B/B 04 · Gene regulation and steroid hormone receptors
A Reporter Assay for a Hormone-Responsive Gene
Use reporter constructs and receptor mutants to work out how a steroid hormone turns on a gene.
Passage
In eukaryotic cells, steroid hormones are lipid-soluble molecules that enter cells by passing through the plasma membrane. Inside the cell, a steroid hormone binds a specific intracellular receptor protein. The hormone–receptor complex moves to the nucleus, where it binds to short DNA sequences called hormone response elements (HREs) near the gene it controls and changes the rate of transcription. Steroid hormone receptors have separate regions, or domains, for binding the hormone (the ligand-binding domain, LBD), binding DNA (the DNA-binding domain, DBD) and interacting with other proteins that affect transcription (the activation domain, AD).
In every eukaryotic gene, general transcription factors and RNA polymerase II assemble at the core promoter, which in many genes contains a TATA box about 25 to 30 base pairs upstream of the start site. Regulatory proteins that bind to other DNA sequences, sometimes thousands of base pairs away, can increase or decrease how efficiently this machinery assembles. A regulatory sequence of this kind is useful experimentally because it can be moved, deleted or mutated independently of the gene it controls.
A researcher studies one gene that is turned on by a steroid hormone. She builds a series of reporter constructs in which the gene’s promoter, with or without its HRE, is placed in front of the coding sequence of luciferase, an enzyme whose activity is easy to measure. The constructs are introduced into cultured cells that make the normal hormone receptor, and luciferase activity is measured with and without the hormone. Activity is expressed relative to the value for the intact construct without hormone, which is set to 1. Table 1 shows the results.
A second reporter gene on a separate piece of DNA is included in every sample as a control for differences in the number of cells that take up DNA. Each luciferase value is divided by the control value before it is compared with the others, and each number is the average of three independent experiments.
Table 1. Relative luciferase activity for different reporter constructs.
| Reporter construct | No hormone | With hormone |
|---|---|---|
| Intact promoter and HRE | 1 | 20 |
| HRE deleted | 1 | 1 |
| HRE replaced by a random sequence of the same length | 1 | 1 |
| HRE moved 2,000 base pairs farther upstream | 1 | 15 |
| HRE inserted in the reverse orientation | 1 | 19 |
| TATA box deleted from the promoter, HRE intact | 0.1 | 0.1 |
In a second experiment, she keeps the intact reporter and changes the receptor. Cells that make no receptor of their own are given a normal receptor or one of three mutant receptors. Hormone binding is tested with a labeled hormone. Binding to the HRE is tested with a gel-shift assay, which detects protein–DNA complexes in the presence of hormone. Table 2 shows the results.
Table 2. Properties of normal and mutant receptors with the intact reporter.
| Receptor | Binds hormone | Binds HRE (with hormone) | Luciferase activity (with hormone) |
|---|---|---|---|
| Normal | Yes | Yes | 20 |
| DBD mutant | Yes | No | 1 |
| LBD mutant | No | No | 1 |
| AD mutant | Yes | Yes | 1 |
The researcher concludes that the luciferase assay can report on whether the receptor regulates the gene, and she plans to use the same assay to test other hormones, including a hormone that is a small peptide.
Questions
Question 1 of 6
Which conclusion about the HRE is best supported by the first two reporter constructs after hormone treatment?
If the hormone acted on luciferase, the deleted construct would also show an increase.
The construct with a random sequence of the same length is also inactive. The specific sequence matters, not just the presence of DNA at that site.
The value without hormone is 1 for both the intact and the deleted constructs, so the HRE has no effect without hormone.
Why the answer is what it is
Deleting the HRE or replacing it with a random sequence of the same length removes the hormone effect (20 falls to 1). The effect therefore depends on the specific sequence, not on length. Without hormone the HRE has no effect on the activity.
Review: Gene Expression & Regulation · Molecular & Cell Biology · Endocrinology
Question 2 of 6
The HRE still works when it is moved 2,000 base pairs upstream or reversed. What does this indicate about the HRE?
Core promoters need a precise position and orientation for the transcription machinery. The HRE tolerates changes in both.
The HRE is a regulatory DNA sequence that is not translated; a coding sequence would not make the construct hormone-dependent.
The passage describes the HRE as bound by the hormone–receptor complex, which then influences transcription.
Why the answer is what it is
Enhancer-type elements can function at a distance and in either orientation. The modest drop (20 to 15 at a greater distance) fits this behavior and rules out a fixed-position promoter element.
Review: Gene Expression & Regulation · Molecular & Cell Biology · Endocrinology
Question 3 of 6
What can be concluded from the construct in which the TATA box is deleted?
Activity is 0.1 with and without hormone, so the hormone has no effect, rather than a lowering effect.
The receptor binds the HRE according to the second experiment. The TATA box is part of the core promoter.
If the HRE could act as the promoter, activity would increase with hormone, but it stays at 0.1.
Why the answer is what it is
Without the TATA box, the basal level falls to 0.1 and the hormone has no effect, which means that the HRE amplifies transcription that is started at the core promoter. It cannot substitute for the core promoter.
Review: Gene Expression & Regulation · Molecular & Cell Biology · Endocrinology
Question 4 of 6
The AD mutant binds the hormone and the HRE but fails to activate the reporter. What is the most likely defect?
It binds the HRE in the gel-shift assay in the presence of hormone, so it is able to reach DNA.
Table 2 shows that the AD mutant binds the hormone normally.
It binds the HRE, as shown by the gel-shift assay.
Why the answer is what it is
The AD mutant has the ligand-binding and DNA-binding functions intact. The remaining function in the passage’s description is interaction with other proteins that affect transcription, so a defect there explains why the bound receptor fails to increase transcription.
Review: Gene Expression & Regulation · Molecular & Cell Biology · Endocrinology
Question 5 of 6
Which conclusion about the receptor’s domains is best supported by the mutants in Table 2?
The AD mutant binds the HRE normally. Its defect is in a later step.
The LBD mutant, which cannot bind hormone, also cannot bind the HRE or activate the reporter. The LBD is necessary.
The AD mutant has an intact DBD and still fails to activate the reporter.
Why the answer is what it is
The DBD mutant binds hormone but not DNA, so the two functions are separable. The LBD mutant, lacking hormone binding, does not bind the HRE and the AD mutant binds both but fails to activate. Each domain is therefore needed for the full response.
Review: Gene Expression & Regulation · Molecular & Cell Biology · Endocrinology
Question 6 of 6
The researcher plans to use the same assay with a small peptide hormone. Why is it unlikely that this peptide would work through the pathway shown in the passage?
Peptide hormones bind receptors with high affinity. Their size is not the problem.
Peptides are not converted to steroids.
Peptide hormones act through receptors and signaling cascades, not by binding DNA.
Why the answer is what it is
The passage describes lipid-soluble hormones that cross the membrane and bind intracellular receptors. Polar peptide hormones cannot cross the plasma membrane, so they bind cell-surface receptors and signal through second messengers, which would not act through an HRE and a nuclear receptor in this way.
Review: Gene Expression & Regulation · Molecular & Cell Biology · Endocrinology · Molecular & Cell Biology
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