Prothymosin α (PTα), a protein associated with cell proliferation and chromatin remodeling, and found to selectively enhance ER transcriptional activity by interacting with a repressor of ER activity, is shown to be a primary response gene to estrogen. Prothymosin α mRNA was rapidly increased by estrogen, followed by a 6-fold increase in prothymosin α protein content in ER-containing breast cancer cells. Analysis of the prothymosin α promoter and 5′-flanking region, and electrophoretic gel mobility shift studies showed the strong inducibility by the estradiol-ER complex to be mediated by two consensus half-palindromic estrogen response elements at -750 and -1051, which directly bind the ER. Estrogenic stimulation of prothymosin α required a DNA binding form of ER with a functional activation function-2 domain. The prothymosin α 5′-regulatory region also contains multiple Sp1 sites. Although addition of Sp1 did not further enhance estradiol-ER stimulated prothymosin α transcriptional activity in breast cancer cells, transfection and response element mutagenesis studies using Drosophila cells, which are deficient in Sp1, revealed that Sp1 and the estradiol occupied-ER can each activate the prothymosin α gene independently of the other and act in an additive manner. These observations, documenting robust prothymosin α up-regulation by the estradiol-ER complex via widely spaced half-palindromic estrogen response element motifs, are reminiscent of those shown previously for the ovalbumin gene and suggest that the use of multiple half response elements may be a more common mode for regulation of gene expression by the ER than previously appreciated. In addition, these observations suggest interrelationships between cell proliferation and gene transcriptional activities and indicate a positive mechanism by which PTα, which increases ER transcriptional effectiveness, is itself up-regulated by the estrogen-ER complex.
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