**4. Stat-DNA promoter-binding motifs**

Defining transcription factor binding sites was critical for revealing the structure of cis-regu‐ latory motifs that regulated transcriptional activity [53]. However, microarray analysis using different cell types determined that although several hundred genes were potential STAT3 target gene sites, only a small fraction of those STAT3-target gene sites turned out to be true direct STAT3-target genes [54].

As previously indicated, p-STAT proteins do not act independently of one another and U- and p-STAT-protein interactions take various forms which enable them to bind efficiently to DNA [55]. These activated STAT-protein interaction types include, 1) the direct binding of activated STAT homodimers to DNA; 2) the interaction of activated STATs with non-STAT proteins to form activated STAT/non-STAT protein complexes which bind to DNA; and 3) activated STAT proteins interacting with other non-STAT transcription factors or co-activator proteins which bind to DNA [16, 53]. In addition, several novel mechanisms were described for the binding of U-STAT3 and U-STAT1 to DNA [54, 55]. In that regard, Cheon et al. [56] showed that U-STAT3 can drive expression of proteins not induced by p-STAT3, whereas U-STAT1 was shown to ex‐ tend and up-regulate the expression of a subset of genes initially responsive to p-STAT1 (e.g., interferon, IL-6), that result in more prolonged antiviral and/or immune responses. Thus, the results of these studies provided novel information regarding the functional significance for U-STAT1 and U-STAT3 acting as transcriptional activators and organizers of chromatin. These events have been shown to be important cellular mechanisms for regulating gene transcription in the nuclei of cells of the immune system and cancer cells.

The results of DNA sequencing studies originally demonstrated specific DNA-binding sites for STAT1 and STAT3 [57]. Boucheron et al. [58] then demonstrated that specific DNA binding sites existed for STAT5A and STAT5B homodimers despite the fact that STAT5A and STAT5B are evolutionarily conserved and encoded by 2 genes with a 91% homology in amino acid structure [59]. Moreover, targeted gene deletion of STAT5A and STAT5B in mice resulted in distinctive phenotypes [60]. This finding suggested a structural dissimilarity in the DNA-bind‐ ing motifs for these two STAT proteins. The results of studies reported in [60] were later con‐ firmed using the IL-3-dependent early pre-B cell line, Ba/F3 [61]. Here it was shown that both STAT isoforms bound to all of the promoters tested, but STAT5A and STAT5B bound with dif‐ fering kinetics [62]. This result suggested that DNA binding activity was likely at the root of any differences in the biological activity of these two STAT protein isoforms.

Ehret et al. [63] compared the specificity of STAT-DNA binding sites in specific STAT gene knockout mice showing distinct phenotypes with the STAT-DNA binding sites in a variety of cultured cells. From the *in vitro* analysis, Ehret et al. [63] also demonstrated that DNA binding site motifs for STAT1, STAT5A, STAT5B and STAT6 were essentially the same with only minor differences in DNA binding site specificity. However, STAT5A DNA-binding specificity was much more similar to STAT6 than was the preferential DNA-binding site for STAT1. Thus, the preferential DNA binding site for STAT6 contained a 4 base pair spacer (i.e. TTCNNNNGAA) (N4) which was defined as the weak DNA binding site. However, ad‐ ditional analyses showed that STAT6 bound to TTCNNNG-AA (N3) sites (i.e. the strong binding site) as well [63]. The binding of STAT1 and STAT5 to the N3 site was distinct from STAT5A which preferred N4. Of note, most of the STAT6 binding sites were found in IL-4 responsive promoters in the N4 sites [64-67]. These results reported by Ehret et al. [63] were extended by the findings of Moucadel and Constantinescu [64] who showed that STAT5B bound to chromatin at both the N3 and the N4 site.
