1 RNA levels. The -69/+20 promoter region contains an overlapping SP1/EGR site. The TPA-responsive element was localized to the overlapping SP1/EGR site by using a synthetic reporter construct. A mutation in this site that inhibited EGR protein binding blocked the -69/+20 MDR1 promoter response to TPA. The expression of a dominant negative EGR protein also blocked the TPA response of the -69/+20 promoter construct. Finally, the expression of EGR1 was sufficient to activate a construct containing tandem MDR1 promoter SP1/EGR sites. These data suggest a role for EGR1 in modulating MDR1 promoter activity in hematopoietic cells.