# Gene transcriptions/Boxes/Zs

"Both miRNAs [miR‐141 and miR‐200c] map closely on human chromosome 12p13.31 and the stem–loop sequences are separated by only a 338‐base‐pair spacer sequence [...]. This spacer and the putative promoter 600 bp upstream from the hsa‐miR‐200c stem–loop contain six putative binding sequences for [zinc‐finger E‐box binding homeobox 1] ZEB1, two of which were restricted to ZEB factors (Z‐box 1 and 2, CAGGTA)."
The "overall miRNA gene structure and the two Z‐boxes, as well as E‐box 2, are highly conserved in evolution from zebrafish to human [...]."
"Mutation of the two highly conserved Z‐boxes showed that Z‐box 2 confers the strongest repressive function by ZEB1 [...] and also made the promoter activity insensitive to stable knockdown of ZEB1 [...]. A direct binding of ZEB1 to the two conserved ZEB1 sites (Z‐boxes) was shown by electromobility shift assay by using recombinant DNA‐binding domain of ZEB1 and nuclear extracts from SW480 colorectal cancer cells [...]. By applying chromatin immunoprecipitation (ChIP) with chromatin from SW480 or HCT116 colorectal cancer cells, we could show that endogenous ZEB1 binds to the native promoter region [...]. These data indicate that the transcriptional repressor ZEB1 can directly suppress expression of both miR‐141 and miR‐200c by binding to their putative common promoter."
"The absolute transcriptional activity of the promoter was 55‐fold higher than the activity of the spacer sequence. [Mutations] (mut) of the Z‐boxes render the promoter less sensitive to suppression by ZEB1."
"Mutation of the two conserved Z‐boxes (Z1, Z2) strongly reduced the specific binding complex [...]."

## Consensus sequences

"Snail-related and E2A factors bind to a consensus E-box promoter element (CANNTG), whereas ZEB factors bind to a related, but not necessarily identical sequence (CAGGTG/A), termed Z-Box. We detected 2 potential binding sites for Snail-related and E2A factors (E-box 1 and 2) and 3 for ZEB factors (E-box 2 and Z-box 1 and 2) in the human lama3a promoter [...]. Furthermore, sequence comparison of 1000 nucleotides upstream of the translation start site of the human and mouse lama3 gene showed that these sites are located in a highly conserved region of approximately 210 nucleotides, whereas further upstream sequences were not conserved [...]. Three of the 4 potential repressive elements were conserved between mouse and human, indicating their functional relevance."

## SIP1-mediated Epithelial Mesenchymal Transition (EMT)

In vivo SIP1, or Zinc finger E-box-binding homeobox 2 (ZEB2), binds "to potential SIP1-binding sites (Z-boxes) located in the vicinity of the cyclin D1 transcription start site."
Three "Z-boxes with coordinates -1014 to -1010 (Z-box 1); -857 to -853 (Z-box 2); and -300 to -290 (Z-box 3) are occupied by SIP1 in [doxorubicin (DOX)] DOX-stimulated cells. In contrast, neither sequences upstream of Z-box 1 [...], nor sequences containing Z-boxes 4 and 5 located at the first exon/intron boundary (-390 to -409) [...] were detected in association with SIP1."
Z-boxes 1-3 contain 5'-AGGTG-3'.
The "mutation of Z-boxes 1–3 markedly activated reporter activity in SIP1-expressing cells [...]."
"SIP1 represses cyclin D1 transcriptional activity via direct interaction with Z-boxes 1–3 in the cyclin D1 promoter."