FIGURE 3.

Characterization of a GC-responsive region 4.6 kb upstream of the human DUSP1 start site. A, Multiz alignment of GRR-4.6 of 28 mammalian species at the UCSC Genome Browser (University of California, Santa Cruz) (66). The positions of GBS-4.6.1, -4.6.2, and -4.6.3 are indicated below the alignment. B, sequences of GBS-4.6.2 of human (Homo sapiens; Hs.), rat (Rattus norvegicus; Rn.), mouse (Mus musculus; Mm.), dog (Canis familiaris; Cfa.), cow (Bos taurus; Bta.), horse (Equus caballus; Eca.), and armadillo (Dasypus novemcintus; Dn.). The conserved GBS sequence GNACANNNNG is indicated by asterisks, and an additional GR half-site is denoted by an arrow. Differences from the human sequence are highlighted. Sequences of mutated versions of GBS-4.6.2 are also shown. C, a 467-bp human genomic fragment centered on GBS-4.6.2 was cloned upstream of the SV40 early promoter in pGL3p. Mutations at GBS-4.6.1, -4.6.2, or -4.6.3 (as indicated in B) were introduced by PCR. Dex responses were assayed as in Fig. 2 and normalized against the response of the empty vector pGL3p. With the exception of pGL3p-GRR-4.6-GBS-4.6.2-m2, all constructs were significantly different from the parental vector pGL3p. Statistical comparisons against pGL3p-GRR-4.6 are indicated. D, orthologous GRR-4.6 fragments from dog, human, mouse, and rat (Cfa., Hs., Mm., and Rn.) were cloned into pGL3p, and responses to Dex were calculated as in C. Statistically significant differences from pGL3p are indicated beside each construct, and additional comparisons are as shown. E, short oligonucleotides containing human or mouse GBS-4.6.2 were subcloned into the firefly luciferase reporter pGL4.26 (for simplicity indicated as pGL4 in the figure). Responses to Dex were calculated as in C. Statistically significant differences from pGL4 are indicated beside each construct, and other statistical comparisons are as indicated.