# Gene transcriptions/Boxes/Ws

"Consistent with its role as a transcriptional repressor, OsWRKY71 binds specifically to functionally defined TGAC-containing W boxes of the Amy32b promoter in electrophoretic mobility shift assays (Zhang et al. 2004)."
"Details of auto-regulation or cross-regulation by WRKY factors were provided for the parsley group I member PcWRKY1 and its ortholog AtWRKY33 [37**,39,41]. In response to PAMP treatment PcWRKY1 transcripts accumulate rapidly and transiently [42]. AtWRKY33 is activated with similar kinetics by defense-related stimuli [18,34,41]. This rapid response is mediated by a conserved arrangement of three synergistically acting W boxes (W$_{ABC}$). Chromatin immunoprecipitation (ChIP) revealed that in vivo these orthologous W boxes are constitutively occupied by WRKY proteins [37**,41]."
"The synthesis of SA and the expression of NPR1, a key regulator of some PAMP-triggered responses, appear to be partly controlled by WRKY factors. NPR1 is regulated by WRKY TFs interacting with two W box elements in its 50UTR [44]. Defense-associated SA production is strongly dependent on pathogen-inducible expression of ICS1 [45]. This gene is a likely target of WRKY TFs, as its promoter is enriched for W boxes."
A "missense mutation within its WRKY domain results in conditional activation of defense responses and loss of in vitro binding to W boxes suggesting a negative role of this factor in defense signaling [49]."
Functionality and conservation of the W-box element across plant species shown by gel shift experiments, random binding site selection, yeast one-hybrid screens and co-transfection assays performed with many different WRKY proteins and In silico-based studies have identified clusters of W-boxes in stress-inducible promoters, where the binding of WRKY proteins to W-boxes is a feature of both biotic and abiotic stress responses, together with other plant processes such as germination. It has also been shown that multiple W-boxes have a synergistic effect on transcription.
Almost all WRKY transcription factors bind preferentially to W-boxes, and since their discovery, this has raised the question as to how they show specificity for the promoters of their target genes. Although the W-box core is required, adjacent sequences also play a role in determining binding-site preference. Recent evidence suggests that the TGAC core is more degenerate, composed of a guanine adenine cytosine (GAC) core, and the upstream thymine and downstream pyrimidine flanking sequences help dictate recognition by specific WRKY factors. Basic residues of the WRKY protein domain also are believed to recognize the phosphate backbone of the cis-element.
The solution structure of the C-terminal WRKY domain of Arabidopsis WRKY4 in complex with the W-box DNA has been determined by NMR. A four-stranded β-sheet enters the major groove of DNA in a structure called the β-wedge, where the sheet is nearly perpendicular to the DNA helical ais: as predicted amino acids in the conserved WRKYGQK signature motif contact the W-box DNA.

## WRKY

WRKY family of genes contain the common degenerate nucleotide sequence: WRKY, or W = A or T (U) [Weak], R = A or G [puRine], K = G or T (U) [Keto], Y = C or T (U) [pYrimidine].
"Plant immune responses are associated with the concerted modulation of a large number of different WRKY transcripts and proteins [15,34–36,37**]. Upon triggering of SA-dependent defenses, at least 49 AtWRKY genes exhibited differential regulation representing separate waves of transcript accumulation or repression [34]. Their promoters are statistically enriched for W boxes, suggesting that they are autoregulated or controlled by other WRKY proteins [34]."
"Some architectural features of the WRKY web are emerging. As motif D containing group I WRKY TFs can be phosphorylated by MAP-kinases, they are likely to serve as the first WRKY proteins activated in response to PAMP-triggered MAPK signaling. Their targets may include the IIe WRKY genes AtWRKY22 and AtWRKY29, which are upregulated by a PAMP-induced MAPK cascade and contain multiple W boxes within their respective promoters [4]."

## Consensus sequences

The "presence of WRKY TF binding sites (C/TTGACC/T, W boxes) in numerous co-regulated Arabidopsis defense gene promoters provided circumstantial evidence that zinc-finger-type WRKY factors play a broad and pivotal role in regulating defenses [10]."
The W box is a DNA cis-regulatory element sequence, (T)TGAC(C/T), which is recognized by the family of WRKY transcription factors.

## Sampling A1BG promoters

For the Basic programs (starting with SuccessablesWbox.bas) written to compare nucleotide sequences with the sequences on either the template strand (-), or coding strand (+), of the DNA, in the negative direction (-), or the positive direction (+), including extending the number of nts from 958 to 4445, the programs are, are looking for, and found:
* negative strand in the negative direction (from ZSCAN22 to A1BG) is SuccessablesWbox--.bas, looking for 3'-(C/T)TGAC(C/T)-5', 5, 3'-CTGACT-5', 17, 3'-TTGACT-5', 130, 3'-TTGACT-5', 307, 3'-CTGACT-5', 1935, 3'-CTGACC-5', 3749,
* negative strand in the positive direction (from ZNF497 to A1BG) is SuccessablesWbox-+.bas, looking for 3'-(C/T)TGAC(C/T)-5', 6, 3'-CTGACC-5', 1662, 3'-CTGACC-5', 2213, 3'-TTGACC-5', 2873, 3'-CTGACT-5', 2945, 3'-TTGACC-5', 4018, 3'-CTGACC-5', 4216,
* positive strand in the negative direction is SuccessablesWbox+-.bas, looking for 3'-(C/T)TGAC(C/T)-5', 1, 3'-CTGACC-5', 734,
* positive strand in the positive direction is SuccessablesWbox++.bas, looking for 3'-(C/T)TGAC(C/T)-5', 3, 3'-TTGACC-5', 1953, 3'-CTGACT-5', 2674, 3'-TTGACT-5', 3735,
* complement, negative strand, negative direction is SuccessablesWboxc--.bas, looking for 3'-(A/G)ACTG(A/G)-5', 1, 3'-GACTGG-5', 734,
* complement, negative strand, positive direction is SuccessablesWboxc-+.bas, looking for 3'-(A/G)ACTG(A/G)-5', 3, 3'-AACTGG-5', 1953, 3'-GACTGA-5', 2674, 3'-AACTGA-5', 3735,
* complement, positive strand, negative direction is SuccessablesWboxc+-.bas, looking for 3'-(A/G)ACTG(A/G)-5', 5, 3'-GACTGA-5', 17, 3'-AACTGA-5', 130, 3'-AACTGA-5', 307, 3'-GACTGA-5', 1935, 3'-GACTGG-5', 3749,
* complement, positive strand, positive direction is SuccessablesWboxc++.bas, looking for 3'-(A/G)ACTG(A/G)-5', 6, 3'-GACTGG-5', 1662, 3'-GACTGG-5', 2213, 3'-AACTGG-5', 2873, 3'-GACTGA-5', 2945, 3'-AACTGG-5', 4018, 3'-GACTGG-5', 4216,
* inverse complement, negative strand, negative direction is SuccessablesWboxci--.bas, looking for 3'-(A/G)GTCA(A/G)-5', 2, 3'-GGTCAG-5', 1353, 3'-GGTCAA-5', 4416,
* inverse complement, negative strand, positive direction is SuccessablesWboxci-+.bas, looking for 3'-(A/G)GTCA(A/G)-5', 6, 3'-AGTCAG-5', 2101, 3'-GGTCAG-5', 2221, 3'-AGTCAG-5', 2608, 3'-AGTCAA-5', 2614, 3'-AGTCAG-5', 2619, 3'-GGTCAG-5', 4270,
* inverse complement, positive strand, negative direction is SuccessablesWboxci+-.bas, looking for 3'-(A/G)GTCA(A/G)-5', 6, 3'-GGTCAG-5', 440, 3'-GGTCAG-5', 577, 3'-GGTCAG-5', 713, 3'-GGTCAG-5', 2249, 3'-GGTCAG-5', 2586, 3'-GGTCAA-5', 4308,
* inverse complement, positive strand, positive direction is SuccessablesWboxci++.bas, looking for 3'-(A/G)GTCA(A/G)-5', 6, 3'-GGTCAG-5', 2025, 3'-AGTCAG-5', 2099, 3'-GGTCAG-5', 2606, 3'-GGTCAG-5', 2997, 3'-GGTCAG-5', 3083, 3'-GGTCAA-5', 3380,
* inverse, negative strand, negative direction, is SuccessablesWboxi--.bas, looking for 3'-(C/T)CAGT(C/T)-5', 6, 3'-CCAGTC-5', 440, 3'-CCAGTC-5', 577, 3'-CCAGTC-5', 713, 3'-CCAGTC-5', 2249, 3'-CCAGTC-5', 2586, 3'-CCAGTT-5', 4308,
* inverse, negative strand, positive direction, is SuccessablesWboxi-+.bas, looking for 3'-(C/T)CAGT(C/T)-5', 6, 3'-CCAGTC-5', 2025, 3'-TCAGTC-5', 2099, 3'-CCAGTC-5', 2606, 3'-CCAGTC-5', 2997, 3'-CCAGTC-5', 3083, 3'-CCAGTT-5', 3380,
* inverse, positive strand, negative direction, is SuccessablesWboxi+-.bas, looking for 3'-(C/T)CAGT(C/T)-5', 2, 3'-CCAGTC-5', 1353, 3'-CCAGTT-5', 4416,
* inverse, positive strand, positive direction, is SuccessablesWboxi++.bas, looking for 3'-(C/T)CAGT(C/T)-5', 6, 3'-TCAGTC-5', 2101, 3'-CCAGTC-5', 2221, 3'-TCAGTC-5', 2608, 3'-TCAGTT-5', 2614, 3'-TCAGTC-5', 2619, 3'-CCAGTC-5', 4270.