**2. The relevance of duplicated GGAA-motifs in the 5′-upstream regions of human genes to the regulation of biological events**

#### **2.1. The transcription factors that recognize and bind to the GGAA-containing motifs**

The most widely known transcription factors (TFs) that selectively recognize the GGAA-corecontaining sequences are the ETS (E26 transformation specific) family proteins, which consist of at least 27 members [10, 11]. Moreover, a genome-wide ChIP-seq analysis estimated that the promoter regions of human genes are very frequently occupied by ETS family or GGAA-binding proteins [12]. The duplication of the GGAA-motif could be advantageous to organisms, as it would allow the transcription of various genes to be controlled in a manner that is mainly dependent on the expression profile of the GGAA-binding proteins in the cells [13, 14]. Besides ETS family proteins, several TFs could bind to the motif. For example, some of the duplicated GGAA-motifs would be identical to IFN-stimulated response element (ISRE), the consensus sequence of which is 5′-GGAAANNGAAACT-3′ [15], if one of the Ns was G. The double-stranded sequence, 5′-AACTTT-3′, which is a core binding motif of the IRF1 [16], could be generated if CT was inserted between GGAA and TTCC. Moreover, NF-κB p65 (RELA) homodimer binds to two symmetric sequences, 5′-GGAATTTCC-3′ and 5′-GGAATTCCC-3′ [17]. IRF8 (ICSBP) either positively or negatively regulates transcription through binding to ISRE [18, 19].

Importantly, the ETS family proteins and other TFs cooperatively regulate the expression of various genes. For instance, STAT1 plays a role in the regulation of the expression of interferon (IFN) stimulated genes (ISGs) with ETS family proteins [20, 21]. Sp1 and Ets1 interact with each other to regulate mouse *Npr1* gene expression [22]. The ETS-binding consensus sequence is frequently found with a second ETS-binding sequence and with the Sp1-binding sequence, but not with a TATA element [23], implying the exclusive role of the GGAA- and TATA elements. The human *VEGFR1* promoter region, which contains overlapping GGAA-motifs, is also regulated by CREB and EGR-binding elements [24]. Furthermore, the mouse *Ppp2r1a* gene promoter, which carries duplicated TTCC motifs, is regulated by Creb, Ets1, Ap2 alpha, and Sp1proteins [25]. Thus, multiple elements adjacent to duplicated GGAA-motifs may recruit various TFs to form a pre-initiation complex on the transcription start site (TSS) of TATA-less promoters. The transcription initiation system might be advantageous for a rapid response to stresses in a TATA-independent manner.
