# Gene transcriptions/Boxes/GCs/Laboratory

A laboratory is a specialized activity, a construct, you create where you as a student, teacher, or researcher can have hands-on, or as close to hands-on as possible, experience actively analyzing an entity, source, or object of interest. Usually, there's more to do than just analyzing. The construct is often a room, building or institution equipped for scientific research, experimentation as well as analysis.
This laboratory is a continuation of the previous laboratory.
In the room next door is an astronaut on the Mars expedition, three months along on the six-month journey. A physician and lab assistants have been performing tests on her. Because she has been in zero gravity for more than three months her body chemistry and anatomy now differ from what it was in the controlled gravity environment of Earth. She has lost about 10 % each of her bone, muscle, and brain mass. Comparisons with gene expression sequences now and when on Earth have found that the gene expression for alpha-1-B glycoprotein is not normal. If a way to correct this expression cannot be found she must be returned to Earth maybe to recover, maybe not!
But, it is unlikely she will survive three more months at zero g either to be returned to Earth or put on Mars. Worse, the microgravity may not be the only culprit. There is also the radiation of the interplanetary medium.
You have been tasked to examine her DNA to confirm, especially with the extended data between ZNF497 and A1BG, the presence or absence of GC boxes regarding the possible expression of alpha-1-B glycoprotein.

## Consensus sequences

"A GC box sequence, one of the most common regulatory DNA elements of eukaryotic genes, is recognized by the Spl transcription factor; its consensus sequence is represented as 5'-G/T G/A GGCG G/T G/A G/A C/T-3' [or 5′-KRGGCGKRRY-3′] (Briggs et al., 1986)."

## Nucleotides

DNA mapping has been performed. Her DNA for A1BG promoters can be found at Gene_transcriptions/A1BG#Nucleotides.

## Programming

Sample programs for preparing test programs are available at Gene transcriptions/A1BG/Programming.

## Hypotheses

* GC boxes are not present in the promoter of A1BG.
* If a GC box is present it does not assist in the transcription of A1BG.

## Core promoters

The core promoter is approximately -34 nts upstream from the TSS.
From the first nucleotide just after ZSCAN22 to the first nucleotide just before A1BG are 4460 nucleotides. The core promoter on this side of A1BG extends from approximately 4425 to the possible transcription start site at nucleotide number 4460.
To extend the analysis from inside and just on the other side of ZNF497 some 3340 nts have been added to the data. This would place the core promoter some 3340 nts further away from the other side of ZNF497. The TSS would be at about 4300 nts with the core promoter starting at 4266.
Def. "the factors, including RNA polymerase II itself, that are minimally essential for transcription in vitro from an isolated core promoter" is called the basal machinery, or basal transcription machinery.

## Proximal promoters

Def. a "promoter region [juxtaposed to the core promoter that] binds transcription factors that modify the affinity of the core promoter for RNA polymerase.$^{[12][13]}$" is called a proximal promoter.
The proximal sequence upstream of the gene that tends to contain primary regulatory elements is a proximal promoter.
It is approximately 250 base pairs or nucleotides, nts, upstream of the transcription start site.
The proximal promoter begins about nucleotide number 4210 in the negative direction.
The proximal promoter begins about nucleotide number 4195 in the positive direction.

## Distal promoters

The "upstream regions of the human [cytochrome P450 family 11 subfamily A] CYP11A and bovine CYP11B genes [have] a distal promoter in each gene. The distal promoters are located at −1.8 to −1.5 kb in the upstream region of the CYP11A gene and −1.5 to −1.1 kb in the upstream region of the CYP11B gene."
"Using cloned chicken β$^A$-globin genes, either individually or within the natural chromosomal locus, enhancer-dependent transcription is achieved in vitro at a distance of 2 kb with developmentally staged erythroid extracts. This occurs by promoter derepression and is critically dependent upon DNA topology. In the presence of the enhancer, genes must exist in a supercoiled conformation to be actively transcribed, whereas relaxed or linear templates are inactive. Distal protein–protein interactions in vitro may be favored on supercoiled DNA because of topological constraints."
Distal promoter regions may be a relatively small number of nucleotides, fairly close to the TSS such as (-253 to -54) or several regions of different lengths, many nucleotides away, such as (-2732 to -2600) and (-2830 to -2800).
The "[d]istal promoter is not a spacer element."
Using an estimate of 2 knts, a distal promoter to A1BG would be expected after nucleotide number 2460.
Any transcription factors before A1BG from the direction of ZN497 may be out to 2300 nts.

## Samplings
### Regarding hypothesis 1
#### GC boxes

"A GC box sequence, one of the most common regulatory DNA elements of eukaryotic genes, is recognized by the Spl transcription factor; its consensus sequence is represented as 5'-G/T G/A GGCG G/T G/A G/A C/T-3' [or 5′-KRGGCGKRRY-3′] (Briggs et al., 1986)."
For the Basic programs (starting with SuccessablesGC.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 (+), the programs are, are looking for out to 4445, and found:
* negative strand in the negative direction is SuccessablesGC--.bas, looking for 3'-G/T G/A GGCG G/T G/A G/A C/T-5', 0,
* negative strand in the positive direction is SuccessablesGC-+.bas, looking for 3'-G/T G/A GGCG G/T G/A G/A C/T-5', 1, 3'-TGGGCGGGAC-5', 409,
* positive strand in the negative direction is SuccessablesGC+-.bas, looking for 3'-G/T G/A GGCG G/T G/A G/A C/T-5', 2, 3'-TGGGCGTGGT-5', 1898, 3'-TGGGCGTGGT-5', 3048 ,
* positive strand in the positive direction is SuccessablesGC++.bas, looking for 3'-G/T G/A GGCG G/T G/A G/A C/T-5', 0,
* complement, negative strand, negative direction is SuccessablesGCc--.bas, looking for 3'-A/C C/T CCGC A/C C/T C/T A/G-5', 2, 3'-ACCCGCACCA-5', 1898, 3'-ACCCGCACCA-5', 3048,
* complement, negative strand, positive direction is SuccessablesGCc-+.bas, looking for 3'-A/C C/T CCGC A/C C/T C/T A/G-5', 0,
* complement, positive strand, negative direction is SuccessablesGCc+-.bas, looking for 3'-A/C C/T CCGC A/C C/T C/T A/G-5', 0,
* complement, positive strand, negative direction is SuccessablesGCc++.bas, looking for 3'-A/C C/T CCGC A/C C/T C/T A/G-5', 1, 3'-ACCCGCCCTG-5', 409,
* inverse complement, negative strand, negative direction is SuccessablesGCci--.bas, looking for 3'-A/G C/T C/T A/C CGCC C/T A/C-5', 1, 3'-ACTCCGCCCA-5', 3092,
* inverse complement, negative strand, positive direction is SuccessablesGCci-+.bas, looking for 3'-A/G C/T C/T A/C CGCC C/T A/C-5', 0,
* inverse complement, positive strand, negative direction is SuccessablesGCci+-.bas, looking for 3'-A/G C/T C/T A/C CGCC C/T A/C-5', 1, 3'-GCTCCGCCTC-5', 1505,
* inverse complement, positive strand, positive direction is SuccessablesGCci++.bas, looking for 3'-A/G C/T C/T A/C CGCC C/T A/C-5', 1, 3'-GCCACGCCCC-5', 491,
* inverse, negative strand, negative direction, is SuccessablesGCi--.bas, looking for 3'-C/T G/A G/A G/T GCGG G/A G/T-5', 1, 3'-CGAGGCGGAG-5', 1505,
* inverse, negative strand, positive direction, is SuccessablesGCi-+.bas, looking for 3'-C/T G/A G/A G/T GCGG G/A G/T-5', 1, 3'-CGGTGCGGGG-5', 491,
* inverse, positive strand, negative direction, is SuccessablesGCi+-.bas, looking for 3'-C/T G/A G/A G/T GCGG G/A G/T-5', 1, 3'-TGAGGCGGGT-5', 3092,
* inverse, positive strand, positive direction, is SuccessablesGCi++.bas, looking for 3'-C/T G/A G/A G/T GCGG G/A G/T-5', 0.

## Verifications

To verify that your sampling has explored something, you may need a control group. Perhaps where, when, or without your entity, source, or object may serve.
Another verifier is reproducibility. Can you replicate something about your entity in your laboratory more than 3 times. Five times is usually a beginning number to provide statistics (data) about it.
For an apparent one time or perception event, document or record as much information coincident as possible. Was there a butterfly nearby?
Has anyone else perceived the entity and recorded something about it?
Gene ID: 1, includes the nucleotides between neighboring genes and A1BG. These nucleotides can be loaded into files from either gene toward A1BG, and from template and coding strands. These nucleotide sequences can be found in Gene transcriptions/A1BG. Copying the above discovered CRE boxes and putting the sequences in "⌘F" locates these sequences in the same nucleotide positions as found by the computer programs.

## Core promoters GC boxes

From the first nucleotide just after ZSCAN22 to the first nucleotide just before A1BG are 4460 nucleotides. The core promoter on this side of A1BG extends from approximately 4425 to the possible transcription start site at nucleotide number 4460.
There are no GC boxes in the core promoter in the negative direction.
From the first nucleotide just after ZNF497 to the first nucleotide just before A1BG are 858 nucleotides. The core promoter on this side of A1BG extends from approximately 824 to the possible transcription start site at nucleotide number 858. Nucleotides (nts) have been added from ZNF497 to A1BG. The TSS for A1BG is now at 4300 nts from just on the other side of ZNF497. The core promoter should now be from 4266 to 4300.
There are no GC boxes in the core promoter in the positive direction.

## Proximal promoter GC boxes

The proximal promoter begins about nucleotide number 4210 in the negative direction.
There is no GC box in the negative direction.
The proximal promoter begins about nucleotide number 4195 in the positive direction.
There is no GC box in the positive direction.

## Distal promoter GC boxes

Using an estimate of 2 knts, a distal promoter to A1BG would be expected after nucleotide number 2460 in the negative direction.
There are two GC boxes in the distal promoter in the negative direction: 3'-ACCCGCACCA-5' at 3048 and 3'-ACTCCGCCCA-5' at 3092 nts, negative strand, and their complements.
Using an estimate of 2 knts, a distal promoter to A1BG would be expected after nucleotide number 2300 in the positive direction.
There are none in the distal promoter in the positive direction.

## Transcribed GC boxes

A Google Scholar search using A1BG and GC box produced one result scoring an interaction between SP2 and A1BG.
"Multiple processive phosphorylation of Sp1 depends on binding of Sp1 to GC box-containing DNA."
"In promoters containing multiple GC boxes but lacking the TATAA box, transcription start sites may be single and specific, as observed in the nerve growth factor receptor gene (42) and the cellular retinol-binding protein gene (37), or there may be multiple heterogeneous start sites, such as those found in the c-myb (4), insulin receptor (45), and c-Ha-ras (21) genes."
"GC boxes are responsible for directing transcription from the major and the minor start sites."
"In the murine terminal deoxynucleotidyltransferase promoter, the sequence immediately surrounding the site of initiation (Inr region) has been found to be the only sequence element required for accurate initiation of transcription (43)."
"Although the arrangement of GC boxes in other non-TATAA gene promoters is quite variable, they are usually found very close to the transcription start site(s)."
"All TATAA-less promoters have at least two GC boxes; this feature may be a functional requirement for inducing initiation in the absence of TATAA or an Inr-like sequence."
"Spl-binding sites clearly regulate transcriptional initiation in a TATAA-less promoter."

## Dihydrofolate reductases

Protein-DNA "interactions at three of the four proximal GC box sequence elements in one such promoter, that of the hamster dihydrofolate reductase gene, control initiation and relative use of the major and minor start sites."
Although "the GC boxes are apparently equivalent with respect to factor binding, they are not equivalent with respect to function. At least two properly positioned GC boxes were required for initiation of transcription. Abolishment of DNA-protein interaction by site-specific mutation of the most proximal GC box (box I) resulted in a five fold decrease in transcription from the major initiation site and a threefold increase in heterogeneous transcripts initiating from the vicinity of the minor start site in vitro and in vivo. Mutations that separately abolished interactions at GC boxes II and III while leaving GC box I intact affected the relative utilization of both the major and minor initiation sites as well as transcriptional efficiency of the promoter template in in vitro transcription and transient expression assays. Interaction at GC box IV when the three proximal boxes were in a wild-type configuration had no effect on transcription of the dihydrofolate reductase gene promoter. Thus, GC box interactions not only are required for eficient transcription but also regulate start site utilization in this TATAA-less promoter."
"A large subclass of polymerase II promoters lacks both TATAA and CCAAT sequence motifs but contains multiple GC boxes. This promoter class includes several housekeeping genes (e.g., the genes encoding dihydrofolate reductase [DHFR] [reference 2 and references therein], hydroxymethylglutaryl coenzyme A reductase [39], hypoxanthine guanine phosphoribosyltransferase [3], and adenosine deaminase [46]) as well as nonhousekeeping genes (e.g., the transforming growth factor alpha [9,23], rat malic enzyme [36], human c-Ha-ras [21], epidermal growth factor receptor [2], and nerve growth factor receptor [42] genes)."
Factors "found to interact with GC boxes [...:] Spl (for a review, see reference 31); more recently, the factors LSF (28), ETF (26), GCF-1 (27), and AP2 (35) have also been shown to interact with GC boxes."
"GC boxes are required for efficient promoter activity in the genes in which they have been analyzed. [...] in all examined cases, more than one GC box is present. Many of these promoters have unique transcription start sites, while others display multiple but specific start sites."
"The hamster DHFR promoter contains neither TATAA nor CCAAT sequence elements (2,34). There are four GC boxes in the 210 bp 5' to the DHFR-coding sequence and two binding sites for the transcription factor E2F immediately 3' to the major transcription start site (8). The DHFR major start site in the hamster gene has been mapped to nucleotide position -63 relative to ATG (34). A minor transcriptional start site that accounts for 15 to 20 % of DHFR transcription is located at position -107 relative to ATG (34). The center of the first GC box is positioned 45 bp upstream of the major transcription start site, while the center of the second GC box is 45 bp upstream of the minor start site. Interestingly, the minor start site is located in the center of the first GC box. This spatial arrangement is conserved among mammalian DHFR genes, suggesting that the relative distance of the initiation site to the center of each upstream GC box is important for specifying the start site or regulating the efficiency of transcription."
For "TATAA-less promoters, [...] a GC box-binding factor is required for transcription and that a truncated promoter containing one GC box is transcriptionally inactive (44)."
The "DNA-protein interactions occurring at the GC boxes in the DHFR promoter are functionally distinct and that factors binding to the GC boxes must interact in a position-dependent manner."
GC box IV starting at -196 is 5'-TGGGCGGGGC-3' ending at -187, GC box III -176 5'-GAGGCGGAGT-3' at -167, GC box II -155 5'-GAGGCGGGGC-3' at -146, and GC box I -112 5'-AGGGCGTGGC-3' at -103, where +1 (the transcription start site) is 5'-ATG-3' at +3, minor start sites are -107 5'-G-3', -66 5'-G-3', -64 5'-A-3', and -63 5'-A-3'.
Two "upstream GC boxes are required for initiation of transcription."
"GC boxes I and II control utilization of the major start site, whereas utilization of the minor start site is controlled by any pairwise combination of GC boxes upstream of the minor start (e.g., boxes I and II or IV or boxes IIand IV)."

## Endothelial tissue-type plasminogen activators

The "t-PA promoter [has] CRE or GC box II or GC box III elements".
Valproic Acid (valproate, 2-propylpentanoic acid, VPA), "VPA-induced t-PA expression is dependent on the proximal GC boxes in the t-PA promoter and may involve interactions with Sp2, Sp4, and KLF5."
The "proximal promoter region of the t-PA gene [includes] the cAMP responsive element- (CRE- [−224 to −217]) like site, the CAAT- binding transcription factor/nuclear factor-1- (CTF/NF1- [−203 to −189]) like binding site, and the three GC box elements (I [−154 to −145], II [−72 to −66], and III [−49 to −43])."
"The t-PA gene is transcribed primarily from a TATA-independent transcription initiation site (TIS) [18, 19]. Three elements in the t-PA promoter, just upstream of the TIS, have previously been reported to be important for t-PA expression: one cyclic adenosine monophosphate (cAMP) responsive element- (CRE-) like site [TGACATCA] and two GC boxes (II [CCCGCCC] and III [CCCACCC]) [16, 17]."

## Laboratory reports

Below is an outline for sections of a report, paper, manuscript, log book entry, or lab book entry. You may create your own, of course.
GC boxes transcription laboratory
by --Marshallsumter (discuss • contribs) 04:52, 21 April 2019 (UTC)

### Abstract

GC boxes are apparently fairly common in gene promoters. If present in the promoters of A1BG, these boxes could make transcription easier. The presence of a GC box may indicate a gene silencer especially when a CAAT box is present. Testing these promoters for a GC box and possible interactions with other transcription factors already found increases the likelihood of multiple transcription pathways. At least one GC box has been found between neighboring gene ZSCAN22 and A1BG. The TATA boxes earlier discovered in the distal promoter on this same side may be indicative of a second transcription start site.

### Introduction

Many transcription factors (TFs) may occur upstream and occasionally downstream of the transcription start site (TSS), in this gene's promoter. The following have been examined so far: (1) AGC boxes (GCC boxes), (2) ATA boxes, (3) CAAT boxes, (4) C and D boxes, (5) CAREs (GA responsive complexes), (6) CArG boxes, (7) CENP-B boxes, (8) CGCG boxes, (9) CRE boxes, (10) DREB boxes, (11) EIF4E basal elements (4EBEs), (12) enhancer boxes (E boxes), (13) E2 boxes, (14) Factor II B recognition elements, (15) GAREs (GA responsive complexes), (16) G boxes, (17) GLM boxes, (18) HNF6s, (19) HY boxes, (20) Metal responsive elements (MREs), (21) Motif ten elements (MTEs), (22) Pyrimidine boxes (GA responsive complexes), (23) STAT5s, (24) TACTAAC boxes, (25) TATA boxes, (26) TAT boxes (GA responsive complexes), (27) TATCCAC box, (28) W boxes (GA responsive complexes), (29) X boxes, and (30) Y boxes.
But, no (3) CAAT box, (7) CENP-B box, (8) CGCG boxes are too close to ZSCAN22, (10) no DREB box, (11) EIF4E basal element), (13) E2 boxes, (15) GARE are too close to ZSCAN22, (16) no G box, (17) GLM box, (21) MTE, (24) TACTAAC box, (26) TAT box, (27) TATCCAC box or TATC box, (29) X box, or (30) Y box occur.
Interactions may occur with (1) an AGC (GCC) box, (2) an ATA box, (4) C boxes, a D box, but the other C-box and D-box have not been tested, (5) CAREs, (6) CArG boxes, (9) a CRE box, (12) enhancer boxes, (14) a BRE$^u$, (18) HNF6s, (19) HY boxes, (20) an MRE, (22) pyrimidine boxes, (23) STAT5s, (25) TATA boxes outside the core promoter, or (28) W boxes.

### Experiments

Regarding hypothesis 1: GC boxes are not present in the promoter of A1BG.
The Basic programs (starting with SuccessablesGCbox.bas) were 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 the extended number of nts from 958 to 4445, looking for GC boxes, their possible complements and inverses, to test the hypothesis that GC boxes are not present in the promoter of A1BG.
Regarding hypothesis 2: If an GC box is present it does not assist in the transcription of A1BG.
Literature searches were performed to determine the likely TFs and possible interactions to transcribe A1BG.

### Results
#### Hypothesis 1

There are no GC boxes in the core promoter in the negative or positive directions.
There is no GC box in the proximal promoter in the negative or positive direction.
There are two GC boxes in the distal promoter in the negative direction: 3'-ACCCGCACCA-5' at 3048 and 3'-ACTCCGCCCA-5' at 3092 nts, negative strand, and their complements.
There are no GC boxes in the distal promoter in the positive direction.

#### Hypothesis 2

If a GC box is present it does not assist in the transcription of A1BG.
A Google Scholar search using A1BG and GC box produced one result scoring an interaction between SP2 and A1BG only.

### Discussions
#### Hypothesis 1 discussion

Regarding hypothesis 1: GC boxes are not present in the promoter of A1BG. There are two GC boxes in the distal promoter in the negative direction: 3'-ACCCGCACCA-5' at 3048 and 3'-ACTCCGCCCA-5' at 3092 nts, negative strand, and their complements. But, there are none on the positive side between ZNF497 and A1BG. Therefore, hypothesis 1 is false. If A1BG can only be transcribed from the positive side promoter, then hypothesis 1 is true.

#### Hypothesis 2 discussion

Regarding hypothesis 2: If an GC box is present it does not assist in the transcription of A1BG. In order to assess whether a GC box assists in the transcription of A1BG, pairings of a GC box with other TFs are examined.

##### "GC box" and "AGC box"

Google search 5 results (0.05 sec):
* A "GC box [is] the consensus sequence for binding of the transcription factor Sp l (30,31) [which] functions to increase transcription levels of promoters containing GC boxes." No AGC box or GCC box is mentioned.
* Some "putative wound-response elements including AGC box-like sequences$^{28}$, TCA motif-like sequences$^{28}$, carrot extensin gene wound-response elements (AT-rich motif, TTTTTTT, TGACGT)$^{29}$, constitutive PAL footprint and elicitor-inducible PAL footprint$^{31}$, and proteinase inhibitor II footprint$^{31}$ have been found in cabch29 promoter. Some cis-elements related to organ and tissue-specific expression such as GATA motif-like sequence, ASF-1 binding site-like elements also existed in 5′ upstream region. Meanwhile, some basic transcriptional regulatory cis-elements including G box-like and GC box-like elements are located in this region."
* "A GC box (-114/-101) and a CCAAT box (-62/-51) binding Sp1 and NF-Y respectively, were shown to underlie both basal and calcitonin induced expression of CYP24 promoter constructs." and "Localised in the -298 bp [region of the proximal promoter of the rat CYP24 promoter] is the proximal vitamin D$_3$ response element (VDRE-1) [CGCCCTCACTCACCT] at position -150/-136 and VDRE-2 [CGCACCCGCTGAACC] at position -258/-244 with respect to the transcriptional start site. [...] Downstream of VDRE-1 at position -128/-119 lies an Ets-1 binding site (EBS) [TGACTCCATCCTCT]. Upstream of VDRE-1, a novel binding sequence at position -171/-168 termed the CYP24 hydroxylase enhancing protein (CHEP) binding site (CBS) [TGTCGGTCA] has been identified. [There] is a putative GC box [CCGCCC] and a CCAAT box [CATTG]".
* A "fragment of 61 bp of the tobacco class I (GLB [β-1,3-glucanase B]) promoter region, bears two copies of the AGC box. When a deletion of this box was carried out, a loss of the gene expression was observed. [...] The AGC box was found in the Solyc04g076630 gene promoter." GC box is not mentioned.
* "The "AGC box", an enhancer element identified in tobacco, was only recognized in the predicted promoter sequence of [actin depolymerizing factor] ADF11." GC box is not mentioned.

##### "GC box" and "ATA box"

Google Scholar search About 21 results (0.09 sec):
* For "direct muscle-specific transcription of chimeric chloramphenicol acetyltransferase (CAT) gene constructs [...] the transcriptional start site [...] lies 37 base pairs (bp) upstream of the 5' end [...] in addition to an ATA and GC box, this region contains domains that have been implicated in the regulation of other muscle-specific genes: a CArG box at -91 bp; myocyte-specific enhancer-binding nuclear factor 1 binding site homologies at -58, -535, and -583 bp; and a muscle-CAAT consensus sequence at -394 bp relative to the cap site. [...] The ATA box [AATAT], GC box [GGCGGG], CArG box [CCTATTATGC], and MCAT [CATTCCT] consensus sequences [are] described".
* "The ATA box [AAATAT], GC box [GGCGGG], CArG box [CCTATTATGCG], [two E boxes CAGTTG] and M-CAT [CATTCCT] consensus sequences are [described from the mouse dystrophin promoter]."
* The "cis-control signals in one of the two promoters of the developmentally regulated rat insulin-like growth factor II gene (rIGF-II) [...] consists of no more than 128 base-pairs, which include an ATA box and four proximal upstream GC boxes binding the general transcription factor Spl. Three of the latter sites deviate from the known Spl consensus recognition sequence. The two types of cis-acting regulatory signals (GCATA motif) of the P2 promoter are inter-dependent and sufficient for transcription."
* The "expression of the S-type cystatin genes (CST1, CST2, and CST4) is under a different control mechanism from that of the cystatin C gene (CST3). These four cystatin genes contain the ATA-box sequence (ATAAA) in their 5'-flanking regions; however, the CAT-box sequence (CAT), a binding site of the transcription factor, CTF, is found only in the 5'-flanking region of the S-type cystatin genes. On the other hand, the GC-box sequence (GGGCGG), a binding site of transcription factor, Spl, is found in the cystatin C gene, the sequence of which is found often in many housekeeping genes."
* Sites "within the [thymidine kinase] TK promoter to which known cellular transcription factors can bind [are]: OTF-1 (ATGCAAAT), Spl (GGGCGG), TFIID (ATA box), and C/EBP (CCAAT box)."

##### "GC box" and "CAAT box"

Google Scholar search About 2,760 results (0.07 sec):
* See number 5 just above.
* "A DNA control sequence $_T$$^G$GGGCGG$_{AAT}$$^{GGC}$, or the “GC” box, has been described in the promoter regions upstream of a number of eukaryotic genes transcribed by polymerase II (for review, see Dynan, W. S. and Tjian, R.,Nature316:774, 1985). The “GC” box can occur in single or multiple copies and is the binding site for a protein factor, Spl, which activates initiation of transcription. [In] the presence of the tk promoter, which was shown to be weaker in vitro than the protamine promoter (12), the difference between the silencing effect of the GC box alone [...] and GC and CAAT box together [...] is more pronounced".

##### "GC box" "C box" "D box"

The other C-box and D-box have not been tested.
Google scholar search results: About 16 results (0.09 sec).
* "The FCD fragment contains 7 conserved sequences which are present in the mPl and mP2 genes: the F, C, and D boxes [...]. The bull protamine 1 (bPl) gene is post-meiotmcally expressed [23, 24] like the mPl gene, but lacks the F and C boxes and [...] has a modified D box [25, 26]. [...] The rP2 gene contains the D box and modified F and C boxes [...]. Part of the 122-bp fragment (containing the C and D boxes [...] but excluding the F fragment, which contains the F box and [...] is necessary for the putative negative factor binding. This indicates that one negative factor binding site is contained, either partially or totally, in the CD region of the FCD fragment." TGGGCA is a B-box, GAGGCCATCT is a C-box, TCTCACATT(A/C)AATAAGTCA is a D-box, CCTCACAGA is an E-box. "There are also several additional nucleotide changes in the rP2 gene, two in the GC box and one between the E and B boxes". "The mP2 EB fragment used for binding was the 118 nucleotide fragment extending from the Dde I site at position -140 to the Dde I site at position -23 [...]. This fragment contains the GC, E, B, CAAT, and TATA boxes."
* The GC boxes are not apparently associated with the A box, B box, C box, and D box.
* The human ribosomal protein L11 gene (HRPL11) has an NF1 beginning at -92 (TGCGCC), an AP2 beginning at -59 (TTAGCC), a GC box beginning at -45 (GAGCCC), a GA-binding protein (GABP) beginning at -13 (CGGAAG), another GC box beginning at +100 (CGCCCGC), an M-CAT beginning at +113 (AGGAATA), another GC box beginning at +124 (GCCCGCA), two potential snRNA-coding sequences in intron 4: the C box beginning at +4131 (GGTGATG), 18S RNA beginning at +4143 (GTTTGCTC), an H box beginning at +4225 (CTAAATC), a D box beginning at +4237 (TCCTG), 28S RNA beginning at +4251 (GAACCTGAAAG), and an ACA box beginning at +4329 (CACA), and no TATA or CAAT boxes (TATA-like element ATAA was found in region −24...–27). "Identification of specific regions (C/D and H/ACA boxes [33]) and sequences homologous to rRNA [34] suggested two possible variants of a snRNA-coding sequence for HRPL11 intron 4 [...]."

##### "GC box" and "CARE"

Google Scholar: Your search - "GC box" "CARE" -"care" - did not match any articles.

##### "GC box" and "CArG box"

* For "direct muscle-specific transcription of chimeric chloramphenicol acetyltransferase (CAT) gene constructs [...] the transcriptional start site [...] lies 37 base pairs (bp) upstream of the 5' end [...] in addition to an ATA and GC box, this region contains domains that have been implicated in the regulation of other muscle-specific genes: a CArG box at -91 bp; myocyte-specific enhancer-binding nuclear factor 1 binding site homologies at -58, -535, and -583 bp; and a muscle-CAAT consensus sequence at -394 bp relative to the cap site. [...] The ATA box [AATAT], GC box [GGCGGG], CArG box [CCTATTATGC], and MCAT [CATTCCT] consensus sequences [are] described".
* "The ATA box [AAATAT], GC box [GGCGGG], CArG box [CCTATTATGCG], [two E boxes CAGTTG] and M-CAT [CATTCCT] consensus sequences are [described from the mouse dystrophin promoter]."

##### "GC box" and "CRE box"

Google scholar search 6 results (0.03 sec):
* "The CpG dinucleotides within a fragment of the [mouse lactate dehydrogenase c gene] mldhc promoter containing a GC box and tandem activating transcription factor/cAMP-responsive element [CRE] binding sites are hypermethylated in somatic tissues and hypomethylated in testis." "Three of the CpGs were located within putative transcription factor binding sites including a GC box located at −70 bp 5′ to the transcription start site and tandem near-consensus activating transcription factor/cAMP-responsive elements (ATF/CREs) at −53 bp and −39 bp. The location of these elements relative to each other and to the transcription start site is of particular interest in light of the recent finding by Iannello et al. [4] that somatic repression of the testis-specific Pdha-2 gene occurs through targeting of an ATF/CRE. The ATF/CRE site within the Pdha-2 promoter is located at −62 bp, similar to the placement of the ATF/CREs in the mldhc promoter. In addition, both the Pdha-2 and mldhc promoters contain a GC box, a consensus binding site for transcription factor Sp1, upstream of their ATF/CREs, separated by 12 bp and 11 bp, respectively." GC box beginning at -70 bp is (GGGTGG) and 5' ATF/CRE beginning at -53 bp is (TGATGT).

##### "GC box" and "E box"

* "The ATA box [AAATAT], GC box [GGCGGG], CArG box [CCTATTATGCG], [two E boxes CAGTTG] and M-CAT [CATTCCT] consensus sequences are [described from the mouse dystrophin promoter]."

##### "GC box" and "BRE$^u$"

Google scholar search About 34 results (0.05 sec):
* "Deletions of the GC‐box (ΔB) and TFIIB recognition element (BREu; ΔD) however, result in a significant reduction of P6 activity in Cor‐1 cells only. There is therefore a cell‐type‐specific requirement for the GC‐box and BREu to drive full DAGLα promoter activity."

##### "GC box" and "HNF6"

Google scholar search About 26 results (0.06 sec):
* HNF4a has a consensus sequence of (CARRGKBCAAAGT­YCA) and HNF6 has a consensus sequence of (NTATYGATCH) for the top "enriched promoter motifs in promoters from TIMP4-regulated genes." The GC box is mentioned in a reference only with respect to "17β-Estradiol sup­presses MHC class I chain-related B gene expression via an intact GC box."
* "Hex expression in avian anterior lateral endoderm is regulated by autocrine [bone morphogenetic protein] BMP signaling. Characterization of the mouse Hex gene promoter identified a 71-nucleotide BMP-responsive element (BRE) that is required for up-regulation of Hex by an activated BMP signaling pathway." "The BRE contains two copies of a GCCGnCGC-like motif that in Drosophila is the binding site for Mad and Madea followed by two CAGAG boxes that are similar to sequences required for transforming growth factor-ß/activin responsiveness of several vertebrate genes." "Analysis of Hex promoter sequence between -493 and -321 revealed several motifs showing homology to previously identified Smads binding elements [...]. Two GC-rich elements, located at -448 (GC1) and -428 (GC2), show homology with the sequence GCCGnCGC that has been shown to bind Drosophila Mad and Madea and mammalian Smad1 and Smad4 (9, 11). GC1 contains two nucleotide differences, and GC2 contains a single nucleotide difference, from the Drosophila consensus sequence. GC2 is also identical to an element that is responsible for BMP responsiveness of the mouse Smad6 promoter (13). Two CAGAG boxes (CA1 and CA2) located at -411 and -385 are similar to Smad binding elements found in human and Xenopus BMP-responsive gene promoters (7, 15, 16)." GC box 1 (GC1) is (GCCGCCCG) and GC box 2 (GC2) is (GCCGGCGGC). "The Hex BRE contains two GC-rich elements and two CAGAG boxes that act in concert to bind Smad4 and complexes of Smad1 and Smad4 to activate transcription." "Hex is required for liver development as knock-out mice show greatly reduced expression of transcription factors such as Hnf3b, Hnf6, Hnf4α, and Hnf1 and fail to express liver-specific genes such as α-fetoprotein or serum albumin (41, 43)."

##### "GC box" and "HY box"

Google scholar search Your search - "GC box" and "HY box" - did not match any articles.

##### "GC box" and "MRE"

Google scholar search About 253 results (0.09 sec):
* In the top above diagram, the labels represent: dBLE, distal basal-level element; pBLE, proximal basal-level element; GC, GC-box element; MRE, metal-responsive element; GRE, glucocorticoid-responsive element; TATA, "TATA box."
* In the top, second down diagram: "Nucleotide sequences of the P. ostreatus poxc (A) and poxa1b (B) promoter regions, extending about 400 nt upstream of the start codon (ATG). Transcription-initiation sites are indicated by vertical arrows. The putative TATA box, GC box, MREs, heat-shock elements (HSEs) and xenobiotic-responsive elements (XREs) are underlined. poxc MREs are named cMREs, whilst poxa1b MREs are named a1bMREs. In each promoter, the MRE sites are numbered according to their proximity to the start codon. The orientation of MREs is indicated by arrows. Oligonucleotides used for probe amplifications are also indicated. MRE sequences identified by footprinting analyses are boxed."

##### "GC box" and "Pyrimidine box"

Google scholar results About 21 results (0.05 sec):
* "Upstream from the transcriptional start site, several motifs were found [...]. A typical TATA box is located at -43. The CAAT consensus sequence cannot be found between -80 and -120; however, two sequence motifs (GCGCCC, GGGCAG), which are homologous to the consensus sequences for the Spl-binding site, GGGCGG (GC box) [19] were found around -114 and -570. The GC box has been found in promoters of many viral and cellular genes [20], and acts as a binding site of a protein, Spl, which is necessary for transcriptional activity. A pyrimidine box (CCTTT) and Box I (GCAGTG) which are part of the GA response complex [21] were found at -208 and -256. Two 8 bp sequences (CACGTCGC, CACGTAAC) which are similar to an ABA response element (ABRE, CACGTGGC) [22] were located at -308, -648 relative to the + 1 site. The core sequence of the ABA response element (ACGT) is the binding site for basic leucine zipper transcriptional factors or common plant regulatory factors (CPRFs) [23]."

##### "GC box" and "STAT"

Google scholar results About 2,170 results (0.08 sec):
* "This gene encodes a member of the STAT-induced STAT inhibitor (SSI), also known as suppressor of cytokine signaling (SOCS), family. SSI family members are cytokine-inducible negative regulators of cytokine signaling. The expression of this gene can be induced by a subset of cytokines, including IL2, IL3 erythropoietin (EPO), CSF2/GM-CSF, and interferon (IFN)-gamma. The protein encoded by this gene functions downstream of cytokine receptors, and takes part in a negative feedback loop to attenuate cytokine signaling. Knockout studies in mice suggested the role of this gene as a modulator of IFN-gamma action, which is required for normal postnatal growth and survival."
* Transcription "of the mouse [STAT-induced STAT inhibitor-1] SSI-1 gene was initiated from six adjoining sites accompanying three GC boxes and a single GC box-like element near them, but not from the TATA box or an initiator sequence." "Consensus sequences of GC box (GGGCGG), GC box-like element (GGGTGG), and GAS element (TTC(N)$_{3–4}$GAA) [occur]. Four GAAA units are indicated by boxes and named G1–G4."

##### "GC box" and "TATA box"

Google scholar search results About 6,270 results (0.09 sec):
* "During productive infection, human cytomegalovirus (HCMV) UL44 transcription initiates at three distinct start sites that are differentially regulated. Two of the start sites, the distal and the proximal, are active at early times [...]. The UL44 early viral gene product is essential for viral DNA synthesis. [...] The UL44 early viral promoters have a canonical TATA sequence, “TATAA.”"

##### "GC box" and "TAT box"

Google scholar 7 results (0.05 sec): but none contain the TAT box, instead misprints; e.g., "TAT?, box", "TAT#, box", or "T TA box".

##### "GC box" and "W box"

Google scholar results About 105 results (0.10 sec):
Characteristics of sequences from some rye simple sequence repeat (SSR) markers :
| SSR marker | Sequence name | Base pairs | Characteristics of sequence | GenBank acc. No. | Material cloned |
| Xgwm259 | pMD259-1000 | 893 | TATA box, CAAT box, GC box, W box, and AATAAA polyadenylation | EF535855 | Secale cereale 'Kustro' |
| Xgwm260 | pMD260-1000-1 | 988 | GC box and W-box | EF535876 | Secale cereale 'Kustro' |
| Xgwm260 | pMD260-1000-2 | 990 | GC box and W-box | EF535877 | Secale cereale 'AR106BONE' |
Apparently, an AGC box, ATA box, CAAT box, C box or D box not tested yet, C and D box, CArG box, CRE (or CRE box), E box, BRE$^u$, HNFs, MRE, pyrimidine box, STATs, TATA box in the distal promoter, and W box can interact with a GC box to transcribe A1BG.

### Conclusions

Because of the presence of at least one GC box between ZSCAN22 and A1BG, A1BG could be transcribed by a GC box with or without other TFs to assist or be assisted.
The key to assisting the recovery of any astronaut now depends on which means of transcription best moderates the effects of microgravity or irradiation. This may require extensive molecular genetic testing.

## Laboratory evaluations

To assess your example, including your justification, analysis and discussion, I will provide such an assessment of my example for comparison and consideration.
Evaluation
No wet chemistry experiments were performed to confirm that Gene ID: 1 may be transcribed from either side using transcription factors in the core, proximal or distal promoters. The NCBI Gene database is generalized, whereas individual human genome testing could demonstrate that A1BG is transcribed from either side using known transcription factors. Sufficient nucleotides have been added to the data sets for the ZNF497 side to confirm likely transcription of A1BG by these known transcription factors.