# Gene transcriptions/HNF6s/Laboratory

A laboratory is a specialized activity where 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, expensive equipment, instruments, and/or machinery are available for taking the entity apart to see and accurately record how it works, what it's made of, and where it came from. This may involve simple experiments to test reality, collect data, and attempts to make some sense out of it.
Expensive equipment can be replaced or substituted for with more readily available tools.

## Notations

You are free to create your own notation or use that already presented. A method to statistically assess your locator is also needed.

## Laboratory control group

A laboratory control group of some large number of laboratory test subjects or results may be used to define normal limits for the presence of an effect.

## Instructions

This laboratory is an activity for you to explore the universe for, to create a method for, or to examine. While it is part of the {{Gene project}}, it is also independent.
Some suggested entities to consider are
* available classification,
* human genes,
* eukaryotes,
* nucleotides,
* classical physics quantities,
* known gene expressions, or
* geometry.
More importantly, there are your entities.
You may choose to define your entities or use those already available.
Usually, research follows someone else's ideas of how to do something. But, in this laboratory you can create these too.
This is a gene project laboratory, but you may create what a laboratory, or a {{Gene project}} is.
This laboratory is structured.
I will provide an example. The rest is up to you.
Questions, if any, are best placed on the Discuss page.
To include your participation in each of these laboratories create a subpage of your user page once you register at wikiversity and use this subpage, for example, your online name/laboratory effort.
Enjoy learning by doing!

## Hypotheses

* HNF6s have a role as downstream signal transducers in A1BG.
* A1BG is not transcribed by any HNF6s.
* HNF6s may assist transcription of A1BG by other transcription factors.

## Introduction

Hepatic nuclear factors (HNFs) bind through their DNA-binding domain (DBD) to consensus elements (A/G/T)(A/T)(A/G)T(C/T)(A/C/G)AT(A/C/G/T)(A/G/T), resulting in gene transcription.
HNF6 is actually produced by GeneID: 3175 ONECUT1 one cut homeobox 1: "This gene encodes a member of the Cut homeobox family of transcription factors. Expression of the encoded protein is enriched in the liver, where it stimulates transcription of liver-expressed genes, and antagonizes glucocorticoid-stimulated gene transcription. This gene may influence a variety of cellular processes including glucose metabolism, cell cycle regulation, and it may also be associated with cancer. Alternative splicing results in multiple transcript variants."
Both "the 2.3 kb and the 160 bp proximal parts of the a1bg promoter direct sex-specific expression of the reporter gene, and that a negative regulatory element resides in the −1 kb to −160 bp region."
"Computer analysis of the 2.3 kb rat a1bg promoter fragment revealed [...] one HNF6/HNF3 binding site at [...] −137/−128 [...]."
The "GH-dependent sexually dimorphic expression conveyed by the 2.3 kb a1bg promoter is enhanced by the HNF6/HNF3 site [...]."
The "binding of [...] HNF6 to the respective site by electromobility shift analysis (EMSA) [was verified] using female-derived [rat] liver nuclear extracts. [...] HNF6 bound to the a1bg HNF6 oligonucleotide, but in this case, the mutated oligonucleotide was able to compete for binding when added in large excess [...]. However, [...] the HNF6 binding capacity of the mutated oligonucleotide was clearly reduced. A 20 molar excess of the mutated oligonucleotide had only a marginal effect on the binding of HNF6 [...], whereas a 20 molar excess of unlabelled probe [...] completely abolished binding. Supershift analysis with an HNF6 antibody revealed a complex with a slightly lower mobility than the HNF6 complex [...]. By extending the electrophoresis run and including nuclear extract from hypophysectomized rats, devoid of GH and thereby lacking HNF6 (Lahuna et al. 1997), the two different complexes were clearly visualized. The complex with the lower mobility is most probably due to the binding of HNF3, in analogy with what was shown by Lahuna et al. for the CYP2C12 HNF6 binding site; HNF3 can bind to the site in the absence of HNF6 (Lahuna et al. 1997). [...] HNF6 could bind to [its] respective site in the a1bg promoter in vitro, and the mutations introduced in respective site abolished binding of the corresponding factor."
The "expression of a −116/−89 deletion construct in which also the HNF6 site was mutated, (−116/−89) delmutHNF6-Luc, [...] the generated luciferase activities were reduced in both sexes [...]. This is in contrast to that mutation/deletion of the sites separately only affected the expression in female livers."
The "−116/−89 region contains a site(s) of importance for the GH-dependent and female-specific expression of the a1bg gene, and that the impact of this region together with the HNF6 site is more complex than mere enhancement of the expression in females."
HNF6 is expressed at higher levels in female than in male rat liver (Lahuna et al. 1997). Indeed, following mutation of the HNF6-binding element, mutHNF6-Luc, the sex-differentiated expression was attenuated due to reduced expression in females. Thus, for a1bg, the sex-related difference in amount of HNF6 is likely to contribute to the sex-differentiated and female characteristic expression."
Nuclear "proteins binding to the a1bg −116/−89 region [are] members of the [nuclear factor 1] NF1 and the [octamer transcription factor] Oct families of transcription factors. NF1 genes are expressed in most adult tissues (Osada et al. 1999). It is not known how NF1 modulates transcriptional activity, and both activation and repression of transcription have been reported (Gronostajski 2000). Cofactors such as [CREB binding protein] CBP/p300 [E1A binding protein p300] and [histone deacetylase] HDAC have been shown to interact with NF1 proteins suggesting modulation of chromatin structure (Chaudhry et al. 1999). NF1 factors have also been shown to interact directly with the basal transcription machinery as well as with other transcription factors, including Stat5 (Kim & Roeder 1994, Mukhopadhyay et al. 2001) and synergistic effects with HNF4 have been reported (Ulvila et al. 2004). In addition to the HNF6, Stat5 and NF1/Oct sites, the a1bg promoter harbours an imperfect HNF4 site at −51/−39 with two mismatches compared with the HNF4 consensus site. HNF4 is clearly important for the expression of CYP2C12 (Sasaki et al. 1999), however, the −51/−39 region in a1bg was not protected in the footprinting analysis and was therefore not analysed further. Like NF1, Oct proteins have been reported to be involved in activation as well as repression of gene expression (Phillips & Luisi 2000). [...] NF1 and Oct-1 have been shown to, reciprocally, facilitate each other’s binding (O’Connor & Bernard 1995, Belikov et al. 2004)."
In the diagram on the right is liver "expression of a1bg-luciferase constructs. (A) Stat5 and HNF6 consensus sequences and corresponding sites in the 2.3 kb a1bg promoter alongside with the used mutations. (B) Female (black bars) and male (open bars) rats [results]."

## Core promoters

[[Imagew:Core_promoter_elements.svg|link=https://en.wikiversity.org/wiki/File:Core_promoter_elements.svg%7Cright%7Cthumb%7C300x300px%7CThe diagram shows an overview of the four core promoter elements B recognition element (BRE), TATA box, initiator element (Inr), and downstream promoter element (DPE), with their respective consensus sequences and their distance from the transcription start site. Credit: Jennifer E.F. Butler & James T. Kadonaga.]] 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

Once you've decided on an entity, source, or object, compose a method, way, or procedure to explore it.
One way is to perceive (see, feel, hear, taste, or touch, for example) if there are more than one of them.
Ask some questions about it.
Does it appear to have a spatial extent?
Is there any change over time?
Can it be profiled with a kind of spectrum for example, by emitted radiation? Sample by plotting two or more apparent variables against each other, like intensity versus wavelength.
Is there some location, time, intensity, where there isn't one?
Regarding hypothesis 1: HNFs have a role as downstream signal transducers in A1BG.
Def. a series of chemical reactions within a cell which start when a transmembrane protein comes into contact with a chemical signal, resulting in a second messenger being triggered is called a signal transduction.
A transcription activator, or activator of transcription, may be a transcription factor that increases gene transcription, where most bind to enhancers or proximal promoter elements.
HNFs may have a downstream proximal promoter element. "Downstream" can refer to downstream from an enhancer but before the transcription start site, downstream from a TATA box or an initiator element but before the transcription start site (TSS), downstream from another promoter element and containing the TSS, or downstream after the TSS.
As is shown in the computer programming sampling is performed to 100 nts past the TSS.
Regarding hypotheses 2: A1BG is not transcribed by any HNF6s.
For the Basic programs (starting with SuccessablesHNF6.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, and found:
* negative strand in the negative direction (from ZSCAN22 to A1BG) is SuccessablesHNF6--.bas, looking for 3'-(A/G/T)(A/T)(A/G)T(C/T)(A/C/G)AT(A/C/G/T)(A/G/T)-5', 3, 3'-GTGTTAATAA-5', 1725, 3'-TAGTTGATAA-5', 3527, 3'-TTATTAATCG-5', 4229,
* negative strand in the positive direction (from ZNF497 to A1BG) is SuccessablesHNF6-+.bas, looking for 3'-(A/G/T)(A/T)(A/G)T(C/T)(A/C/G)AT(A/C/G/T)(A/G/T)-5', 3, 3'-ATGTCCATGG-5', 3581, 3'-TTATTAATCA-5', 4147, 3'-TTATTGATTA-5', 4164,
* positive strand in the negative direction is SuccessablesHNF6+-.bas, looking for 3'-(A/G/T)(A/T)(A/G)T(C/T)(A/C/G)AT(A/C/G/T)(A/G/T)-5', 1, 3'-AAATTGATAA-5', 3361,
* positive strand in the positive direction is SuccessablesHNF6++.bas, looking for 3'-(A/G/T)(A/T)(A/G)T(C/T)(A/C/G)AT(A/C/G/T)(A/G/T)-5', 1, 3'-GAGTCCATTG-5', 3732,
* complement, negative strand, negative direction is SuccessablesHNF6c--.bas, looking for 3'-(A/C/T)(A/T)(C/T)A(A/G)(C/G/T)TA(A/C/G/T)(A/C/T)-5', 1, 3'-TTTAACTATT-5', 3361,
* complement, negative strand, positive direction is SuccessablesHNF6c-+.bas, looking for 3'-(A/C/T)(A/T)(C/T)A(A/G)(C/G/T)TA(A/C/G/T)(A/C/T)-5', 1, 3'-CTCAGGTAAC-5', 3732,
* complement, positive strand, negative direction is SuccessablesHNF6c+-.bas, looking for 3'-(A/C/T)(A/T)(C/T)A(A/G)(C/G/T)TA(A/C/G/T)(A/C/T)-5', 3, 3'-CACAATTATT-5', 1725, 3'-ATCAACTATT-5', 3527, 3'-AATAATTAGC-5', 4229,
* complement, positive strand, positive direction is SuccessablesHNF6c++.bas, looking for 3'-(A/C/T)(A/T)(C/T)A(A/G)(C/G/T)TA(A/C/G/T)(A/C/T)-5', 3, 3'-TACAGGTACC-5', 3581, 3'-AATAATTAGT-5', 4147, 3'-AATAACTAAT-5', 4164,
* inverse complement, negative strand, negative direction is SuccessablesHNF6ci--.bas, looking for 3'-(A/C/T)(A/C/G/T)AT(C/G/T)(A/G)A(C/T)(A/T)(A/C/T)-5', 2, 3'-ACATGGACAT-5', 802, 3'-TAATGAACTT-5', 1301,
* inverse complement, negative strand, positive direction is SuccessablesHNF6ci-+.bas, looking for 3'-(A/C/T)(A/C/G/T)AT(C/G/T)(A/G)A(C/T)(A/T)(A/C/T)-5', 1, 3'-TTATTGATTA-5', 4164,
* inverse complement, positive strand, negative direction is SuccessablesHNF6ci+-.bas, looking for 3'-(A/C/T)(A/C/G/T)AT(C/G/T)(A/G)A(C/T)(A/T)(A/C/T)-5', 3, 3'-AAATTGATAA-5', 3361, 3'-TCATCAACTA-5', 3525, 3'-TTATTAATTC-5', 4542,
* inverse complement, positive strand, positive direction is SuccessablesHNF6ci++.bas, looking for 3'-(A/C/T)(A/C/G/T)AT(C/G/T)(A/G)A(C/T)(A/T)(A/C/T)-5', 2, 3'-CCATTGACTC-5', 3736, 3'-ATATTAACAA-5', 4172,
* inverse, negative strand, negative direction, is SuccessablesHNF6i--.bas, looking for 3'-(A/G/T)(A/C/G/T)TA(A/C/G)(C/T)T(A/G)(A/T)(A/G/T)-5', 3, 3'-TTTAACTATT-5', 3361, 3'-AGTAGTTGAT-5', 3525, 3'-AATAATTAAG-5', 4542,
* inverse, negative strand, positive direction, is SuccessablesHNF6i-+.bas, looking for 3'-(A/G/T)(A/C/G/T)TA(A/C/G)(C/T)T(A/G)(A/T)(A/G/T)-5', 2, 3'-GGTAACTGAG-5', 3736, 3'-TATAATTGTT-5', 4172,
* inverse, positive strand, negative direction, is SuccessablesHNF6i+-.bas, looking for 3'-(A/G/T)(A/C/G/T)TA(A/C/G)(C/T)T(A/G)(A/T)(A/G/T)-5', 2, 3'-TGTACCTGTA-5', 802, 3'-ATTACTTGAA-5', 1301,
* inverse, positive strand, positive direction, is SuccessablesHNF6i++.bas, looking for 3'-(A/G/T)(A/C/G/T)TA(A/C/G)(C/T)T(A/G)(A/T)(A/G/T)-5', 1, 3'-AATAACTAAT-5', 4164.
Regarding hypothesis 3: HNF6s may assist transcription of A1BG by other transcription factors.
"NF1 factors have also been shown to interact directly with the basal transcription machinery as well as with other transcription factors, including Stat5 (Kim & Roeder 1994, Mukhopadhyay et al. 2001) and synergistic effects with HNF4 have been reported (Ulvila et al. 2004)."
"NF1 and Oct-1 have been shown to, reciprocally, facilitate each other’s binding (O’Connor & Bernard 1995, Belikov et al. 2004)."

## 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 HNF6s and putting the sequences in "⌘F" locates these sequences in the same nucleotide positions as found by the computer programs.

## Core promoters HNF6s

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 HNF6s in the core promoter between 4425 and 4460.
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 HNF6s in the core promoter between 4266 and 4300.

## Proximal promoter HNF6s

The proximal promoter begins about nucleotide number 4210 in the negative direction.
There is one HNF6 in the proximal promoter between 4210 and 4460: 3'-TTATTAATCG-5' at 4229.
The proximal promoter begins about nucleotide number 4050 in the positive direction.
There two HNF6s in the proximal promoter between 4050 and 4300, 3'-TTATTGATTA-5' at 4164 and 3'-TATAATTGTT-5' at 4172.

## Distal promoter HNF6s

Using an estimate of 2 knts, a distal promoter to A1BG would be expected after nucleotide number 2460.
There are the following HNF6s on the negative strand, negative direction: 3'-TAGTTGATAA-5' at 3527 and 3'-TTTAACTATT-5' at 3361. And, their complements on the positive strand, negative direction: 3'-AAATTGATAA-5' at 3361, 3'-AAGGGACTT-5' at 3782.
Distal HNF6s in the positive direction, if they exist, would be inside ZNF497 or beyond, e.g., 3'-ATGTCCATGG-5' at 3581, with complements 3'-GAGTCCATTG-5' at 3732 and 3'-GGTAACTGAG-5' at 3736, on the positive strand.

## Transcribed HNF6s

"The locations of [nuclear factor I] NFI-binding sites, [glucocorticoid response elements] GREs, and [mammary gland factor] MGF (HNF6)-binding sites in the distal promoter region are as described in references 28 and 29. The underlined nucleotides indicate the palindromic nature of the MGF-binding sites."
"The distal region (−830 to −720 bp) of the rat whey acidic protein (WAP) gene contains a composite response element (CoRE), which has been demonstrated previously to confer mammary gland-specific and hormonally regulated WAP gene expression. Point mutations in the binding sites for specific transcription factors present within this CoRE have demonstrated the importance of both nuclear factor I (NFI) and STAT5 as well as cooperative interactions with the glucocorticoid receptor (GR) in the regulation of WAP gene expression in the mammary gland of transgenic mice."

## 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.
HNF6 transcription laboratory
by --Marshallsumter (discuss • contribs) 02:47, 28 October 2017 (UTC)

### Abstract

Three hypotheses have been examined: (1) HNF6s have a role as downstream signal transducers in A1BG, (2) alpha-1-B glycoprotein (A1BG) is not transcribed by any HNF6s, and (3) HNF6s may assist transcription of A1BG by other transcription factors. These have been tested by literature searching articles that report HNF6s in the promoter region of a particular human gene and by using a simple computer program to look for HNF6s in the nucleotide sequences on either side of the A1BG gene. Both the template DNA strand and the coding strand have been checked. To show that these HNF6s can be used during or for transcription of A1BG at least one transcription factor has been found.

### Introduction

According to one source, A1BG is transcribed from the direction of ZNF497: 3' - 58864890: CGAGCCACCCCACCGCCCTCCCTTGG$_{+1}$GGCCTCATTGCTGCAGACGCTCACCCCAGACACTCACTGCACCGGAGTGAGCGCGACCATCATG : 58866601-5', per Michael David Winther, Leah Christine Knickle, Martin Haardt, Stephen John Allen, Andre Ponton, Roberto Justo De Antueno, Kenneth Jenkins, Solomon O. Nwaka, and Y. Paul Goldberg, Fat Regulated Genes, Uses Thereof and Compounds for Mudulating Same, US Patent Office, July 29, 2004, at http://www.google.com/patents?hl=en&lr=&vid=USPATAPP10416914&id=7iaVAAAAEBAJ&oi=fnd&printsec=abstract#v=onepage&q&f=false where the second 'G' at left of four Gs in a row is the TSS. Transcription was triggered in cell cultures and the transcription start site was found using reverse transcriptase. But, the mechanism for transcription is unknown.
Controlling the transcription of A1BG may have significant immune function against snake envenomation. A1BG forms a complex that is similar to those formed between toxins from snake venom and A1BG-like plasma proteins. These inhibit the toxic effect of snake venom metalloproteinases or myotoxins and protect the animal from envenomation.
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) CArG boxes, (4) enhancer boxes, (5) HY boxes, (6) metal responsive elements (MREs), and (7) STAT5s.

#### AGC boxes (GCC boxes)

An AGC box was found in the distal promoter of either gene ZSCAN22 or A1BG on both the template and coding strands. But, as the only known transcription of A1BG occurs between Gene ID: 162968 ZNF497 and Gene ID: 1 A1BG, it is unlikely that this AGC box is naturally used to transcribe A1BG.
A full web search produced several references including a GeneCard for "zinc finger protein 497" and "GCC box", including "May be involved in transcriptional regulation." Zinc fingers are mentioned in association with GCC boxes in plants. It seems unlikely that an AGC box is involved in any way with the transcription of A1BG.
An extension of the nucleotide data for the positive direction from ZNF475 toward A1BG from 958 nts to 4445 nts has not discovered any AGC boxes even in the distal promoter just beyond ZNF497.

#### ATA boxes

Regarding hypothesis 1: there are no ATA boxes in the core promoter of A1BG from either direction or strand.
This hypothesis has been shown to be true.
A corollary hypothesis might be 1.1: there are no ATA boxes in the proximal promoter of A1BG from either direction or strand.
This corollary hypothesis may be true. "The analysis of the promoter region indicated that a putative ATA box is located 54 nucleotides upstream from the transcription start site". There is one inverse and inverse complement ATA box in the proximal promoter in the positive direction between 4050 and 4300: 3'-AAATAA-5' at 4142, and 3'-TTTATT-5' at 4142. As the TSS is at 4300 nts, this ATA box is some 158 nts away, where with the smaller data set 3'-TTTATT-5' was at 703. As the TSS is at 858 nts, this ATA box is some 155 nts away, which is approximately the same number of nts from the TSS but not close enough to be in the core promoter and not 54 nts upstream from the TSS or to match other such genes with an ATA box.
But the ATA box at 2347 is likely involved in transcription of A1BG in analogy to the rat. Although this has not been confirmed as involved, the existence of this ATA box likely proves hypothesis 1 false.
Regarding hypothesis 2: ATA boxes have a role as downstream signal transducers in A1BG.
There is the following inverse ATA box on the negative strand, negative direction: 3'-AAATAA-5' at 4537. On this strand, in this direction the TSS is at 4460 nts from ZSCAN22. This ATA box is 77 nts downstream. So far no published research has been found to verify this type of downstream promoter or enhancer ATA box. There may be another isoform TSS nearby. As such, hypothesis 2 may be true.
Regarding hypothesis 3: ATA boxes may assist transcription of A1BG by other transcription factors.
This hypothesis has been shown by literature search to be true. But, none of the ATA boxes for A1BG are close enough to any STAT5 promoter to match known transcription initiation.

#### CArG boxes

By combining a literature search with computer analysis of each promoter between ZSCAN22 and A1BG and ZNF497 and A1BG, CArG boxes have been found. To show that these CArG boxes may be used during or for transcription of A1BG at least one transcription factor has been affirmed.
A literature search of more recent results discovered: "Of the [Flowering Locus C] FLC binding sites, 69% contained at least one CArG-box motif with the core consensus sequence CCAAAAAT(G/A)G and an AAA extension at the 3′ end [. Three] other MADS-box flowering-time regulators, SOC1, SVP, and AGAMOUS-LIKE 24 (AGL24), bind to two different CArG-box motifs at 502 bp (CTAAATATGG) and 287 bp (CAATAATTGG) upstream of the translation start in the SEP3 gene (24), consistent with different specificities for the different MADS-box proteins."
These together with the core motif CC(A/T)$_6$GG suggest a more general CArG-box motif of (C(C/A/T)(A/T)$_6$(A/G)G). Subsequent computer-program testing revealed two more general CArG boxes: 3'-CAAAAAAAAG-5' at 1399 nts from ZSCAN22 and 3'-CATTAAAAGG-5' at 3441 nts from ZSCAN22, but none within 4300 nts toward A1BG from ZNF497.
These results show that the presence of CArG boxes on the ZSCAN22 side of A1BG implies their use when transcribing A1BG, although they may be pointing toward ZSCAN22. These suggest that the hypothesis (A1BG is not transcribed by a CArG box) is false. Regarding the second hypothesis (The lack of a CArG box on either side of A1BG does not prove that it is not actively used to transcribe A1BG), the presence of more general CArG boxes in the distal promoter tentatively confirms this hypothesis.
CArG boxes do occur in the distal promoter of A1BG on the ZSCAN22 side only. And, it is likely that a CArG box is involved in some way with the transcription of A1BG.

#### Enhancer boxes

The presence of many enhancer boxes on both sides of A1BG demonstrate that the hypothesis: "A1BG is not transcribed by an enhancer box", is false.
The finding by literature search of evidence verifying that at least one transcription factor can enhance or inhibit the transcription of A1BG using one or more enhancer boxes disproves the hypothesis: "Existence of an enhancer box on either side of A1BG does not prove that it is actively used to transcribe A1BG".
Enhancer boxes do occur in the proximal and distal promoters of A1BG. And, it is likely that an enhancer box is involved in some way with the transcription of A1BG.

#### HY boxes

HY boxes were not found in either core promoters or the proximal promoters in either direction. However, HY boxes were found in the distal promoters on both sides of A1BG. No genes are described in the literature so far as transcribed from HY boxes in any distal promoters.
Either A1BG can be transcribed by HY boxes in the distal promoter, or A1BG is not transcribed by HY boxes. As the literature appears absent from a Google Scholar advanced search to confirm possible transcription from distal promoters, wet chemistry experiments are needed to test the possibility.

#### Metal responsive elements

By combining a literature search with computer analysis of the promoter between ZSCAN22 and A1BG and ZNF497 and A1BG, metal responsive elements have been found. Literature search has also discovered at least three post-translational isoforms including the unaltered precursor. Although no metal responsive elements overlap any enhancer boxes in the distal promoter, there are elements in the distal promoter.
"The human genome is estimated to contain 700 zinc-finger genes, which perform many key functions, including regulating transcription. [Four] clusters of zinc-finger genes [occur] on human chromosome 19".
Nearby zinc-fingers on chromosome 19 include ZNF497 (GeneID: 162968), ZNF837 (GeneID: 116412), and ZNF8 (GeneID: 7554).
"In rodents and in humans, about one third of the zinc-finger genes carry the Krüppel-associated box (KRAB), a potent repressor of transcription (Margolin et al. 1994), [...]. There are more than 200 KRAB-containing zinc-finger genes in the human genome, about 40% of which reside on chromosome 19 and show a clustered organization suggesting an evolutionary history of duplication events (Dehal et al. 2001)."
ZNF8 is in cluster V along with A1BG.
"In contrast to the four clusters considered [I through IV], one that occurs at the telomere of chromosome 19, which we will call cluster V, has been very stable [over mouse, rat, and human]."
"Apart from the somewhat unexpected location of Zfp35 on mouse chromosome 18 and of the AIBG orthologs on mouse chromosome 15 and rat chromosome 7, there has been little rearrangement."
So far no article has reported any linkage between zinc, including various zinc fingers, or cadmium, and A1BG.
Regarding additional isoforms, mention has been made of "new genetic variants of A1BG."
"Proteomic analysis revealed that [a circulating] set of plasma proteins was α 1 B-glycoprotein (A1BG) and its post-translationally modified isoforms."
Pharmacogenomic variants have been reported. There are A1BG genotypes.
A1BG has a genetic risk score of rs893184.
"A genetic risk score, including rs16982743, rs893184, and rs4525 in F5, was significantly associated with treatment-related adverse cardiovascular outcomes in whites and Hispanics from the INVEST study and in the Nordic Diltiazem study (meta-analysis interaction P=2.39×10$^{−5}$)."
"rs893184 causes a histidine (His) to arginine (Arg) [nonsynonymous single nucleotide polymorphism (nsSNP), A (minor) for G (major)] substitution at amino acid position 52 in A1BG."
For example, GeneID: 9 has isoforms: a, b, X1, and X2. Each of these (a and b) have variants. Variants 1-6 and 9 all encode the same isoform (a).
Variants 7, 8 and 10 all encode isoform b. Isoforms X1 and X2 are predicted.
Variants can differ in promoters, untranslated regions, or exons. For GeneID: 9: This variant (1) represents the longest transcript but encodes the shorter isoform (a). This variant is transcribed from a promoter known as P1, promoter 2, or NATb promoter.
This variant (2, also known as Type IID) lacks an alternate exon in the 5' UTR, compared to variant 1. This variant is transcribed from a promoter known as P1, promoter 2, or NATb promoter.
This variant (9, also known as Type IA) has a distinct 5' UTR and represents use of an alternate promoter known as the NATa or P3 promoter, compared to variant 1.
But, A1BG in NCBI Gene lists only one isoform, the gene locus itself, and the protein transcribed is a precursor subject to translational or more likely post-translational modifications.
The presence of multiple MREs coupled with experimental results from the literature indicating post-translational isoforms tends to confirm the existence of two or more isoforms for A1BG.
It isn't known which, if any, assist in locating and affixing the transcription mechanism for A1BG. This examination is the first to test one such DNA-occurring TF: the HNF6s.
The presence of multiple MREs coupled with experimental results from the literature indicating post-translational isoforms tends to confirm the existence of two or more isoforms for A1BG and likely transcription from either side.

#### STAT5s

All three hypotheses have been addressed.
Regarding hypothesis 1: STAT5s have a role as downstream signal transducers in A1BG, where the murine downstream promoter element is only 11 nts displaced from the human one. This suggests a STAT5 participation in human gene transcription of A1BG in the proximal promoter downstream between any other promoter and the TSS on the ZNF497 side of A1BG.
Regarding hypothesis 2: A1BG is not transcribed by any STAT5s is clearly disproved by the STAT5 transcription factor in the proximal promoter on the ZNF497 side of A1BG.
And, regarding hypothesis 3: STAT5s may assist transcription of A1BG by other transcription factors, literature search has found that STAT5s assist transcription of A1BG by other transcription factors. The proximal STAT5 promoter is -58 to -50 from A1BG TSS. If another STAT5 promoter is at -2.3 kb, it is about -1.4 kb inside ZNF497 which is 3212 nts long. Per analogy to the rat this would be expected. A STAT5 transcription site lies at 3'-TTCCGGGAA-5' at 4247 in the proximal promoter, i.e. from 4242 (-58) to 4250 (-50). This suggests that STAT5 assists in the transcription of A1BG.

### Experiments

Computer programs were written and run on the positive and negative strands between ZSCAN22 or ZNF497 and A1BG.
Regarding hypothesis 1: HNF6s have a role as downstream signal transducers in A1BG.
HNF6s may have a downstream proximal promoter element if the computer nts sampling is additionally, approximately at least 250 nts downstream of the transcription start site. "Downstream" can refer to downstream from an enhancer but before the transcription start site, downstream from a TATA box or an initiator element but before the transcription start site (TSS), downstream from another promoter element and containing the TSS, or downstream after the TSS. The computer programs written to test for HNF6 promoters were limited to 100 nts below the apparent TSSs.
Regarding hypotheses 2: A1BG is not transcribed by any HNF6s.
Here, the experiments have two parts: (1) are there any HNF6 promoters? and (2) are any of these used to transcribe A1BG?
The Basic programs (starting with SuccessablesHNF6.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 (+) looking for 16 possible types of promoters.
Regarding hypothesis 3: HNF6s may assist transcription of A1BG by other transcription factors.
An extensive literature search was performed to find even one example of a HNF6 assist of an A1BG transcription.

### Results

Regarding hypothesis 1: HNF6s have a role as downstream signal transducers in A1BG.
(1) "Downstream" can refer to downstream from an enhancer but before the transcription start site.
There is a HNF6 on the negative strand in the positive direction (from ZNF497 to A1BG) of 3'-TTCCGGGAA-5' at 808 in the proximal promoter, where the TSS is at 858 nts from ZNF497.
There is no such "downstream" promoter between ZSCAN22 and A1BG.
(2) Downstream from a TATA box or an initiator element (Inr) but before the transcription start site (TSS).
Both a TATA box or an Inr are within the core promoter. There are no HNF6s within any core promoters per the computer program sampling from ZNF497 or ZSCAN22 and A1BG.
(3) Downstream from another promoter element and containing the TSS. There are no HNF6s within any core promoters per the computer program sampling from ZNF497 or ZSCAN22 and A1BG containing either TSS.
(4) Downstream after the TSS. No HNF6s were detected at least to 100 nts downstream of each TSS.
Regarding hypotheses 2: A1BG is not transcribed by any HNF6s.
There is a HNF6 on the negative strand in the positive direction (from ZNF497 to A1BG) of 3'-TTCCGGGAA-5' at 808 in the proximal promoter, where the TSS is at 858 nts from ZNF497. This direction is the only confirmed transcription of A1BG; therefore, it is likely A1BG is transcribed using this HNF6 transcription factor.
There are two HNF6s on the negative strand in the negative direction, 3'-AAGCAACTT-5' at 3506 and 3'-AAGGGACTT-5' at 3782. Both of these are in the distal promoter between ZSCAN22 and A1BG.
Regarding hypothesis 3: HNF6s may assist transcription of A1BG by other transcription factors.
Both "the 2.3 kb and the 160 bp proximal parts of the a1bg promoter direct sex-specific expression of the reporter gene, and that a negative regulatory element resides in the −1 kb to −160 bp region."
"Computer analysis of the 2.3 kb rat a1bg promoter fragment revealed two putative HNF6 sites and one [hepatic nuclear factor 6] HNF6/HNF3 binding site at −2077/−2069, −69/−61 and −137/−128 respectively [...]."
The "GH-dependent sexually dimorphic expression conveyed by the 2.3 kb a1bg promoter is enhanced by the HNF6/HNF3 site [...]."
"HNF6 bound to the a1bg HNF6 oligonucleotide, but in this case, the mutated oligonucleotide was able to compete for binding when added in large excess [...]. However, [...] the HNF6 binding capacity of the mutated oligonucleotide was clearly reduced. A 20 molar excess of the mutated oligonucleotide had only a marginal effect on the binding of HNF6 [...], whereas a 20 molar excess of unlabelled probe [...] completely abolished binding. Supershift analysis with an HNF6 antibody revealed a complex with a slightly lower mobility than the HNF6 complex [...]. By extending the electrophoresis run and including nuclear extract from hypophysectomized rats, devoid of GH and thereby lacking HNF6 (Lahuna et al. 1997), the two different complexes were clearly visualized. The complex with the lower mobility is most probably due to the binding of HNF3, in analogy with what was shown by Lahuna et al. for the CYP2C12 HNF6 binding site; HNF3 can bind to the site in the absence of HNF6 (Lahuna et al. 1997). [...] HNF6 could bind to their respective site in the a1bg promoter in vitro, and the mutations introduced in respective site abolished binding of the corresponding factor."
The "expression of a −116/−89 deletion construct in which also the HNF6 site was mutated, (−116/−89) delmutHNF6-Luc, [...] the generated luciferase activities were reduced in both sexes [...]. This is in contrast to that mutation/deletion of the sites separately only affected the expression in female livers."
The "−116/−89 region contains a site(s) of importance for the GH-dependent and female-specific expression of the a1bg gene, and that the impact of this region together with the HNF6 site is more complex than mere enhancement of the expression in females."
Following "mutation of the HNF6-binding element, mutHNF6-Luc, the sex-differentiated expression was attenuated due to reduced expression in females. Thus, for a1bg, the sex-related difference in amount of HNF6 is likely to contribute to the sex-differentiated and female characteristic expression."
Nuclear "proteins binding to the a1bg −116/−89 region [are] members of the [nuclear factor 1] NF1 and the [octamer transcription factor] Oct families of transcription factors. NF1 genes are expressed in most adult tissues (Osada et al. 1999). It is not known how NF1 modulates transcriptional activity, and both activation and repression of transcription have been reported (Gronostajski 2000). Cofactors such as CBP/p300 and HDAC have been shown to interact with NF1 proteins suggesting modulation of chromatin structure (Chaudhry et al. 1999). NF1 factors have also been shown to interact directly with the basal transcription machinery as well as with other transcription factors, including Stat5 (Kim & Roeder 1994, Mukhopadhyay et al. 2001) and synergistic effects with HNF4 have been reported (Ulvila et al. 2004). In addition to the HNF6, Stat5 and NF1/Oct sites, the a1bg promoter harbours an imperfect HNF4 site at −51/−39 with two mismatches compared with the HNF4 consensus site. HNF4 is clearly important for the expression of CYP2C12 (Sasaki et al. 1999), however, the −51/−39 region in a1bg was not protected in the footprinting analysis and was therefore not analysed further. Like NF1, Oct proteins have been reported to be involved in activation as well as repression of gene expression (Phillips & Luisi 2000). [...] Moreover, NF1 and Oct-1 have been shown to, reciprocally, facilitate each other’s binding (O’Connor & Bernard 1995, Belikov et al. 2004)."
In the diagram on the right is liver "expression of a1bg-luciferase constructs. (A) Stat5 and HNF6 consensus sequences and corresponding sites in the 2.3 kb a1bg promoter alongside with the used mutations. (B) Female (black bars) and male (open bars) rats [results]."

### Discussion

Regarding hypothesis 1: HNF6s have a role as downstream signal transducers in A1BG.
The only known TSS for A1BG lies at 4300 nts from just beyond ZNF497 toward A1BG. There two HNF6s in the proximal promoter between 4050 and 4300, 3'-TTATTGATTA-5' at 4164 and 3'-TATAATTGTT-5' at 4172, i.e. outside from 4242 (-58) to 4250 (-50). This suggests that HNF6 assists in the transcription of A1BG, but not downstream of the TSS.
"Computer analysis of the 2.3 kb rat a1bg promoter fragment revealed [a] HNF6 [site] at [...] −69/−61 [...]."
The murine downstream promoter element is only 11 nts displaced from the human one. This suggests a HNF6 participation in human gene transcription of A1BG.
Regarding hypothesis 2: A1BG is not transcribed by any HNF6s.
"Computer analysis of the 2.3 kb rat a1bg promoter fragment revealed two putative HNF6 sites [...] at −2077/−2069 [and] −69/−61 [...]."
There are two HNF6s on the negative strand in the negative direction, 3'-AAGCAACTT-5' at 3506 (-954) and 3'-AAGGGACTT-5' at 3782 (-678) in the distal promoter between ZSCAN22 and A1BG. Although much closer than their likely murine counterparts, they are on the other side of A1BG from the HNF6 site confirming hypothesis 1. If active in humans or murine-like HNF6s occur within or beyond ZNF497 in this distal promoter, then human A1BG is transcribed using HNF6 promoters disproving hypothesis 2.
A Google Scholar search using ZNF497 with HNF6 found no articles discussing HNF6 sites inside or associated with ZNF497. To confirm they exist, a data file going 4300 nts to just beyond ZNF497 has been created and tested for a distal promoter on this side. Distal HNF6s in the positive direction, if they exist, would be inside ZNF497 or beyond, e.g., 3'-ATGTCCATGG-5' at 3581 was found.
Regarding hypothesis 3: HNF6s may assist transcription of A1BG by other transcription factors.
Literature search has found that HNF6s assist transcription of A1BG by other transcription factors. The proximal HNF6 promoter is -58 to -50 from A1BG TSS. If another HNF6 promoter is at -2.3 kb, it is about -1.4 kb inside ZNF497 which is 3212 nts long. Per analogy to the rat this would be expected.
Per earlier laboratories transcription factors may occur in the distal promoters on the ZNF497 side of A1BG for
* ATA boxes 3'-AATAAA-5' occurs at 3427,
* CArG boxes,
* Enhancer boxes,
* HY boxes,
* MREs and
* STAT5s 3'-TTCCATGAA-5' occurs at 128.
The HNF6 promoter on the other side of A1BG (at about +3 kb is way beyond -2.1 through ZNF497 unless the DNA is folded to allow the HNF6 on the ZSCAN22 side to be used in analogy to the HNF6 on the same side as in the rat.

### Conclusions

All three hypotheses have been addressed. Regarding hypothesis 1: HNF6s have a role as downstream signal transducers in A1BG, where the murine downstream promoter element is only 11 nts displaced from the human one. This suggests a HNF6 participation in human gene transcription of A1BG in the proximal promoter downstream between any other promoter and the TSS on the ZNF497 side of A1BG. Regarding hypothesis 2: A1BG is not transcribed by any HNF6s is clearly disproved by the HNF6 transcription factor in the proximal promoter on the ZNF497 side of A1BG. And, regarding hypothesis 3: HNF6s may assist transcription of A1BG by other transcription factors, literature search has found that HNF6s assist transcription of A1BG by other transcription factors. The proximal HNF6 promoter is -58 to -50 from A1BG TSS. If another HNF6 promoter is at -2.3 kb, it is about -1.4 kb inside ZNF497 which is 3212 nts long it may be STAT5 at 128 nts. Per analogy to the rat this would be expected.

## 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 is transcribed from either side using HNF6s, especially in the distal promoters. The NCBI database is generalized, whereas individual human genome testing could demonstrate that A1BG is transcribed from either side. Sufficient nts have been to the data sets for the ZNF497 side to confirm likely transcription of A1BG per analogy to the rat.