Previous studies defined a DNA element necessary for glucocorticoid repression of the pro‐opiomelanocortin (POMC) gene. The glucocorticoid receptor (GR) binds this negative glucocorticoid response element (nGRE) with an in vitro affinity similar to that of GR for positive GREs. However, whereas GR binds GREs as homodimers, a novel GR complex which forms with nGRE appears to contain three GR molecules. Biochemical characterization of this complex as well as equilibrium binding studies suggest that it is formed by sequential binding of a GR homodimer followed by binding of a GR monomer on the opposite side of the double helix. The DNA‐binding domain (DBD) of GR is sufficient for differential binding of GRE and nGRE, as bacterially‐expressed DBD formed unique nGRE complexes that contain three GR polypeptides. Thus, the POMC nGRE provides the first example of an interaction between GR and DNA in which GR binds otherwise than as a homodimer. Despite its high affinity for GR, the nGRE differs significantly from GREs in that it does not activate transcription in any context. As the nGRE appears insufficient on its own to confer hormone responsiveness, other POMC promoter elements are likely to be required to mediate glucocorticoid repression.

Two closely related homeobox transcription factors, Pitx1 and Pitx2, have been implicated in patterning of lateral plate mesoderm derivatives: Pitx1 for specification of hindlimb identity and Pitx2 for determination of laterality. We show that, together, Pitx1 and Pitx2 are required for formation of hindlimb buds and, when present in limited doses, for development of proximal (femur) and anterior (tibia and digit 1) hindlimb structures. Although Pitx1 is expressed throughout developing hindlimb buds, Pitx2 is not expressed in limb bud mesenchyme itself, but is coexpressed with Pitx1 in the presumptive hindlimb field before bud growth. Thus, Pitx1 and Pitx2 genes are required for sustained hindlimb bud growth and formation of hindlimbs.

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Homeoproteins and basic helix-loop-helix (bHLH) transcription factors are known for their critical role in development and cellular differentiation. The pituitary pro-opiomelanocortin (POMC) gene is a target for factors of both families. Indeed, pituitary-specific transcription of POMC depends on the action of the homeodomain-containing transcription factor Pitx1 and of bHLH heterodimers containing NeuroD1. We now show lineage-restricted expression of NeuroD1 in pituitary corticotroph cells and a direct physical interaction between bHLH heterodimers and Pitx1 that results in transcriptional synergism. The interaction between the bHLH and homeodomains is restricted to ubiquitous (class A) bHLH and to the Pitx subfamily. Since bHLH heterodimers interact with Pitx factors through their ubiquitous moiety, this mechanism may be implicated in other developmental processes involving bHLH factors, such as neurogenesis and myogenesis.Cell-specific transcription results from complex molecular interactions that involve the synergistic action of multiple transcription factors. Taken together, these interactions provide the molecular basis for the complex program of cell differentiation and development. Factors of two classes of transcription factors often involved in developmental processes, the homeodomain (HD) and the basic helix-loop-helix (bHLH) factors, were found to form the basis for cell-specific transcription of pituitary pro-opiomelanocortin (POMC) gene expression. We have used this system to define the molecular mechanism by which these two classes of transcription factors can synergistically interact to control transcription. More specifically, our studies reveal a specific interaction between the HD of the Pitx (Ptx) subfamily of homeobox proteins and the widely expressed class A bHLH transcription factors.

The pro-opiomelanocortin (POMC) gene is specifically expressed in corticotroph cells of the anterior pituitary. To define the POMC promoter sequences responsible for tissue-specific expression, we assessed POMC promoter activity by gene transfer into POMC-expressing pituitary tumor cells and fibroblast L cells. The rat POMC promoter was only efficiently utilized and correctly transcribed in AtT-20 cells. 5'-End deletion analysis revealed two promoter regions required for activity in AtT-20 cells. When tested by fusion to a heterologous promoter, DNA fragments corresponding to both regions exhibited tissue-specific activity, suggesting the presence of at least two tissue-specific DNA sequence elements within the promoter. In summary, POMC promoter sequences from -480 to -34 base pairs appear sufficient to mimic the specificity of anterior pituitary expression.Control of eucaryotic transcription results from the interaction of multiple DNA sequence elements that are presumably recognized by as many trans-acting factors (reviewed in references 21 and 33). In gene transfer experiments with tissue culture cells or transgenic animals, it was shown that various DNA sequence elements are responsible for tissuespecific expression of many cellular genes (2,3,9,16,24,29,31,39,45,46). Tissue-specific sequence elements were identified in the 5'-flanking region of the genes coding for immunoglobulins (16,24,31), insulin (46), elastase (45), otl-antitrypsin (9), and many others. Some sequences conferring tissue specificity have enhancer properties and are located either upstream of the site of transcription initiation or elsewhere in the gene (1,20,37,42). In many cases, multiple DNA sequence elements are required to confer tissue specificity (8,13,16,24,31,48), includi1ag promoterproximal and -distal sequences.The pituitary gland represents an interesting system to investigate the role of tissue-specific DNA sequence elements in the expression of cell-specific genes. Pituitary hormone-secreting cells have a common embryological origin; however, each pituitary hormone gene is expressed in a distinct cell type (17). To identify DNA sequences required for tissue-specific expression in the pituitary, we studied the activity of promoter sequences present in the rat proopiomelanocortin (POMC) gene. The POMC gene is present in a single copy per haploid genome (12); it is expressed in two different pituitary cell types, the anterior pituitary corticotrophs, in which POMC is processed to adrenocorticotropin, and the pars intermedia melanotrophs, in which the same POMC protein precursor is processed to o-melanotropin (14). Regulation of POMC gene expression in these two tissues is also cell specific (18; J.-P. Gagner and J. Drouin, Mol. Endocrinol., in press), highlighting the functional and developmental differences between the two secreting cell types. MATERIALS AND METHODSPlasmid constructions. Recombinant plasmids were constructed by standard procedures (30). Plasmid DNAs used for electroporation were purified by banding twice on...