(Written for the CSH course manual "Early Development of Xenopus laevis" by P. Kolm 7/97)
- General Idea
- What types of proteins can be made inducible?
- Which receptor ligand binding domain should be added?
- Where should the ligand binding domain be added?
- Is hormone-induced activity reversible?
- Does the transactivation function of the ligand binding domain interfere with functionality?
Ectopic expression of genes in a temporally controlled manner is useful for analyzing gene function during development, particularly when events during gastrula or neurula stages are to be examined. Injection of RNAs into cleavage-stage embryos results in widespread and high level expression, but injected RNAs are generally translated immediately after their introduction, resulting in expression many hours earlier than the normal period of gene function with potentially misleading results. While expression later during development can be obtained by expressing genes from promoter-driven plasmid DNA, such constructs are expressed in only a fraction of cells that contain the DNA (Vize, et al., 1991) .
We have found that a steroid hormone-inducible system allows high levels of expression, in addition to temporal control of protein activity. This system utilizes fusions between the hormone-binding domain (hbd) of steroid receptors and a heterologous proteins (reviewed in (Mattioni et al., 1994)). In the absence of hormone, the fusion protein is held in an inactive state, presumably due to complex formation with hsp 90 (Scherrer et al., 1993). Addition of hormone causes a conformational change that dissociates hsp90, resulting in the rapid activation of the fusion protein (Tsai and O'Malley, 1994). There are several advantages to this system:
1) In vitro transcribed RNA can be injected at cleavage stages for widely distributed high levels of protein.
2) Addition of the hormone ligand binding domain can stabilize the protein relative to the wild type protein (Kolm and Sive, 1995; Tada et al., 1997), allowing activation though late neurula/early tailbud stages (Gammill and Sive, 1997).
3) Steriod hormones are small lipophilic molecules that can diffuse through the epithelial layer that surrounds the embryo. Activation of the protein can be achieved by incubation of the embryos in standard culture medim containing the hormone of choice.
4) Hormone addition rapidly activates the protein, so that increases in the levels of downstream targets can be seen in two hours (L. Gammill unpublished). This makes hormone inducible proteins ideal for the isolation of downstream targets of transcription factors (Braselmann et al, 1992).
What types of proteins can be made inducible?
A wide variety of different types of protein-hbd fusions have been reported, including a number of types of DNA binding protein and RNA binding proteins, kinases, and enzymes (Table 1). The primary consideration is that the protein in question have a known function, so it can be determined that the addition of the ligand binding domain does not alter protein function. Additionally, a general knowledge of the domain structure of the protein is useful, so that insertion of the ligand binding domain does not disrupt any important functional domain.
Which receptor ligand binding domain should be added?
Hormone binding domains from both the steroid and thyroid hormone families of receptors can be used to regulate protein function. The exact hormone binding domain that is used is dependent on two factors:
- The ligand used should not be present in the embryo at the stages examined. Of course, this can be exploited to restrict protein activity to regions of the embryo where ligand is present.
We have found that the glucocorticoid receptor (GR) ligand binding domain is very tightly regulated in Xenopus embryos (Kolm and Sive, 1995), even though there is GR RNA present in the embryo as early as gastrula stages (Gao et al., 1994). A potential disadvantage of GR is that the isolated hbd has a relatively low affinity for ligand, requiring high levels of hormone in the medium (Mattioni et al., 1994). In contrast, the estrogen receptor (ER) hbd is somewhat leaky when used in whole embryos, but is regulated quite tightly in animal caps (Kolm and Sive, 1995). Our preliminary data suggests that the thyroid hormone receptor (TR) hbd may also be useful during gastrula and neurula stages (unpublished). Other potential ligand binding domains include the mineralocorticoid receptor (MR) and the androgen receptor (AR).
As an alternative, hbds specific for ligands that are not endogenous to Xenopus embryos can be used. There are a number of hbds with point mutations that specifically bind synthetic hormones, rather than the normal endogenous ligand (Table 2). This include a mutant ER (ER*) that makes it tamoxifen regulated and a mutant progesterone receptor (PR*) that makes it RU486 regulated. It must be noted, however, that Tada et al. have reported that ER* is also leaky in Xenopus embryos.
Additionally, tissue culture data suggests that the Drosophila ecdysone recptor (EcR) hbd may be used to make myristerone-inducible proteins (Christopherson et al., 1992; No et al., 1996). However, a drawback of this system is that myristerone-inducibility appears dependent on the coexpression of a heterodimeric partner, USP or RXR (No et al., 1996; Takebayashi et al., 1996).
- Addition of the hormone should not affect gene expression or development on its own. There are many hormones that, when added to Xenopus gastrula or neurula embryos, have no discernible affect on development, including dexamethasone (Gao et al., 1994; Kolm and Sive, 1995), b-estradiol (Baker and Tata, 1990; Kolm and Sive, 1995), tri-iodothyronine (Old et al., 1992; Smith et al., 1994), and myristerone (Kolm, Patel unpublished).
Where should the ligand binding domain be added?
For maximal tightness of regulation the hbd should be inserted relatively close to the functional domain to be regulated (Mattioni et al., 1994) . Additionally, insertion of the hbd, normally at the C-terminus, at the N-terminus can lead to initiation from an internal AUG, leading to proteins produced without this regulatory domain (Mattioni et al., 1994) . Of course it is also critical to insert the hbd in a region of the protein where the structure of functional domains won't be disrupted. Insertion into several different regions of the protein can ensure that one of them will retain the activity of the native protein.
Is hormone-induced activity reversible?
It has been demonstrated that removal of hormone from the medium can reverse the activity of hbd fusion proteins (Jackson et al., 1993; Mattioni et al., 1994; Spitkovsky et al., 1994). However, these assays have been done in tissue culture over a period of days. It is not clear whether simple removal of ligand can reverse protein activity in the short time period necessary to make this useful during Xenopus development. An alternative, possibility is the addition of antagonists to inhibit normal lbd function (Boehmelt et al., 1992).
Does the transactivation function of the ligand binding domain interfere with functionality?
The ligand binding domain of most nuclear receptors contains a transactivation function, termed TAF-2. This can certainly be a problem when the hbd is attached to a weak transactivator (Schuermann et al., 1993). A current technique to test the requirement for transcriptional regulators in gene expression is to fuse the repressor region of the Drosophila engrailed protein (EnR) (Jaynes and O'Farrell, 1991) to the DNA binding domain of the transcriptional activator to be examined (Conlon et al., 1996; Fan and Sokol, 1997). Addition of a strong activation domain could cancel out the repressor activity of these proteins. This problem could be prevented by using mutant forms of GR or ER that inactivate the TAF-2 activity (Mattioni et al., 1994; Lyon and Watson, 1996). Indeed, tamoxifen-inducible ER that does not have TAF-2 function has been shown to effectively regulate the activity of a Myb-EnR fusion protein (Lyon and Watson, 1996).