Molecular Endocrinology, doi:10.1210/me.2005-0383
Molecular Endocrinology 20 (10): 2326-2342
Copyright © 2006 by The Endocrine Society
Dosage-Sensitive Sex Reversal Adrenal Hypoplasia Congenita Critical Region on the X Chromosome, Gene 1 (DAX1) (NR0B1) and Small Heterodimer Partner (SHP) (NR0B2) Form Homodimers Individually, as Well as DAX1-SHP Heterodimers
Anita K. Iyer,
Yao-Hua Zhang and
Edward R. B. McCabe
Department of Human Genetics (A.K.I., E.R.B.M.), David Geffen School of Medicine at University of California Los Angeles (UCLA); Department of Pediatrics (Y.-H.Z., E.R.B.M.), David Geffen School of Medicine at UCLA; UCLA Molecular Biology Institute (E.R.B.M.); and Mattel Children’s Hospital at UCLA (E.R.B.M.), Los Angeles, California 90095
Address all correspondence and requests for reprints to: E. R. B. McCabe, M.D., Ph.D., Department of Pediatrics, 22-412 MDCC, David Geffen School of Medicine at University of California Los Angeles, 10833 Le Conte Avenue, Los Angeles, California 90095-1752. E-mail: emccabe{at}mednet.ucla.edu.
 |
ABSTRACT
|
|---|
Dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX1) (NR0B1), and small heterodimer partner (SHP) (NR0B2) are atypical nuclear receptor superfamily members that function primarily as corepressors through heterodimeric interactions with other nuclear receptors. Mutations in DAX1 cause adrenal hypoplasia congenita, and mutations in SHP lead to mild obesity and insulin resistance, but the mechanisms are unclear. We investigated the existence and subcellular localization of DAX1 and SHP homodimers and the dynamics of homodimerization. We demonstrated DAX1 homodimerization in the nucleus and cytoplasm, and dissociation of DAX1 homodimers upon heterodimerization with steroidogenic factor 1 (SF1) or ligand-activated estrogen receptor-
(ER). DAX1 homodimerization involved an interaction between its amino and carboxy termini involving its LXXLL motifs and activation function (AF)-2 domain. We observed SHP homodimerization in the nucleus of mammalian cells and showed dissociation of SHP homodimers upon heterodimerization with ligand-activated ER. We observed DAX1-SHP heterodimerization in the nucleus of mammalian cells and demonstrated the involvement of the LXXLL motifs and AF-2 domain of DAX1 in this interaction. We further demonstrate heterodimerization of DAX1 with its alternatively spliced isoform, DAX1A. This is the first evidence of homodimerization of individual members of the unusual NR0B nuclear receptor family and heterodimerization between its members. Our results suggest that DAX1 forms antiparallel homodimers through the LXXLL motifs and AF-2 domain. These homodimers may function as holding reservoirs in the absence of heterodimeric partners. The formation of DAX1 and SHP homodimers and DAX1-SHP and DAX1-DAX1A heterodimers suggests the possibility of novel functions independent of their coregulator roles, suggesting additional complexity in the molecular mechanisms of DAX1 and SHP action.
|
INTRODUCTION
|
|---|
THE NUCLEAR RECEPTOR superfamily consists of a series of transcription factors that modulate gene expression in response to a variety of physiological cues and have critical roles in cellular homeostasis and development (1, 2, 3). Superfamily members have a characteristic domain structure consisting of four main functional modules: an amino-terminal (N-terminal) modulator domain that may contain a hormone-independent transactivation domain [activation function 1 (AF-1)]; a DNA-binding domain (DBD) that allows the receptor to bind cognate response elements in the promoters of target genes; a hinge region between the DBD and the ligand binding domain (LBD); and an LBD that mediates ligand binding, dimerization, and nuclear localization, and contains the activation function (AF)-2 ligand-dependent transactivation domain. Nuclear receptors are typically activated by a ligand and function by binding a response element on DNA and associating with transcriptional coregulatory proteins to regulate transcription of target genes.
The NR0B family of nuclear receptors consists of the two orphan receptors DAX1 [dosage-sensitive sex reversal, adrenal hypoplasia congenita (AHC) critical region on the X chromosome, gene 1], encoded by NR0B1, and SHP (small heterodimer partner), encoded by NR0B2 (2, 3). The protein domain structures of DAX1 and SHP are unusual when compared with other members of the superfamily (2, 3, 4). They contain a carboxy-terminal domain (CTD) homologous to the LBD of superfamily members and also contain the AF-2 transactivation domain. This CTD has strongest amino acid similarity to testis receptor and chicken ovalbumin upstream promoter transcription factor (4, 5). DAX1 and SHP are unusual in the sense that they lack the canonical DBD, AF-1 modulator domain, and hinge region. DAX1 instead has an N-terminal domain (NTD) consisting of 3.5 alanine/glycine-rich repeats of a novel 65- to 70-amino acid motif. SHP contains a very short 70-residue NTD that is homologous to the DAX1 NTD (6). A classical receptor function has not been demonstrated for either of these receptors; rather, they have been shown to inhibit the action of many nuclear receptors primarily through heterodimeric interactions (3, 7).
Mutations in DAX1 cause X-linked AHC, a disorder characterized by underdevelopment of the adrenal cortex (8, 9, 10, 11). Patients with AHC due to DAX1 mutations also develop hypogonadotropic hypogonadism caused by hypothalamic and pituitary defects in gonadotropin secretion (8, 12). DAX1 is expressed throughout the developing and adult hypothalamic-pituitary-adrenal-gonadal (HPAG) axis; therefore, the complex endocrine phenotype caused by DAX1 defects suggests a role for DAX1 in the normal development and function of this axis (13, 14, 15). This tissue expression profile is very similar to that of steroidogenic factor-1 (SF1), encoded by NR5A1, a nuclear receptor essential for normal HPAG development and steroid biosynthesis (16). SF1 knockout mice show a complex endocrine phenotype very similar to that of AHC patients with DAX1 mutations, suggesting that SF1 and DAX1 cooperatively regulate transcription of genes critical for proper development of the HPAG axis. However, DAX1 has been shown, paradoxically, to inhibit SF1-mediated transcriptional transactivation of steroidogenic genes (17, 18). How disruption of SF1, or of the antagonist of its action, DAX1, lead to similar phenotypes poses a functional conundrum that has not yet been resolved and suggests additional complexity and pleiotropy in molecular mechanisms of DAX1 action (7). DAX1 is now thought to have a broader functional role in HPAG axis development and adult function as a repressor through heterodimeric interactions with the androgen receptor (AR; NR3C4), estrogen receptor (ER; NR3A1–2), and progesterone receptor (PR; NR3C3), and also liver receptor homolog-1 (LRH1; NR5A2), nerve growth factor-inducible gene B (NGFI-B; NR4A1), and ER-related receptor 
(NR3B3) (7, 19, 20), although the significance of this repressor function in development is poorly understood (7). A function for DAX1 independent of steroidogenesis is supported by observations of DAX1 expression in early embryonic development before the presence of the steroidogenic axis (21), and in bone cell development (22).
SHP has a more ubiquitous pattern of expression compared with DAX1, as expression has been shown in heart, brain, liver, adipose tissue, adrenal gland, small intestine, and pancreas (6, 23, 24, 25). SHP has been shown to have a role in many metabolic processes including cholesterol and bile acid homeostasis as well as glucose metabolism (24, 26, 27) and is thought to be involved in the regulation of genes in these pathways by functioning through heterodimeric interactions as a transcriptional repressor of retinoic X receptor (RXR), hepatocyte nuclear factor 4, glucocorticoid receptor, liver X receptor, LRH-1, and pregnane X receptor (26, 27, 28, 29, 30, 31). SHP also has been shown to inhibit the action of activated AR, ER, retinoic acid receptor, and thyroid hormone receptor and is thought to control and dampen the cellular responses to various hormones (6, 23, 32, 33, 34). A function for SHP in hepatic apoptosis as a repressor of NGFI-B has also been proposed (35). Mutations in the NR0B2 gene are associated with insulin resistance, mild obesity, and high birth weight in some populations (36), and although this clinical phenotype could involve the repressor function of SHP in various metabolic pathways, it is still unclear how mutations in SHP lead to mild obesity. This observation, and the fact that a function for SHP in the adrenal gland, where DAX1 is also expressed, has not been elucidated, suggests additional complexity in the molecular action of SHP.
DAX1 and SHP repress the action of their target nuclear receptors via a variety of mechanisms, but DAX1-mediated repression of SF1, and both DAX1- and SHP-mediated repression of ER, are thought to occur by a similar two-step mechanism of coactivator competition followed by direct transcriptional repression (18, 23, 33, 34, 37). Transcriptional coactivators contain LXXLL motifs, or nuclear receptor boxes, through which they bind to the AF-2 domain of nuclear receptors to activate transcription. DAX1 contains three and SHP contains two nuclear receptor boxes, and both receptors are considered LXXLL-containing corepressors, because they repress the action of their target receptors through direct protein-protein interactions involving the LXXLL motifs of DAX1 or SHP and the AF-2 domain of the nuclear receptors. This has been shown for SHP to result in a competition for coactivator binding to the target receptor (23), and such a competition is hypothesized for DAX1 (7). Both DAX1 and SHP can then directly repress transcription through a C-terminal silencing domain critical for repressor function (38, 39). This domain is thought to recruit other corepressor proteins. DAX1 has been shown to interact with corepressor proteins nuclear receptor corepressor and Alien through its transcriptional silencing domain (40, 41), and SHP has been shown to associate with histone deacetylases through its repression domain (42).
DAX1A is an alternatively spliced isoform of DAX1 (43, 44). The DAX1A protein contains the first 389 amino acids of DAX1 followed by a novel 12-amino acid motif, and thus lacks the last 70 amino acids of the DAX1 CTD, which includes part of the transcriptional silencing domain and the AF-2 domain. This isoform is expressed in a variety of tissues, including the adrenal gland, brain, ovary, pancreas, and testis, and is thought to antagonize the repressor activity of DAX1 on SF1-mediated transactivation by competing with DAX1 for binding to SF1. The existence of this isoform creates additional complexity in the molecular mechanisms of DAX1 action.
Nuclear receptors function as transcription factors in monomeric, homodimeric, or heterodimeric forms, and some function in more than one dimeric combination type to generate functional complexity in nuclear receptor signaling in various developmental and physiological contexts (1, 3, 45). DAX1 and SHP function as corepressors of their nuclear receptor targets through heterodimeric interactions (3, 7). It is possible that these unusual receptors may form and have specific functions as homodimers independent of other nuclear receptors.
In this study, we demonstrate homodimerization of DAX1 in yeast in vitro and in both the nucleus and the cytoplasm of mammalian cells. We show dissociation of the homodimer upon heterodimerization with SF1 or ligand-activated ER, and we provide evidence for the formation of an antiparallel homodimer through an interaction between the LXXLL motifs and AF-2 domain. We also demonstrate homodimerization of SHP in the nucleus of mammalian cells and show dissociation of the homodimer upon heterodimerization with ligand-activated ER. In addition, we provide evidence for the formation of DAX1-SHP heterodimers and also heterodimerization of DAX1 with DAX1A. This is the first report of homodimerization of members of the unusual NR0B family of nuclear receptors, or of the existence of DAX1-SHP or DAX1-DAX1A heterodimers, and these observations suggest additional complexity in the molecular mechanisms of DAX1 and SHP action.
|
RESULTS
|
|---|
DAX1 Homodimerizes in the Yeast Two Hybrid (Y2H) System
DAX1 homodimerization was first investigated in the Y2H system. The known interaction between DAX1 and SF1 (17, 18) was confirmed as a positive control, because cotransformation of Gal4DB-DAX1 and Gal4AD-SF1 resulted in activation of the HIS3 reporter gene, whereas each construct alone did not activate the reporter gene (Fig. 1A
). Similar results were observed for cotransformation of Gal4DB-SF1 and Gal4AD-DAX1 (data not shown). Cotransformation of Gal4DB-DAX1 and Gal4AD-DAX1 resulted in activation of the HIS3 reporter, whereas each construct alone did not (Fig. 1A). To eliminate the possibility of a false positive in the Y2H system, activation of a second reporter gene, ß-galactosidase, was evaluated using a quantitative liquid assay. Cotransformation of Gal4DB-DAX1 and Gal4AD-DAX1 resulted in a 7-fold greater activation of the ß-galactosidase reporter than each construct alone (Fig. 1B) (P < 0.0001), suggesting a putative homodimerization of DAX1.
View larger version (24K):
[in this window]
[in a new window]
|
Fig. 1. DAX1 Homodimerizes in the Y2H System
A, Y2H assay testing for activation of the HIS3 reporter gene. Gal4DB and Gal4AD fusion constructs of indicated combinations were transformed into yeast strain AH109. Growth (++) on media lacking leucine and tryptophan indicate cotransformants. Growth (++) on media lacking leucine, tryptophan, and histidine indicate cotransformants showing reporter activation. Tr, Trace. B, Y2H assay testing for activation of the ß-galactosidase reporter. Constructs of indicated combinations were transformed into yeast strain Y187. The ß-galactosidase activity of 10 independent cotransformants was measured in triplicate by quantitative liquid assay. Values shown are mean ± SD. *, P < 0.0001 vs. each DAX1 fusion construct alone.
|
|
DAX1 Homodimerization Occurs in Vitro and in Mammalian Cells
Homodimerization of DAX1 was confirmed in vitro using an antibody-mediated coimmunoprecipitation (co-IP) system involving in vitro translated differentially epitope-tagged proteins. Using a myc antibody, hemagglutinin (HA)-DAX1 was efficiently coimmunoprecipitated in the presence, but not in the absence, of myc-DAX1 (Fig. 2A). Similar results were seen with myc-SF1 and HA-DAX1. These observations suggest that DAX1 homodimerizes in solution as a result of a direct protein-protein interaction.
View larger version (35K):
[in this window]
[in a new window]
|
Fig. 2. Homodimerization of DAX1 in Vitro and in Mammalian Cells
A, DAX1 homodimerizes in solution as shown by in vitro Co-IP. Combinations of in vitro translated myc- and HA-epitope-tagged proteins were immunoprecipitated with a myc antibody; 50% of the IP reaction was resolved by SDS-PAGE and analyzed by Western blotting (WB) with HA antibody. B and C, Co-IP assays in mammalian cells. HEK293 cells were transfected with the indicated combinations of FLAG- and myc-epitope-tagged expression constructs (2 µg each plasmid). Plasmid levels were balanced with empty vector. Whole-cell extracts were subjected to IP with FLAG antibody; 25% of the IP reaction was resolved by SDS-PAGE and analyzed by Western blotting with FLAG and myc antibodies. B, Interaction between myc-DAX1 and FLAG-DAX1 in mammalian cells was evidenced by co-IP (C). The known interaction of DAX1 with SF1 was observed in mammalian cells by co-IP.
|
|
To determine whether DAX1 can homodimerize in mammalian cells, co-IP assays were performed using whole-cell extracts of human embryonic kidney (HEK)293 cells expressing FLAG-DAX1 and myc-DAX1. Using a FLAG antibody, myc-DAX1 was coimmunoprecipitated in the presence, but not in the absence, of FLAG-DAX1 (Fig. 2B
), with similar results observed with FLAG-DAX1 and myc-SF1 as a positive control (Fig. 2C). These interactions were also observed by co-IP using a myc antibody, and also using whole-cell extracts of transfected HeLa cells (data not shown).
The DAX1 Homodimer Exists in Both the Nuclear and Cytoplasmic Subcellular Compartments
We hypothesized that the subcellular localization of the DAX1 homodimer might provide insight regarding its function. To determine whether DAX1 homodimerized in the nucleus, the cytoplasm, or in both compartments, nuclear and cytoplasmic fractions were prepared from HEK293 cells expressing FLAG-DAX1 and myc-DAX1. Western blot analysis showed that heat shock protein (Hsp)90, a cytoplasmic marker (46), was present predominantly in the cytoplasmic fraction, and endogenous c-myc, a nuclear protein (47), was present mostly in the nuclear fraction, ensuring a relatively clean fractionation procedure (Fig. 3A). Analyses using FLAG and myc antibodies showed that DAX1 was expressed in both the cytoplasmic and nuclear fractions, with slightly higher levels in the nucleus (Fig. 3A). A similar localization pattern was observed in HeLa cells (data not shown). These results confirmed previous studies in HeLa cells and in cell lines that endogenously expressed DAX1 and SF1 (48, 49). The localization of myc-SF1 in cells coexpressing FLAG-DAX1 and myc-SF1 (Fig. 3A), or myc-SF1 alone (data not shown), was almost exclusively nuclear.
View larger version (28K):
[in this window]
[in a new window]
|
Fig. 3. DAX1 Homodimerizes in the Nucleus and the Cytoplasm
A, Nuclear and cytoplasmic fractions of HEK293 cells transfected with the indicated combinations of FLAG- and myc-epitope-tagged constructs (2 µg each plasmid) were subjected to Western blot analysis with FLAG, myc, and hsp90 antibodies. Plasmid levels were balanced with empty vector. Hsp90 and endogenous c-myc protein were used as cytoplasmic and nuclear markers, respectively. B and C, Nuclear and cytoplasmic fractions of transfected HEK293 cells were immunoprecipitated with FLAG antibody followed by Western blot (WB) analysis with FLAG and myc antibodies. B, Co-IP showed that DAX1 interacted with SF1 primarily in the nucleus. C, Co-IP showed that myc-DAX1 and FLAG-DAX1 interactions occurred in the nucleus and the cytoplasm. Western analysis in panel A represented 1% Co-IP input. Cyto, Cytoplasm; Nucl, nucleus.
|
|
Co-IP of cytoplasmic and nuclear fractions of cells coexpressing FLAG-DAX1 and myc-SF1 using a FLAG antibody showed co-IP of myc-SF1 with FLAG-DAX1 almost exclusively in the nuclear fraction, indicating that DAX1-SF1 heterodimers were present in the nucleus (Fig. 3B). Co-IP of the cytoplasmic and nuclear fractions of cells coexpressing FLAG-DAX1 and myc-DAX1 using a FLAG antibody showed co-IP of myc-DAX1 with FLAG-DAX1 in both compartments (Fig. 3C), indicating that DAX1 existed as a homodimer in the nucleus as well as the cytoplasm.
The DAX1 Homodimer Dissociates upon Heterodimerization with SF1
To study the dynamics of the DAX1 homodimer in relation to SF1, a protein interaction competition assay was used to test whether the DAX1 homodimer dissociates upon heterodimerization with SF1. A constant amount of FLAG-DAX1 and myc-DAX1 and increasing amounts of HA-SF1 were transfected into HEK293 cells. Western blot analysis of whole-cell extracts using FLAG, myc, and HA antibodies verified equal expression of FLAG-DAX1 and myc-DAX1 across all samples and increasing expression of HA-SF1 (data not shown). Co-IP analyses were performed with the FLAG antibody using whole-cell extracts of these transfected cells. Relative to FLAG-DAX1, immunoprecipitation (IP) followed by Western blotting with HA antibody permitted detection of DAX1-SF1 heterodimer, whereas Western blotting with myc antibody permitted detection of DAX1 homodimer. The amount of HA-SF1 pulled down by FLAG-DAX1 increased slightly and then plateaued due to saturation of the FLAG-DAX1. The amount of myc-DAX1 pulled down by FLAG-DAX1 decreased in the presence of increasing amounts of HA-SF1 (Fig. 4A). This suggested that the DAX1 homodimer dissociated to form heterodimers with SF1 and that the DAX1 homodimer might function as a holding reservoir when not complexed with SF1.
View larger version (23K):
[in this window]
[in a new window]
|
Fig. 4. DAX1 Homodimer Formation Decreases upon Heterodimerization with SF1 or Activated ER
A, Protein interaction competition assay. HEK293 cells were transfected with 1 µg each of plasmids expressing FLAG-DAX1 and myc-DAX1, and 0, 2.5, 4, and 5 µg of HA-SF1 expression plasmid. Plasmid levels were balanced with empty vector. Whole-cell extracts were immunoprecipitated with FLAG antibody followed by Western blot (WB) analysis with FLAG, and also myc and HA antibodies for homodimer and heterodimer detection, respectively. B, DAX1 interaction with ER was enhanced in the presence of ligand. Whole-cell extracts of HEK293 cells transfected with the indicated combinations of plasmids (2 µg each plasmid) in the absence or presence of 1 µM estradiol (E2) were immunoprecipitated with FLAG antibody followed by Western blot analysis with FLAG and HA antibodies. C, Protein interaction competition assay as in panel A instead with 0, 2.5, and 4 µg of HA-ER expression plasmid in the absence or presence of 1 µM E2.
|
|
The DAX1 Homodimer Dissociates upon Heterodimerization with ER in the Presence of Ligand
DAX1 has been shown to repress the action of ER by a mechanism involving a heterodimeric interaction (37) similar to its repression of SF1 (18). The dynamics of the DAX1 homodimer in relation to ER could involve dissociation of the DAX1 homodimer as with SF1. Co-IP studies using whole-cell extracts of HEK293 cells transfected with FLAG-DAX1 and HA-ER show that using a FLAG antibody, HA-ER is coimmunoprecipitated in the presence, but not in the absence, of FLAG-DAX1, and that this interaction is enhanced in the presence of estradiol (Fig. 4B), consistent with previous studies (18, 37).
The protein interaction competition assay as described above for SF1 was used to study the dynamics of the DAX1 homodimer in relation to ER. A constant amount of FLAG-DAX1 and myc-DAX1 and increasing amounts of HA-ER were transfected into HEK293 cells in the absence or presence of estradiol. Western blot analysis of whole-cell extracts using FLAG, myc, and HA antibodies verified equal expression of FLAG-DAX1 and myc-DAX1 across all samples and increasing expression of HA-ER (data not shown). Co-IP analyses using a FLAG antibody in the absence of estradiol showed only a slight decrease in the amount of myc-DAX1 pulled down by FLAG-DAX1 with increasing amounts of HA-ER but, in the presence of estradiol, showed a very dramatic and efficient decrease in the amount of myc-DAX1 coimmunoprecipitated by FLAG-DAX1 (Fig. 4C). These results suggest that the DAX1 homodimer dissociates upon heterodimerizing with ligand-activated ER, similar to the dynamics with SF1, and provide further support of a holding reservoir function for the DAX1 homodimer.
DAX1 May Form an Antiparallel Homodimer
To determine the domains involved in DAX1 homodimerization, constructs of the NTD and CTD of DAX1, DAX1-N, and DAX1-C, respectively (Fig. 5A), were used in various combinations in the Y2H assay utilizing the HIS3 reporter. Cotransformation of Gal4DB-DAX1-N and Gal4AD-DAX1-C, and the reverse combination, resulted in the strongest activation of the HIS3 reporter (Fig. 5B). Cotransformation of Gal4DB-DAX1-N and Gal4AD-DAX1-N resulted in weaker reporter activation, and the combination of Gal4DB-DAX1-C and Gal4AD-DAX1-C did not result in significant reporter activation (Fig. 5B). These results show that DAX1 can homodimerize through an interaction between its NTD and CTD, with perhaps a weak interaction between the NTDs. We conclude that DAX1 homodimerization involves formation of an antiparallel homodimer.
View larger version (28K):
[in this window]
[in a new window]
|
Fig. 5. DAX1 Homodimerization Involves Interaction between Its NTD and CTD
A, Schematic of DAX1 constructs used in the Y2H assay. DAX1-N contains residues 1–198, including the 3.5 repeat domains (arrows) and the LXXLL motifs (black bars). DAX1-C contains residues 207–470, including the conserved LBD-like domain and the AF-2 transactivation domain. B, Y2H assay testing for activation of the HIS3 reporter gene. Gal4DB and Gal4AD fusion constructs of indicated combinations were transformed into yeast strain AH109. Growth (++) on media lacking leucine and tryptophan indicate cotransformants. Growth on media lacking leucine, tryptophan, and histidine was scored based on number of colonies and colony size, and indicate cotransformants showing reporter activation. Tr, Trace.
|
|
LXXLL Motifs and AF-2 Domain Are Involved in DAX1 Homodimerization
DAX1 heterodimerizes through its LXXLL motifs with the AF-2 domains of SF1 and ER (18), and we provided evidence above for the formation of an antiparallel DAX1 homodimer. We hypothesized that DAX1 homodimerization could involve an interaction between its LXXLL motifs and AF-2 domain. DAX1 contains three LXXLL motifs in its NTD (Figs. 5A and 6A). To study the involvement of these LXXLL motifs in DAX1 homodimerization, we introduced point mutations of the last two highly conserved hydrophobic residues of all three motifs into FLAG-DAX1-N (FLAG-m123:DAX1-N) analogous to the work of Suzuki et al. (18) (Fig. 6A). Constructs expressing either the wild-type or mutant FLAG-DAX1-N were cotransfected with myc-DAX1 into HEK293 cells. Western blot analysis of whole-cell extracts verified equal protein expression across all samples (data not shown). Co-IP using a FLAG antibody showed an 85% decrease in the amount of myc-DAX1 pulled down by FLAG-m123:DAX1-N (Fig. 6B). These results indicate that the LXXLL motifs are involved in DAX1 homodimerization.
View larger version (31K):
[in this window]
[in a new window]
|
Fig. 6. LXXLL Motifs Are Involved in DAX1 Homodimerization
A, Schematic of FLAG- (F) and myc- (M) DAX1 constructs used in Co-IP assays. DAX1-N residue numbers are as in Fig. 5. LXXLL residue numbers are in parentheses. LXXLL point mutations are in bold and underlined, and black and white bars represent wild-type and mutant LXXLL motifs, respectively. B, Co-IP assay. HEK293 cells were transfected with the indicated combinations of FLAG- and myc-epitope-tagged expression constructs (2 µg each plasmid). Plasmid levels were balanced with empty vector. Whole-cell extracts were subjected to IP with FLAG antibody; 25% of the IP reaction was resolved by SDS-PAGE and analyzed by Western blotting (WB) with FLAG and myc antibodies. Signal was quantitated by laser densitometry. Data for mutants are presented as percent of wild-type protein (100%) bound. Values shown are mean ± SD.
|
|
DAX1 contains an AF-2 domain similar to other members of nuclear receptor superfamily in its CTD (Figs. 5A and 7A). To study the involvement of the AF-2 domain in DAX1 homodimerization, we introduced point mutations into the first two highly conserved hydrophobic residues of the AF-2 domain (1) into myc-DAX1 and FLAG-DAX1-C (myc-mAF-2:DAX1 and FLAG-mAF-2:DAX1-C, respectively) (Fig. 7A). Constructs expressing FLAG-DAX1-N and wild-type or mutant myc-DAX1 were cotransfected into HEK293 cells. Western blot analysis confirmed equal protein expression across all samples (data not shown). Co-IP with a FLAG antibody showed nearly a 65% decrease in the amount of myc-mAF-2:DAX1 pulled down with FLAG-DAX1-N compared with wild-type myc-DAX1 (Fig. 7B). Conversely, coexpression of FLAG-DAX1-C with myc-mAF-2:DAX1 followed by Co-IP with a FLAG antibody showed that FLAG-DAX1-C interacted with myc-mAF-2:DAX1 almost as strongly as wild-type myc-DAX1. These results provide further evidence for DAX1 homodimerization occurring as a result of an interaction of the NTD with the CTD. Similarly, coexpression of wild-type or mutant FLAG-DAX1-C with myc-DAX1 followed by Co-IP with a FLAG antibody showed nearly a 50% decrease in the amount of myc-DAX1 pulled down by FLAG-mAF-2:DAX1-C compared with wild type. These results indicate that the AF-2 domain is involved in DAX1 homodimerization.
View larger version (31K):
[in this window]
[in a new window]
|
Fig. 7. AF-2 Domain Is Involved in DAX1 Homodimerization
A, Schematic of FLAG- (F) and myc- (M) DAX1 constructs used in Co-IP assays. DAX1-N and DAX1-C residue numbers are as in Fig. 5. AF-2 residues are in superscript. AF-2 point mutations are in bold and underlined, and black and white boxes represent wild-type and mutant AF-2 domain, respectively. B, Co-IP assay. HEK293 cells were transfected with the indicated combinations of FLAG- and myc-epitope tagged expression constructs (2 µg each plasmid). Plasmid levels were balanced with empty vector. Whole-cell extracts were subjected to IP with FLAG antibody; 25% of the IP reaction was resolved by SDS-PAGE and analyzed by Western blotting (WB) with FLAG and myc antibodies. Signal was quantitated by laser densitometry. Data for mutants are presented as percent of wild-type protein (100%) bound. Values shown are mean ± SD.
|
|
SHP Forms Homodimers in the Nucleus of Mammalian Cells
Our observation of DAX1 homodimerization led us to hypothesize that its related NR0B family member SHP might also exist as a homodimer. To determine whether SHP formed homodimers in mammalian cells, co-IP assays were performed on whole-cell extracts of HEK293 cells transfected with FLAG-SHP and myc-SHP. Using a FLAG antibody, myc-SHP was coimmunoprecipitated in the presence, but not in the absence, of FLAG-SHP (Fig. 8A), indicating homodimerization of SHP in these cells. The subcellular localization of the SHP homodimer was investigated through co-IP analyses of nuclear and cytoplasmic fractions of transfected HEK293 cells. Consistent with previous studies (23, 35), Western blot analysis with FLAG and myc antibodies showed that the subcellular localization of FLAG-SHP and myc-SHP was mostly nuclear (Fig. 8B). Hsp90 localized primarily in the cytoplasmic fraction, and endogenous c-myc was almost exclusively nuclear, indicating a relatively clean fractionation procedure (Fig. 8B). Co-IP of the cytoplasmic and nuclear fractions of cells coexpressing FLAG-SHP and myc-SHP using a FLAG antibody showed co-IP of myc-SHP with FLAG-SHP in the nuclear compartment (Fig. 8C), indicating that SHP homodimers existed almost exclusively in the nucleus.
View larger version (34K):
[in this window]
[in a new window]
|
Fig. 8. SHP Homodimerizes in the Nucleus of Mammalian Cells
A, SHP forms homodimers in mammalian cells. HEK293 cells were transfected with indicated combinations of FLAG- and myc-tagged expression constructs (2 µg each plasmid). Plasmid levels were balanced with empty vector. Whole-cell extracts were subjected to IP with FLAG antibody; 25% of the IP reaction was resolved by SDS-PAGE and analyzed by Western blotting with FLAG and myc antibodies. B, Nuclear and cytoplasmic fractions of HEK293 cells transfected with the indicated combinations of FLAG- and myc-epitope-tagged constructs (2 µg each plasmid) were subjected to Western blot (WB) analysis (1% of co-IP input) with FLAG, myc, and hsp90 antibodies. Plasmid levels were balanced with empty vector. Hsp90 and endogenous c-myc protein were used as cytoplasmic and nuclear markers, respectively. C, Nuclear and cytoplasmic fractions of transfected HEK293 cells were immunoprecipitated with FLAG antibody followed by Western blot analysis with FLAG and myc antibodies. The primary site of FLAG-SHP and myc-SHP interaction was in the nuclear fraction. Cyto, Cytoplasm; Nucl, nucleus.
|
|
The SHP Homodimer Dissociates upon Heterodimerization with ER in the Presence of Ligand
SHP has been shown to repress the action of ligand-activated ER through a mechanism involving a heterodimeric interaction similar to the DAX1 repression of ER (18, 23, 33, 34, 37). Because DAX1 homodimers dissociated in the presence of ligand-activated ER, it was possible that SHP homodimers could exhibit similar dynamics in the presence of ER. We first confirmed the ligand dependence of SHP-ER heterodimerization. In the presence of estradiol, HA-ER was coimmunoprecipitated in the presence, but not the absence, of FLAG-SHP (Fig. 9A). The dynamics of the SHP homodimer in relation to this heterodimeric interaction with ER were investigated through the protein interaction competition assay as described above for DAX1. A constant amount of FLAG-SHP and myc-SHP and increasing amounts of HA-ER were transfected into HEK293 cells in the absence or presence of estradiol. Western blot analysis of whole-cell extracts using FLAG, myc, and HA antibodies verified equal expression of FLAG-SHP and myc-SHP across all samples and increasing expression of HA-ER (data not shown). Co-IP using a FLAG antibody showed an efficient decrease in the amount of myc-SHP coimmunoprecipitated by FLAG-SHP in the presence of increasing amounts of HA-ER in a ligand-dependent manner (Fig. 9B). These results suggest that the SHP homodimer, similar to DAX1, dissociates upon heterodimerizing with ligand-activated ER, and these observations support a holding reservoir function for the SHP homodimer.
DAX1 and SHP Form Heterodimers in the Nucleus of Mammalian Cells
Heterodimeric interactions between nuclear receptor family members are known: the glucocorticoid and mineralocorticoid receptors have been shown to heterodimerize (50), and ER and ERß have been shown to form heterodimers that have distinct gene-regulatory functions (51). Because DAX1 and SHP were known to be coexpressed in certain tissues such as the adrenal gland (23), and both homodimerized and exhibited similar dynamics in the presence of a common nuclear receptor target, ER, we hypothesized that DAX1 and SHP might heterodimerize. Co-IP analyses on HEK293 whole-cell extracts expressing FLAG-DAX1 and myc-SHP using a FLAG antibody showed co-IP of myc-SHP in the presence, but not in the absence, of FLAG-DAX1 (Fig. 10A), demonstrating that DAX1 and SHP heterodimerize in addition to forming homodimers in mammalian cells.
View larger version (39K):
[in this window]
[in a new window]
|
Fig. 10. DAX1 and SHP Heterodimerize in the Nucleus of Mammalian Cells
A, Co-IP assays involved transfection of HEK293 cells with indicated combinations of FLAG- and myc-tagged expression constructs (2 µg each plasmid). Plasmid levels were balanced with empty vector. Whole-cell extracts were subjected to IP with FLAG antibody followed by Western blotting (WB) with FLAG and myc antibodies. B, Nuclear and cytoplasmic fractions of HEK293 cells transfected as in panel A were immunoprecipitated with FLAG antibody followed by Western blot analysis with FLAG and myc antibodies. The primary site of FLAG-DAX1 and myc-SHP interaction was in the nuclear fraction.
|
|
The subcellular localization of the DAX1-SHP heterodimer was investigated through co-IP analyses of nuclear and cytoplasmic fractions of transfected HEK293 cells. Western blot analysis confirmed that, as presented earlier, FLAG-DAX1 localized to both the nucleus and cytoplasm, myc-SHP was mostly nuclear, Hsp90 localized primarily in the cytoplasmic fraction, and endogenous c-myc was almost exclusively nuclear (data not shown). Co-IP of the cytoplasmic and nuclear fractions of cells coexpressing FLAG-DAX1 and myc-SHP using a FLAG antibody showed co-IP of myc-SHP with FLAG-DAX1 in the nuclear compartment (Fig. 10B), indicating that DAX1-SHP heterodimers existed almost exclusively in the nucleus.
LXXLL Motifs and AF-2 Domain of DAX1 Are Involved in DAX1-SHP Heterodimerization
Because DAX1 homodimerization involved the LXXLL motifs and AF-2 domain of DAX1, we hypothesized that DAX1-SHP heterodimerization could occur in a similar manner. Constructs expressing wild-type or LXXLL mutant (m123) FLAG-DAX1-N were cotransfected with myc-SHP (Fig. 11A) into HEK293 cells. Western blot analysis confirmed equal protein expression across all samples (data not shown). Co-IP using a FLAG antibody showed an 80% decrease in the amount of myc-SHP pulled down with FLAG-m123:DAX1-N compared with wild-type FLAG-DAX1-N (Fig. 11B). Constructs expressing wild-type or AF-2 mutant FLAG-DAX1-C were cotransfected with myc-SHP into HEK293 cells. Western blot analysis confirmed equal protein expression (data not shown). Co-IP using a FLAG antibody showed nearly a 60% decrease in the amount of myc-SHP pulled down with FLAG-mAF-2:DAX1-C compared with wild-type FLAG-DAX1-C (Fig. 11C). These results indicate that the LXXLL motifs and AF-2 domain of DAX1 are involved in DAX1-SHP heterodimerization.
View larger version (27K):
[in this window]
[in a new window]
|
Fig. 11. LXXLL Motifs and AF-2 Domain of DAX1 Are Involved in DAX1-SHP Heterodimerization
A, Schematic of FLAG- (F) DAX1 and myc- (M) SHP constructs used in Co-IP assays. LXXLL and AF-2 point mutations are in bold and underlined, black and white bars represent wild-type and mutant LXXLL motifs, respectively, and black and white boxes represent wild-type and mutant AF-2 domain, respectively. B and C, LXXLL motif (B) and AF-2 domain (C) involvement in DAX1-SHP heterodimerization. HEK293 cells were transfected with the indicated combinations of FLAG- and myc-epitope-tagged expression constructs (2 µg each plasmid). Plasmid levels were balanced with empty vector. Whole-cell extracts were subjected to IP with FLAG antibody; 25% of the IP reaction was resolved by SDS-PAGE and analyzed by Western blotting (WB) with FLAG and myc antibodies. Signal was quantitated by laser densitometry. Data for mutants are presented as percent of wild-type protein (100%) bound. Values shown are mean ± SD.
|
|
DAX1 Forms Heterodimers with Alternatively Spliced Isoform DAX1A
An alternatively spliced isoform of DAX1, DAX1A (43, 44), has been reported to antagonize DAX1-mediated repression of SF1 transactivation through an interaction with SF1. Because we demonstrate homodimerization of DAX1, it is possible that DAX1A may homodimerize or form heterodimers with DAX1. Constructs expressing FLAG-DAX1 or FLAG-DAX1A (Fig. 12A) were cotransfected with myc-DAX1A into HEK293 cells. Co-IP with a FLAG antibody showed that myc-DAX1A is pulled down in the presence, but not in the absence, of FLAG-DAX1, indicating that DAX1 heterodimerizes with DAX1A (Fig. 12B). However, less than 1% input of myc-DAX1A is pulled down with FLAG-DAX1A, indicating only a weak DAX1A homodimerization.
View larger version (33K):
[in this window]
[in a new window]
|
Fig. 12. Alternatively Spliced Isoform DAX1A Forms Heterodimers with DAX1
A, Schematic comparing DAX1 and DAX1A. Note the lack of AF-2 domain in the CTD. DAX1A residues 390–400 make up a novel 10-amino acid motif (gray bar). B, Co-IP assays involved transfection of HEK293 cells with indicated combinations of FLAG- and myc-tagged expression constructs (2 µg each plasmid). Plasmid levels were balanced with empty vector. Whole-cell extracts were subjected to IP with FLAG antibody followed by Western blotting (WB) with FLAG and myc antibodies.
|
|
|
DISCUSSION
|
|---|
DAX1 and SHP Form Homodimers and Heterodimerize with Each Other and Their Nuclear Receptor Targets in Mammalian Cells
DAX1 and SHP function as repressors through heterodimeric interactions with other nuclear receptors (3, 7). In this study, we demonstrate that DAX1 homodimerizes in the cytoplasm and nucleus and that SHP exists as a homodimer in the nucleus of mammalian cells. This is the first report of homodimerization of members of the NR0B family, thus forming a new interactional paradigm for these unusual nuclear receptors. Homodimerization of DAX1 and SHP and also DAX1-SHP and DAX1-DAX1A heterodimerization represent additional potential complexity in the molecular mechanisms of action of this family of atypical nuclear receptors because it suggests the possibility of novel function(s) independent of other nuclear receptors.
DAX1 Homodimerization in the Cytoplasm and Nucleus May Serve Different Functions
Studies of DAX1 expression in cultured cells, in the developing mouse pituitary, and in embryonic stem cells show that DAX1 is present in the nucleus and cytoplasm (21, 48, 49, 52, 53), and we demonstrate DAX1 homodimerization in both of these subcellular compartments. Therefore, DAX1 could have unique functions in these two compartments as a homodimer independent of its target nuclear receptor heterodimeric partners, perhaps through the proposed RNA binding and posttranscriptional function of DAX1 (48), the regulation of cytoplasmic signaling pathways (54), binding a novel DNA response element to repress transcription (3, 55), and/or by acting as a transcriptional activator via its AF-2 domain, as has been shown for the corepressors chicken ovalbumin upstream promoter transcription factor and SHP (24, 45). DAX1 shuttles between the nucleus and cytoplasm (48), and this may occur via active transport (53). It is unclear whether DAX1 is transported through the nuclear pore complex as a monomer or homodimer.
DAX1 Homodimerization Alters Concepts Regarding Its Heterodimeric Interactions
DAX1 inhibits the action of SF1 and ER by a direct protein-protein interaction followed by the recruitment of corepressor proteins (17, 18, 37, 40, 41). It becomes important to understand the dynamics of the DAX1 homodimer with respect to its known mechanisms of transcriptional repression and heterodimeric interactions. Our observation of decreased DAX1 homodimer formation in the presence of increasing amounts of SF1 suggests that DAX1 represses SF1 as a monomer. In other words, the DAX1 homodimer would dissociate to form heterodimers with SF1 to repress its transactivation. These data suggest that the DAX1 homodimer may function as a holding reservoir for the DAX1 protein in the absence of its nuclear receptor targets (Fig. 13A). SF1 transactivation is stimulated by protein kinase A signaling, and the DAX1-SF1 interaction may be weakened by stimulation, which would favor SF1 transactivation (56). Based on our results, the alternative hypothesis must be considered that protein kinase A signaling, in addition to promoting dissociation of DAX1-SF1 heterodimers, could also promote the formation of DAX1 homodimers.
View larger version (34K):
[in this window]
[in a new window]
|
Fig. 13. DAX1 and SHP Homodimers May Function as Holding Reservoirs in the Absence of Nuclear Receptor Targets
A and B, DAX1 exists as a homodimer in the cytoplasm and the nucleus (left). The DAX1 homodimer dissociates and forms heterodimers with SF1 (A) or ligand-activated ER (B) likely in the nucleus to carry out its repressor function (right). C, SHP homodimers exist primarily in the nucleus (left) and dissociate in the presence of ligand-activated ER to function as a repressor (right). This suggests a holding reservoir function for the DAX1 and SHP homodimers. DAX1 and SHP homodimers may also have novel functions independent of their nuclear receptor targets. E2, Estradiol.
|
|
Because the dynamics of the DAX1 homodimer could be different with respect to its action on nuclear receptors that bind DNA and activate transcription as homodimers (e.g. ER) vs. monomers (e.g. SF1), we tested whether DAX1 homodimer formation decreases in the presence of ER. We demonstrate similar dynamics as with SF1. In the presence of ligand, the DAX1 homodimers dissociate to form heterodimers with activated ER, providing further evidence for a holding reservoir function for the DAX1 homodimer. It is possible that one DAX1 molecule could interact with the ER homodimer, forming a ternary complex, or one DAX1 molecule could interact with each unit of the ER homodimer forming a tetrameric complex (Fig. 13B).
DAX1-mediated repression of its other recognized targets, LRH1, AR, PR, NGFI-B, and ER-related receptor , could also involve dissociation of the DAX1 homodimer. However, because repression of these receptors may occur via mechanisms distinct from that of SF1 and ER, and could possibly involve different heterodimerization interfaces (7, 19, 20), the dynamics of the DAX1 homodimer may differ in these situations.
The AF-2 domain has been reported to influence the nuclear localization of DAX1 (49, 53), where deletion of the AF-2 and many point mutations within the AF-2 cause DAX1 subcellular localization to be predominantly cytoplasmic. This altered localization is thought to be the cause of the diminished repressor activity reported for these mutants. Lehmann et al. (57) report a detailed analysis of the nuclear localization and repressor activity of a series of DAX1 AF-2 mutants. The AF-2 mutant used in our studies, M461A/M462A, was shown to have reduced transcriptional repression ability of SF1 while maintaining a predominantly nuclear localization and wild-type expression levels. We showed that this mutant exhibits decreased homodimerization. It is possible that the reduced repression observed by Lehmann et al. (57) could be due to the diminished homodimerization of this mutant. Further detailed analysis is required to investigate this hypothesis.
SHP Homodimerization in the Nucleus Alters Concepts Regarding Its Heterodimeric Interactions and Mechanisms of Repression
SHP homodimerization in the nucleus suggests that it may have a novel function independent of its heterodimeric partners that may involve binding an uncharacterized DNA response element or possibly functioning as a transcriptional activator via its AF-2 domain. As with DAX1, the dynamics of the SHP homodimer now need to be considered when dissecting molecular mechanisms of SHP-mediated repression. With respect to SHP-mediated repression of ER, our competition assay shows dissociation of the SHP homodimer upon heterodimerization with ligand-activated ER, suggesting that SHP represses ER as a monomer and provides evidence for a holding reservoir function for the SHP homodimer (Fig. 13C). SHP-mediated repression of LRH-1, glucocorticoid receptor, RXR, and hepatocyte nuclear factor 4 occurs through a mechanism similar to repression of ER and involves similar heterodimerization interfaces (26, 29, 30, 58). Therefore, repression of these receptors by SHP may likely involve dissociation of the SHP homodimer. SHP-mediated repression of pregnane X receptor, liver X receptor, AR, NGFI-B, and also the basic helix-loop-helix transcription factor BETA2/NeuroD (25, 28, 31, 32, 35) appears to be more complex with respect to domains of the receptors involved in repression and heterodimerization. Repression of these receptors may involve different dynamics of the SHP homodimer, and whether SHP represses these receptors as a monomer or homodimer remains to be determined.
Heterodimerization of DAX1 and SHP Suggests Further Functional Complexity
Nuclear receptors function in different dimeric combinations, and heterodimers of nuclear receptor family members may have functional roles distinct from homodimers (51). DAX1 and SHP are both expressed in the fetal and adult adrenal glands (23, 59) and have also been shown to be coexpressed in an adrenocortical carcinoma cell line (59), although a spatio-temporal expression analysis has not been performed. Because DAX1 and SHP form homodimers and also heterodimerize with other nuclear receptors, we tested whether DAX1 and SHP could heterodimerize. We demonstrate the formation of DAX1-SHP heterodimers in the nucleus of mammalian cells. The function of the DAX1-SHP heterodimer remains to be determined but is likely beyond that of a holding reservoir.
Reduced Interaction of SHP with DAX1 LXXLL and AF-2 Mutants Suggests a Role for These Motifs in DAX1-SHP Heterodimerization
DAX1 heterodimerization with SF1 or ER has been shown to take place between the LXXLL motifs in DAX1 and the C-terminal AF-2 domain in SF1 and ER (18, 33, 37). The competition of DAX1 homodimer formation with SF1 or activated ER and our Y2H results showing a strong interaction between the DAX1 NTD and CTD are consistent with DAX1 forming an antiparallel homodimer. We further demonstrate that DAX1 homodimerization involves an interaction of the LXXLL motifs in the NTD with the AF-2 domain in the CTD. Thus, residues involved in heterodimerization with SF1 and ER are involved in DAX1 homodimerization, and that DAX1 homodimerizes differently from many other members of the superfamily, which form parallel homodimers through contacts in both the DBD and LBD (1, 3). This is not unprecedented in the nuclear receptor superfamily because the AR has been shown to form an antiparallel homodimer via similar motifs (60). A weak interaction detected between two DAX1 NTDs suggests that contacts in the NTD may be involved in stabilization of the homodimer. In addition, whereas mutation of the LXXLL motifs nearly abolishes DAX1 homodimerization, mutation of the AF-2 domain does not completely abolish DAX1 homodimerization, suggesting the involvement of other residues in the CTD in stabilization of the homodimer.
DAX1 contains three LXXLL motifs in its NTD, and it is possible that one or more of these motifs are primarily involved in DAX1 homodimerization. Suzuki et al. (18) report that the first and third motifs are primarily involved in DAX1-SF1 heterodimerization. It is possible that these same motifs could be involved in DAX1 homodimerization or DAX1-SHP heterodimerization, or the motifs could exhibit different target specificities depending on the homodimeric or heterodimeric partner.
We showed that DAX1 also heterodimerizes with SHP through its LXXLL motifs and AF-2 domain. SHP heterodimerizes through its LXXLL motifs with the AF-2 domain of ER (33). Similar to DAX1, SHP also contains an AF-2 domain. Because we observe a competition of SHP homodimer formation with ER in our assay similar to DAX1, and demonstrate the involvement of the DAX1 LXXLL motifs and AF-2 domain in DAX1-SHP heterodimerization, it is very likely that SHP homodimerization and heterodimerization with DAX1 could occur as a result of an interaction of its LXXLL motifs and the AF-2; therefore, SHP residues involved in heterodimerization with ER are very likely involved in SHP homodimerization and DAX1-SHP heterodimerization.
DAX1 Homodimerization Suggests Additional Functional Complexities
Additional complexity in the mechanisms of DAX1 action has been suggested through the discovery of an alternatively spliced isoform of DAX1, DAX1A (43, 44). DAX1A does not repress SF1 on its own but can antagonize the repressive action of DAX1 on SF1-mediated transcriptional transactivation, possibly as a result of DAX1A competing with DAX1 for binding to SF1 (44). Different isoforms of nuclear receptors have been shown to heterodimerize with each other, such as PR-A and PR-B (61). We show that DAX1 and DAX1A form DAX1-DAX1A heterodimers. The antagonistic action of DAX1A on the repressor activity of DAX1 could thus be due to increased DAX1-DAX1A heterodimer formation, with DAX1A interfering with the interaction between DAX1 and SF1. This would be somewhat analogous to one of the mechanisms proposed to explain the effect of DAX1 on the SF1/WT1 synergistic association (62). The DAX1-DAX1A interaction probably results from the LXXLL motifs in DAX1A interacting with the AF-2 domain of DAX1. A significant homodimeric interaction for DAX1A was not observed and is likely due to the lack of an AF-2 domain in DAX1A. The DAX1-DAX1A heterodimer suggests another layer in the complexity of DAX1 action.
Because the DAX1-DAX1 and SHP-SHP interactions detected in our studies involved at least two receptor molecules, we refer to them as homodimers because the vast majority of nuclear receptors exist and/or function as homo- or heterodimers. However, there are exceptions; for example, RXR has been shown to form tetramers (63), and thus we are not able to exclude formally the possibility of the formation of higher order DAX1 or SHP multimeric complexes in the cell.
These novel interactions within and between the two members of the NR0B family provide mechanistic insight into the complexity of molecular pathogenesis of disorders involving mutations of DAX1 and SHP. Our group has previously studied the role of hierarchical scale-free networks in the molecular mechanisms involving AHC as a consequence of DAX1 mutations (59, 64). This network was constructed based on direct and indirect interactions of DAX1 with its network partners, as we have described for proteomic networks more generally (65), and is therefore relevant to SHP and its network interactions. The work reported here indicates interactions between the DAX1 and SHP networks, as well as new interactions within each of these networks in the form of the respective homodimers. The results we present here show novel interactions between and within these NR0B transcriptional networks and therefore provide molecular insights into the pathogenic mechanisms underlying DAX1-associated AHC and SHP-associated mild obesity and insulin resistance.
Disruption of the known actions of DAX1 as a repressor of nuclear receptor transactivation in the HPAG axis does not sufficiently explain the phenotype of AHC/hypogonadotropic hypogonadism patients, because the significance of this repressor function in the normal development of the HPAG axis is unclear. SHP is involved in many metabolic pathways, but how mutations lead to mild obesity and insulin resistance is not fully understood. Homodimerization of DAX1 and SHP adds complexity to their known mechanisms as transcriptional corepressors. In addition, our results indicate the exciting possibility of a novel function for these unusual members of the nuclear receptor superfamily.
|
MATERIALS AND METHODS
|
|---|
Plasmids
pGBKT7 and pGADT7 expression constructs were used for Yeast Two Hybrid assays (MATCHMAKER Yeast Two Hybrid System 3, CLONTECH Laboratories, Inc., Mountain View, CA). pGBKT7 constructs encode Gal4 DBD (Gal4DB) fusions, and pGADT7 constructs encode GAL4 activation domain (Gal4AD) fusions. Human DAX1 full-length cDNA (DAX1) was cloned by PCR (Platinum Pfx DNA Polymerase; Invitrogen, San Diego, CA) into the EcoRI and BamHI sites of both pGBKT7 and pGADT7 to generate Gal4DB-DAX1 and Gal4AD-DAX1, respectively. The amino-terminal half of DAX1, DAX1-N (residues 1–198), was cloned by PCR into the EcoRI and BamHI sites of both pGBKT7 and pGADT7 to generate Gal4DB-DAX1-N and Gal4AD-DAX1-N, respectively. The carboxy-terminal half of DAX1, DAX1-C (residues 207–470), was cloned by PCR into the NdeI and BamHI sites of both pGBKT7 and pGADT7 to generate Gal4DB-DAX1-C and Gal4AD-DAX1-C, respectively. Human SF1 full-length cDNA (SF1) was cloned into the EcoRI and BamHI sites of both pGBKT7 and pGADT7 to generate Gal4DB-SF1 and Gal4AD-SF1, respectively. The full-length DAX1 and SF1 constructs were also used in the in vitro co-IP assays, because transcription and translation in vitro from the T7 promoter in pGBKT7 and pGADT7 leads to production of myc- and HA-epitope-tagged proteins, respectively, without the GAL4 domain. For mammalian cell expression, pShuttle-Flag3 (66) was used to express FLAG-tagged proteins, pCMVTag3C (Stratagene, La Jolla, CA) to express myc-tagged proteins, and pCruzHA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) to express hemagglutinin (HA)-tagged proteins. DAX1 was subcloned from Gal4DB-DAX1 by PCR into the BamHI and AflII sites of pShuttle-Flag3, and the BamHI and EcoRI sites of pCMVTag3C. DAX1-N and DAX1-C were subcloned from Gal4DB-DAX1 by PCR into the BamHI and AflII sites of pShuttle-Flag3. SF1 was subcloned from Gal4DB-SF1 by PCR into the BamHI and KpnI sites of pShuttle-Flag3, the BamHI and EcoRI sites of pCMVTag3C, and the KpnI and EcoRV sites of pCruzHA. Human small heterodimer partner (SHP) full-length cDNA (Origene Technologies, Rockville, MD) was subcloned by PCR into the BamHI and KpnI sites of pShuttle-Flag3 and the BamHI and EcoRI sites of pCMVTag3C. Human ER full-length cDNA (Origene Technologies) was subcloned by PCR into the KpnI and EcoRV sites of pCruzHA. DAX1A was cloned by RT-PCR from an ovary cDNA library (CLONTECH) into pCR 2.1 (Invitrogen) and subcloned by PCR into the BamHI and AflII sites of pShuttle-Flag3, and the BamHI and EcoRI sites of pCMVTag3C. All clones were verified by sequencing. Mutations in DAX1 were created using the QuikChange Site-Directed Mutagenesis System (Stratagene) using PCR primers containing the indicated mutations. Mutations were verified by sequencing.
Y2H Assays
Yeast strain AH109 (MATCHMAKER Yeast Two Hybrid System 3) was used for qualitative Y2H assays utilizing growth selection of the auxotrophic HIS3 reporter. For plasmid selection in yeast, Gal4DB fusion constructs carried the TRP1 gene and Gal4AD fusion constructs carried the LEU2 genes. Yeast were transformed using the YEASTMAKER Yeast Transformation System 2 (CLONTECH) with combinations of Gal4DB and Gal4AD constructs as indicated in figure legends and plated on Sabouraud dextrose (SD) agar plates lacking leucine and tryptophan to select for cotransformants, and on SD agar plates lacking leucine, tryptophan, and histidine, to select for cotransformants and the expression of the HIS3 reporter gene. Plates were incubated at 30 C for 6 d. Y2H assays utilizing DAX1-N and DAX1-C constructs were performed in the presence of 2.5 mM 3-amino-1,2,4-triazole, a HIS3 inhibitor, to eliminate background growth. For quantitative Y2H assays, yeast strain Y187, which contains a ß-galactosidase reporter, was transformed with combinations of Gal4DB and Gal4AD fusion constructs, plated on SD agar plates lacking leucine and tryptophan, and incubated at 30 C for 6 d. Ten cotransformed colonies of each combination were then grown in liquid SD media lacking leucine and tryptophan and assayed in triplicate in a liquid ß-galactosidase assay using o-nitrophenyl-ß-D-galactopyranoside as a substrate (Yeast Protocols Handbook, CLONTECH). Activity was normalized to cell culture density (OD600) and assay time (Yeast Protocols Handbook, CLONTECH). Statistical analyses were performed using a paired t test with P < 0.05 to indicate significance. Values represent the mean ± SD.
In Vitro Co-IP Assays
In vitro transcription and translation of Gal4DB-DAX1, Gal4AD-DAX1, and Gal4DB-SF1 were performed using the TnT Quick Coupled Transcription/Translation System (Promega Corp., Madison, WI) to generate myc-DAX1, HA-DAX1, and myc-SF1, respectively. In vitro co-IP assays were performed using the MATCHMAKER Co-IP Kit (CLONTECH) with some modifications. Co-IP assays were performed with 1 µg of c-myc monoclonal antibody (clone 9E10, Santa Cruz Biotechnology). Bound proteins were washed five times with 300 µl Wash Buffer 1 and twice with 300 µl Wash Buffer 2, eluted by boiling in 2x Laemmli buffer, and 50% of the IP reaction was analyzed by 12% SDS-PAGE. The proteins were transferred to a polyvinylidine difluoride (PVDF) membrane followed by Western analysis with anti-HA-horseradish peroxidase (HRP) (clone HA-7; Sigma Chemical Co., St. Louis, MO) and visualization with ECL Plus Western Blotting Detection System (Amersham Biosciences, Piscataway, NJ).
Mammalian Cell Culture and Transfections
Human embryonic kidney 293 (HEK293) cells (BD Biosciences, San Jose, CA) were cultured in DMEM containing 10% fetal bovine serum (FBS) and antibiotics at 37 C in 5% CO2. Transfections were performed in six-well plates. Cells were plated at 70–80% confluence 24 h before transfection. Cells were transfected with appropriate expression plasmids (as indicated in figure legends) with quantities according to the manufacturer’s recommendations using Lipofectamine 2000 (Invitrogen). For transfections involving ER, cells were plated 24 h before transfection in phenol red-free DMEM containing 10% charcoal/dextran-treated FBS and antibiotics. Five hours after transfection, the medium was removed and replaced with phenol red-free DMEM with charcoal/dextran-treated FBS in the absence (EtOH) or presence of 1 µM 17ß-estradiol (Sigma). Whole-cell extracts were prepared 36 h post-transfection in RIPA lysis buffer [0.05 M Tris HCl (pH 7.4), 0.15 M NaCl, 1% Nonidet P-40, 1 mM EDTA] in the presence of protease inhibitors (Halt Protease Inhibitor Cocktail EDTA-free; Pierce Biotechnology, Rockford, IL). Nuclear and cytoplasmic fractions were prepared 36 h post-transfection using the NE-PER Nuclear and Cytoplasmic Extraction system (Pierce). Protein concentration was measured using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Inc., Hercules, CA). Extracts and fractions were then analyzed for protein expression by Western blotting or used in co-IP assays.
Western Blotting
Whole-cell extracts and nuclear and cytoplasmic fractions were resolved by 10% SDS-PAGE (DAX1, SF1, and ER detection) or 13% SDS-PAGE (SHP detection), transferred to PVDF membrane, and subjected to Western analysis using anti-FLAG M2 Peroxidase Conjugate (Sigma), anti-c-myc-HRP (clone 9E10, Santa Cruz Biotechnology), anti-HA-HRP (Sigma), anti-Hsp90 (clone AC88, Stressgen Biotech Corp., Victoria, British Columbia, Canada), and antimouse IgG-HRP (Stressgen) as indicated in the figures. Membranes were visualized with ECL Plus Western Blotting Detection System (Amersham).
Co-IP Assays in Mammalian Cells
HEK293 whole-cell extracts (1 mg) or nuclear and cytoplasmic fractions (800 µg) were incubated with 20 µl anti-FLAG-M2 affinity gel (Sigma) overnight at 4 C. The resin was then washed three times with PBS, and bound proteins were eluted by boiling in 2x Laemmli buffer for 5 min; 25% of the IP reaction was resolved by SDS-PAGE. Proteins were transferred to PVDF membranes and analyzed by Western blotting. Signal quantitation was performed by laser densitometry (Molecular Dynamics, Inc., Sunnyvale, CA) with analysis using ImageQuant software (Amersham) on three independent experiments.
|
ACKNOWLEDGMENTS
|
|---|
We thank Peter Tontonoz for mammalian expression plasmids, and Pascal Bernard and Eric Vilain for valuable insights and helpful discussions.
|
FOOTNOTES
|
|---|
This work was supported by United States Public Health Service National Research Service Award GM07104 (to A.K.I.) and National Institutes of Health Grant R01 HD39322 (to E.R.B.M.).
Author Disclosure Summary: A.K.I., Y.-H. Z., and E.R.B.M. have nothing to declare.
First Published Online May 18, 2006
Abbreviations: AF-2, Activation function 2; AHC, adrenal hypoplasia congenita; AR, androgen receptor; co-IP, coimmunoprecipitation; CTD, C-terminal domain; DAX1, dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome, gene 1; DBD, DNA-binding domain; ER, estrogen receptor; FBS, fetal bovine serum; HA, hemagglutinin; HEK, human embryonic kidney; HPAG, hypothalamic-pituitary-adrenal-gonadal; HRP, horseradish peroxidase; Hsp, heat shock protein; IP, immunoprecipitation; LBD, ligand-binding domain; LRH, liver receptor homolog; NGFI-B, nerve growth factor-inducible gene B; NTD, N-terminal domain; PR, progesterone receptor; PVDF, polyvinylidine difluoride; RXR, retinoic X receptor; SF1, steroidogenic factor 1; SD, Sabouraud dextrose; SHP, small heterodimer partner; Y2H, yeast two hybrid.
Received for publication September 16, 2005.
Accepted for publication May 8, 2006.
|
REFERENCES
|
|---|
- Aranda A, Pascual 2001 A Nuclear hormone receptors and gene expression. Physiol Rev 81:1269–1304[Abstract/Free Full Text]
- Burris TP 2001 The nuclear receptor superfamily. In: Burris TP, McCabe ERB, eds. Nuclear receptors and genetic disease. San Diego: Academic Press; 1–57
- Giguere V 1999 Orphan nuclear receptors: from gene to function. Endocr Rev 20:689–725[Abstract/Free Full Text]
- Burris TP, Guo W, McCabe ER 1996 The gene responsible for adrenal hypoplasia congenita, DAX-1, encodes a nuclear hormone receptor that defines a new class within the superfamily. Recent Prog Horm Res 51:241–259; discussion 259–260[Medline]
- Zhang Z, Burch PE, Cooney AJ, Lanz RB, Pereira FA, Wu J, Gibbs RA, Weinstock G, Wheeler DA 2004 Genomic analysis of the nuclear receptor family: new insights into structure, regulation, and evolution from the rat genome. Genome Res 14:580–590[Abstract/Free Full Text]
- Seol W, Choi HS, Moore DD 1996 An orphan nuclear hormone receptor that lacks a DNA binding domain and heterodimerizes with other receptors. Science 272:1336–1339[Abstract]
- Iyer AK, McCabe ER 2004 Molecular mechanisms of DAX1 action. Mol Genet Metab 83:60–73[CrossRef][Medline]
- McCabe ERB 2001 Adrenal hypoplasias and aplasias. In: Scriver CR, Beaudet AL, Sly WS, Valle D, Childs B, Vogelstein B, eds. The metabolic and molecular bases of inherited disease. New York: McGraw-Hill; 4263–4274
- Zanaria E, Muscatelli F, Bardoni B, Strom TM, Guioli S, Guo W, Lalli E, Moser C, Walker AP, McCabe ER, Meitinger T, Monaco AP, Sassone-Corsi P, Camerino G 1994 An unusual member of the nuclear hormone receptor superfamily responsible for X-linked adrenal hypoplasia congenita. Nature 372:635–641[CrossRef]
TOP
ABSTRACT
INTRODUCTION
