Abstract
The tissue-specific basic helix-loop-helix protein Hand1 is essential for the formation of trophoblast giant cells of the murine placenta. In humans, Hand1 is detectable in trophoblastic tumour cells suggesting an equivalent role in trophoblast differentiation. To understand its mode of expression we have cloned and characterized the human Hand1 gene promoter. Primer extension analyses suggest that transcription initiates 19 nucleotides downstream of the TATA element of the proximal 5′ flanking region. Expression of luciferase reporter constructs harboring deletions of the 9.5 kb Hand1 5′ flanking sequence defines a promoter region within 274 bp upstream of the transcriptional start site. Compared to a reporter bearing only the TATA box, the proximal promoter activates transcription up to 30-fold. However, transcriptional activity of the region was observed in both Hand1-expressing and non-expressing cell lines. Sequencing, DNAseI footprint analyses and electrophoretic mobility shift assays reveal the presence of four GC-rich sequences, which show different affinities to the endogenous specificity proteins (Sp), and a CCAAT box. In vitro, the Sp-elements mainly interact with Sp1 and Sp3 while the CCAAT element is recognized by the α CAAT binding factor protein. Mutant luciferase reporters bearing single active or inactive recognition sites demonstrate that two of the four Sp-binding sites (I and IV) contribute little to the overall transcription rate. The two other Sp-cognate sequences, II and III, downregulate and activate reporter expression 2.3- and 2.6-fold, respectively. Co-transfections of Sp1/Sp3 expression vectors and mutated reporter constructs in Sp-deficient SL2 cells indicate that the Sp-binding site II and III indeed function as repressing and activating enhancer sequences. In summary, the data suggest that constitutive expression of the Hand1 gene in cultured cells is regulated by a complex interplay of Sp-proteins interacting with activator and repressor elements.
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