A Complex Deoxyribonucleic Acid Response Element in the Rat Ca2+/Calmodulin-Dependent Protein Kinase IV Gene 5'-Flanking Region Mediates Thyroid Hormone Induction and Chicken Ovalbumin Upstream Promoter Transcription Factor 1 Repression

Yan-Yun Liu and Gregory A. Brent

Molecular Endocrinology Laboratory, Veterans Affairs Greater Los Angeles Healthcare System, Departments of Medicine and Physiology, UCLA School of Medicine, Los Angeles, California 90073

Address all correspondence and requests for reprints to: Gregory A. Brent, M.D., Molecular Endocrinology Laboratory Building 114, Room 230, 11301 Wilshire Boulevard, Los Angeles, California 90073. E-mail: gbrent{at}ucla.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) is regulated by T3 in a time- and concentration-dependent manner in the developing rat brain and plays an important role in neuronal-specific gene regulation. T3 treatment, but not retinoic acid (RA), stimulated endogenous CaMKIV mRNA 5-fold in mouse embryonic stem (ES) cells differentiated into neurons. We localized a region -750 to -700 in the CaMKIV gene 5'-flanking region that conferred T3 responsiveness and bound thyroid hormone receptor (TR), retinoic acid receptor (RAR), and chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TF1). T3 and RA treatment stimulated the CaMKIV hormone response element. Cotransfection of a COUP-TF1 expression vector repressed the T3 response and augmented the RA response. Mutational analysis identified three half-sites arranged in a direct repeat (AB) and overlapping inverted repeat (BC), required for functional induction and receptor binding. TR and RAR bound predominantly to the BC portion of the element and COUP-TF1 to the AB region, with a close correlation of binding and functional studies. COUP-TF1 binding did not influence TR/retinoid X receptor binding but modestly augmented RAR/retinoid X receptor binding. A single element confers T3 and COUP-TF1 regulation of CaMKIV expression.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
T3IS ESSENTIAL for normal neural development, as shown by the severe neurological consequence of thyroid hormone deficiency (1, 2, 3). Iodine deficiency results in both maternal and fetal hypothyroidism, and the neurological manifestations of mental retardation and motor defects, as well as growth retardation, are irreversible after birth (4, 5). Retinoic acid (RA) is required for normal development of the mammalian brain (6, 7). Premature exposure to RA results in a high incidence of birth defects, especially in neural tube-derived structures (8). T3 and RA are known to regulate neuronal outgrowth and synapse formation in vivo and in cell culture systems (9, 10).

A number of T3-responsive genes have been identified in the cerebellum, including calbindin, inositol 1,4,5-triphosphate receptor, Purkinje cell protein-2 (PCP-2), and myelin basic protein, and share a remarkably similar pattern of expression (2). In hypothyroid animals, expression of these genes in the postnatal period is delayed, but they are ultimately expressed at wild-type levels by postnatal d 30–45 (2, 11). The cerebellar structure, however, is abnormal indicating that correct timing of expression of these genes is essential for normal development. The postnatal increase in T3 responsiveness in the cerebellum correlates with reduced expression of chicken ovalbumin upstream-transcription factor 1 (COUP-TF1) (12, 13). A site in the PCP-2 gene promoter has been identified that binds COUP-TF1 and in in vitro studies mediates inhibition of T3-stimulated expression by COUP-TF1 (12, 13). These studies indicate that COUP-TF1 modulates T3 action in the developing cerebellum, exerting primarily an inhibitory action.

Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) is a multifunctional enzyme that plays an important role in neuronal Ca2+ signaling and gene regulation (14). There are two isoforms of CaMKIV, {alpha} (67 kDa) and ß (65 kDa) (15). The ß-form differs from the {alpha}-form by the presence of a 28-amino acid N-terminal extension (16, 17). The {alpha}-form is expressed early in development, and it is the only CaMKIV isoform detected in E15 rat brain (18).

CaMKIV is abundant in developing brain (especially in the granule cells of the cerebellum), thymus, and testis (19, 20, 21). CaMKIV protein is predominantly localized in the nucleus, consistent with a role in the transcriptional regulation of Ca2+-activated genes (20). CaMKIV mediates Ca2+-dependent transcriptional activation primarily through phosphorylating transcription factors such as CREB (cAMP-response element binding protein) (22, 23, 24, 25), activating transcription factor-1 (26), serum response factor (27), nuclear factor-{kappa}B (28), and CREB binding protein (25). CREB binding protein contains a transcriptional activation domain that is regulated by nuclear calcium, cAMP, and CaMKIV (25). Recent studies in hippocampal neurons indicate that CaMKIV is the primary in vivo catalyst for CREB phosphorylation (29). CaMKIV-dependent activation of CREB is a critical step for mediating many genes involved in the process of T cell activation and neuronal plasticity (30, 31). CaMKIV plays a role in long-term potentiation in the rat hippocampus (32). CaMKIV is involved in neuronal communication by regulating neurotransmitter cytoskeletal components (33, 34, 35). In addition to phosphorylation of transcription factors, several studies have reported that CaMKIV is directly involved in regulating gene expression of the tumor necrosis factor family (36), CD5 signaling (37), type I adenylyl cyclase, a neuron specific enzyme promoting synaptic plasticity (38), and enhancing retinoic acid receptor (RAR)-related orphan receptor (ROR) transcription (39).

CaMKIV gene knockout mice have abnormalities in neurological, lymphoid, and reproductive tissues (40). The neurological deficits in CaMKIV-deficient mice include defects in two forms of synaptic plasticity: long-term potentiation in hippocampal CA1 neurons and a late phase of long-term depression in cerebellar Purkinje neurons (29). CaMKIV-deficient mice also exhibit impaired neuronal CREB phosphorylation and Ca2+/CREB-dependent gene expression (29).

CaMKIV is expressed in spermatogonia and spermatids, and targeted germ cells in rat and mouse testes. In germ cells, CaMKIV is associated with the chromatin, suggesting that CaMKIV is involved in chromatin remodeling of spermatids. Supporting evidence from CaMKIV-deficient mice showed that the chromatin condensation of spermatids is impaired (33, 41).

In situ hybridization studies indicate that CaMKIV is regulated by thyroid hormone in a time- and concentration-dependent manner in the developing rat brain (18). In cultured rat fetal telencephalon cells, CaMKIV is induced by addition of T3 (42). We have demonstrated regulation of endogenous CaMKIV by T3 in embryonic stem (ES) cells differentiated into neurons and identified a single complex element in the rat CaMKIV 5'-flanking region that confers this regulation. The isolated element is also stimulated by RA. COUP-TF1, which suppresses the T3 response and augments the RA response in vitro, binds to an overlapping sequence in the same site.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Endogenous Regulation of CaMKIV in Mouse ES Cells
We have previously reported on differentiation of mouse ES cells into neurons using T3 and RA (43, 44). ES cells were differentiated by treatment for 5 d with T3, RA, or T3/RA. Endogenous CaMKIV mRNA, as quantified by ribonuclease (RNase) protection assay, was increased 5-fold by T3 treatment (Fig. 1Go). There was no significant induction of CaMKIV mRNA from RA stimulation alone, although combined RA/T3 treatment induced to the level of T3 alone (Fig. 1Go). The specificity of T3 induction of CaMKIV mRNA expression in this differentiation system is demonstrated by a marked reduction of T3 induction of endogenous CaMKIV mRNA in ES cells with a thyroid hormone receptor (TR){alpha} gene dominant negative knock-in mutation (44).



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Figure 1. T3 and RA Regulation of the Endogenous CaMKIV Gene in Mouse ES Cells

Mouse ES cells were differentiated into neurons (as described in Materials and Methods) with T3 (1nM), RA (1 µM), or RA/T3 treatment for 5 d. Cells were harvested and total RNA extracted. The RNase protection assay (RPAIII kit, Ambion, Inc.), was performed with a PCR amplified CaMKIV probe, radiolabeled with 32P-uridine triphosphate, and hybridized with total RNA. The hybridized fragments were then recovered by precipitation and analyzed on 5% polyacrylaminde gel (A). The density of the CaMKIV and control (ß-actin) bands was determined for each condition (B). Results are the average of at least three experiments and shown as mean ± SD. *, P < 0.05 compared with untreated control.

 
T3 and RA Induction of CaMKIV 5'-Flanking Region in ES Cells
Reporter constructs containing the region to -1060 of the CaMKIV 5'-flanking region and progressive 5'-deletions (-460 and -350), were transiently transfected into ES cells (Fig. 2Go). ES cells contain endogenous TR and RAR sufficient for a T3 and RA response (43). T3 treatment induced the -1060 CaMKIV-chloramphenicol acetyltransferase (CAT) construct about 3-fold. Deletions to -450 and -360 eliminated the T3 response, indicating that the critical sequence for conferring T3 induction was in the region -1060 to -450. Similar studies with RA treatment showed no significant induction.



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Figure 2. Hormone Induction of Rat CaMKIV 5'-Flanking Region Reporter Constructs in Mouse ES Cells

Reporter constructs containing progressive 5' deletions of the rat CaMKIV 5'-flanking region upstream of a CAT reporter (A) were transfected into mouse ES cells. Fold induction (hormone-treated induction/without hormone) is shown for each construct 48 h after treatment with T3 (1 nM), RA (1 µM), or RA/T3, with and without cotransfected COUP-TF1 expression vector. ES cells were plated in a 24-well dish, and 0.4 µg of each construct, or empty vector were used in each transfection. CAT activity was analyzed 48 h after transfection. Results are the average of at least three experiments and shown as mean ± SD. *, P < 0.05 compared with untreated control.

 
COUP-TF1 has been shown to repress T3 induction of several genes expressed in the cerebellum (2). Cotransfection of COUP-TF1 completely repressed T3 induction of the -1060 CaMKIV-CAT construct but augmented the RA response (Fig. 2Go). COUP-TF1 induction of an RA response has been reported in other systems (45, 46).

To further test the influence of COUP-TF1 on the T3 and RA response, the -1060 CaMKIV-CAT-reporter construct was transfected into ES cells with combinations of hormonal stimulation (T3, RA, or T3/RA), with and without cotransfected vectors expressing COUP-TF1 and TR{alpha} (Fig. 3Go). Cotransfection of a TR{alpha}-expressing vector reversed COUP-TF1-dependent repression of the T3 response but did not significantly influence COUP-TF1-dependent augmentation of the RA response. We postulated that a single element could be conferring these actions of T3, RA, and COUP-TF1. Although RA was not shown to regulate the endogenous CaMKIV gene under the conditions studied, given the close connection between T3- and RA-regulated genes (46) and the RA induction seen in the presence of additional COUP-TF1, we characterized both the T3 and RA response of the element. Based on the endogenous T3 response of the CaMKIV gene and avid binding of TR/retinoid X receptor (RXR) (see below), we have characterized the element as a thyroid hormone response element (TRE).



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Figure 3. Hormone Induction of Rat CaMKIV -1060 Reporter Construct in Mouse ES Cells

The CaMKIV -1060 CAT reporter construct was transfected into mouse ES cells with hormone treatment and with and without cotransfection of COUP-TF1 and TR{alpha} expression vectors. Absolute expression (A) and fold induction (B) are shown for each construct 48 h after treatment with T3, RA, or RA. Methods are as described in the Fig. 2Go legend.

 
Functional Characterization of T3 and RA Induction of the CaMKIV Thyroid Response Element
Sequence inspection of the 5'-flanking region, -1060 to -450, showed only one region (-750 to -700), with consensus hexameric AGGTCA sequences for steroid-like receptor binding (47). There were three hexameric sequences arranged as an overlapping direct repeat with a 3-bp gap (AB) and an inverted repeat with a 4-bp gap (BC) pattern. The three hexamers match 5/6, 6/6, and 5/6 (opposite strand) to the AGGTCA hexameric sequence, respectively. A series of mutations were designed to map the critical bases for regulation by T3 and RA (Fig. 4Go). The elements were named with an asterisk after the mutated domain, as shown. The C hexamer designation could be shifted 3 bp in the 5' direction, based on sequence inspection, so mutational analysis was performed on the upstream C1* and downstream C2* residues.



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Figure 4. Sequence of CaMKIV 5'-Flanking Region -750 to -700 and Oligonucleotide Mutations

The wild-type sequence, ABC, is shown and the various mutant oligonucleotides with only the mutated base shown. The designation of binding domains is based on sequence inspection and mutational analysis. The mutant oligonucleotides are named based on an asterisk after the domain that is mutated.

 
Oligonucleotides containing the wild-type sequence and mutations were ligated upstream of a heterologous promoter in a luciferase reporter construct and transfected into ES cells (Fig. 5Go). The wild-type element (ABC) conferred regulation by T3, 5.8-fold, greater than with the -1060 promoter construct. Up-regulation of the isolated element (ABC) was also seen in response to RA (2.8-fold), less than the induction seen with T3. Mutation of the middle "B" (AB*C) domain reduced T3 induction to 2.5-fold. Mutations of the downstream C domain alone (ABC*1, ABC*2, or ABC*1,2), or in combination with B domain mutations (AB*C*2 or AB*C*1,2), eliminated the T3 response. RA induction had a slightly different pattern as a consequence of mutations, compared with the T3 response. Mutations of the middle "B" domain (AB*C) as well as the C domain (ABC*2 and ABC*1,2), eliminated RA induction. The ABC*1 mutation, however, retained some RA induction.



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Figure 5. Hormone Induction of Rat CaMKIV Response Element and Mutations Upstream of Reporter Constructs in Mouse ES Cells

Reporter constructs containing CaMKIV hormone response element with mutations upstream of a luciferase reporter construct were transfected into mouse ES cells. Fold induction (hormone-treated induction/without hormone) is shown for each construct 48 h after treatment with T3, RA, or RA/T3.

 
Functional Testing of COUP-TF1 Action
The influence of COUP-TF1 cotransfection on T3 and RA induction of the CaMKIV ABC element was studied (Fig. 5Go). Repression of T3 induction and augmentation of the RA-response was seen with the wild-type ABC-element, as was demonstrated with the full -1060 5'-flanking region construct. A further COUP-TF1 effect was not seen with the B and C domain mutations that eliminated T3 and RA induction.

COUP-TF1 has been shown to bind as a dimer to a direct hexameric repeat with variable spacing, with greatest binding to a DR1 arrangement (46, 48). COUP-TF1 can also heterodimerize with TR, RAR, and RXR, depending on the nature of the DNA element (46, 48, 49). Binding studies demonstrated COUP-TF1 binding almost exclusively to the AB site (see below). The functional analysis of the domain mutants in regard to COUP-TF1 repression was difficult to interpret. Selective mutations of the A domain resulted in significantly increased basal expression in the presence of unliganded thyroid hormone or RAR, such that ligand-induced expression could not be reliably demonstrated (data not shown). Because the measurable action of COUP-TF1 in this system is repression of the T3 response or augmentation of RA induction, it was not possible to selectively impair COUP-TF1 action on mutated elements with preserved T3 and RA response.

TR Binding to the CaMKIV Hormone Response Element
The ability of the CaMKIV element to bind TR and TR/RXR heterodimers was tested by gel retardation assay. TR{alpha} and RXRß were expressed in reticulocyte lysate system and bound to a labeled ABC wild-type CaMKIV element (-750 to -700) (Fig. 6Go). TR bound alone and was supershifted with a TR{alpha} antibody. Combined TR/RXR bound with much greater intensity compared with TR alone and was also shifted by addition of TR{alpha} antibody. The identified element was shown to bind TR and TR/RXR heterodimers, as well as confer T3 induction to a heterologous promoter.



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Figure 6. Binding of TR and RXR to the CaMKIV TRE

TR and RXR were expressed in a reticulocyte lysate system and bound to 32P-labeled wild-type CaMKIV hormone response element. Anti-TR{alpha} antibody was included in binding reaction (lanes 2 and 4) to supershift the bound complex. Arrows show the position of complexes: a, TR; b, TR/TR antibody; c, TR/RXR; and d, TR/RXR/TR antibody.

 
Binding of TR, RAR, and COUP-TF1 to the CaMKIV TRE
The direct binding of receptors to the functionally defined hormone response element was evaluated by gel retardation assay. Receptor was synthesized in a reticulocyte lysate system and bound to the labeled wild-type element -750 to -700. The mutated elements (Fig. 4Go) were used to compete with receptor binding to the labeled wild-type element (ABC). In this type of assay, reduced competition by the mutated oligonucleotide, compared with wild-type, indicates that the mutated bases are critical for receptor binding. Effective competition by the mutated oligonucleotide, similar to ABC, indicates that the mutated bases are not important for receptor binding. ABC competed very effectively for TR and RAR binding (Fig.7Go, A–C). In general, the binding results for TR and RAR closely matched the functional analysis for T3 and RA induction. In the case of TR binding, the AB*C, ABC*2, ABC*1,2, and AB*C*2 showed a similar level of reduced competition, with the ABC*1 mutation only modestly impairing TR competition. The AB*C*1,2 mutation competed for TR only at the very highest concentrations. Similarly for RAR, the AB*C, ABC*1,2, and AB*C*2 had a similar reduction in competition, and the AB*C*1,2 mutant oligonucleotide competed only at the highest ratios. The ABC*1 element impaired competition, indicating that it is involved in RAR binding.



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Figure 7. Gel Retardation Analysis of TR and RAR Bound to CaMKIV Response Element

TR{alpha}1 and RARß proteins were prepared by in vitro transcription/translation (Promega Corp. TNT kit) and tested for binding to 32P-labeled ABC oligonucleotide. The binding assay included 4–6 µl of in vitro translated protein and 32P-labeled oligo-TRE (10,000 cpm) in a 20-µl reaction at room temperature for 15 min. The reactions were loaded on a 5% nondenaturing polyacrylamide gel (29:1) made in 1x TBE buffer and electrophoresed at constant 150 voltage for 2 h. The gel was dried, exposed to a PhosphorImager screen, and the protein/DNA complex quantified by PhosphorImager. Results are shown for TR (A), RAR (B), and the quantification of density (C). In all conditions, the wild-type element was labeled, and the competing oligonucleotide was the wild-type or mutant oligonucleotides. In this assay, impaired competition indicates that the mutated bases are important for receptor binding.

 
The same studies were performed with the addition of RXR (Fig. 8Go). The CaMKIV element bound much more TR/RXR compared with RAR/RXR. The competition studies showed similar binding patterns to TR or RAR alone, with a few exceptions. The B domain mutations were even more potent inhibitors of TR/RXR binding compared with TR alone and suggests that the B domain plays a central role in binding the TR/RXR complex. The ABC*1 mutation that impaired competition for RAR did not appear to interfere with RAR/RXR binding.



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Figure 8. Gel Retardation Analysis of TR, RAR, and RXR Bound to CaMKIV Response Element

TR{alpha}1, RARß, and RXRß proteins were prepared by in vitro transcription/translation (Promega Corp. TNT kit) and tested for binding to 32P-labeled ABC oligonucleotide (as described in Fig. 7Go). Results are shown for TR/RXR (A), RAR/RXR (B), and the quantification of density (C). In all conditions, the wild-type element was labeled, and the competing oligonucleotide was the wild-type or mutant oligonucleotides. In this assay, impaired competition indicates that the mutated bases are important for receptor binding.

 
COUP-TF1 Binding to the CaMKIV TRE
The functional analysis showed that COUP-TF1 repressed T3 induction and augmented RA induction. We first determined if COUP-TF1 directly bound DNA in the region of the TRE. COUP-TF1 directly bound to the TRE, and was competed by excess wild-type element (Fig. 9Go). The pattern of binding, however, was significantly different from TR and RAR. Mutations that included the upstream elements, A*B*C, A*BC, and A*B*C, completely inactivated the ability of the elements to compete COUP-TF1. Mutations of the downstream hexamer, ABC*1,2, had no effect on competition. These results indicates that COUP-TF1 binds predominantly to a direct repeat element AB separated by a 3-bp gap, and shares an overlapping hexamer B with the predominant TRE/RA response element, BC.



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Figure 9. Gel Retardation Analysis of COUP-TF1 Bound to CaMKIV Response Element

COUP-TF1 protein was prepared by in vitro transcription/translation (Promega Corp. TNT kit) and tested for binding to 32P-labeled ABC oligonucleotide (performed as described in Fig. 7Go legend). Results are shown for COUP-TF1 binding (A) and the quantification of density (B). In all conditions, the wild-type element was labeled, and the competing oligonucleotide was the wild-type or mutant oligonucleotides. In this assay, impaired competition indicates that the mutated bases are important for receptor binding.

 
COUP-TF1 Interactions with TR and RAR
We next determined the influence of COUP-TF1 on TR/RXR and RAR/RXR binding and the nature of the bound complexes (Fig. 10Go). COUP-TF1 bound as a dominant single band that was specifically eliminated by addition of anti-COUP-TF1 antibody (Fig. 10Go, A and B). TR bound the element as a single band and addition of RXR produced an intense RXR/TR homodimer. COUP-TF1 bound the element with a distinct band competed by addition of the anti COUP-TF1 antibody but did not influence the TR/RXR heterodimer (Fig. 10AGo). RAR bound the element as a single band with a shift upwards and increase in intensity with addition of RXR but significantly less than the intensity of the TR/RXR band. COUP-TF1 addition produced a modest increase in the intensity of the band that was diminished with addition of anti-COUP-TF1 antibody (Fig. 10BGo).



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Figure 10. Gel Retardation Analysis of COUP-TF1 Bound to CaMKIV Response Element with and without TR/RXR and RAR/RXR

TR, RAR, RXR, and COUP-TF1 proteins were prepared by in vitro transcription/translation (Promega Corp. TNT kit) and tested for binding to the wild-type 32P-labeled ABC oligonucleotide. The binding assay was performed as described in Fig. 7Go. Anti-COUP-TF1 antibody was added to selected lanes as shown.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Despite the profound influences of thyroid hormone on neurological development and function, only a modest number of genes have been identified that are directly regulated by thyroid hormone (1, 2). We have demonstrated endogenous regulation of the CaMKIV gene in neurologically differentiated ES cells and mapped an element that binds TR and confers T3 induction. The T3 induction of CaMKIV in a neuronal system confirms earlier findings in developing rat brain models (18, 42, 44).

The expression of the -1060 CaMKIV construct in ES cells was significantly higher than that reported previously in HeLa cells and allowed the mapping of the TRE (50). In HeLa cells, the overall expression of the -1060, -500, and -460 5'-flanking region constructs was very low, but the -350 construct was much greater (50). These findings suggest that there is an enhancer present or inhibitor absent in ES cells compared with HeLa cells. This is consistent with a fairly restrictive tissue pattern of expression of the CaMKIV gene (15).

The finding of RA induction and RAR binding to the same CaMKIV element as is induced by T3 is not surprising, given the closely related sequence and overlap of T3 and RA response elements (51). Although the absence of endogenous regulation by RA in our system suggests that RA may not be relevant for CaMKIV regulation, the augmentation of the RA response by COUP-TF1 indicates a possible role in regulation under specific circumstances. In general, the early phase of neural development is RA sensitive, and later stages are T3 sensitive (52). The early development stage is a time of higher COUP-TF1 levels, and later stages are associated with low COUP-TF1 levels (2, 12). The CaMKIV hormone response element, therefore, could promote RA or T3 regulation, depending on COUP-TF1 levels. The spacing of the half-sites influences coactivator recruitment and RA induction, with the greatest induction seen with a DR5 and least with DR1 (53, 54).

The CaMKIV hormone response element is similar to other complex elements that confer T3 response containing three to four interacting hexameric domains, including elements from the rat GH promoter (55), sarcoplamic endoplasmic reticulum calcium/adenosine triphosphatase (56), and PCP-2 (57) genes. The findings in most of these elements is that all sites interact to promote the T3 response. Although mutations of the BC inverted repeat domain with a 4-bp gap significantly impaired T3 induction and TR binding, there was complete inhibition only with mutations in both the 4-bp gap (C*2) and 1-bp gap (C*1) configurations. The binding pattern of RAR was similar to, but not identical to TR, with stronger binding to the BC 1-bp gap configuration (C*1). Mutations in the B domain influenced binding of TR/RXR more than T3 induction.

The element identified is similar to the PCP-2 element that confers T3 response (57). Studies of in vivo expression of PCP-2 (as well as other cerebellar genes) show that postnatal expression in hypothyroid animals is delayed, at a time when COUP-TF1 expression is high, but expression is restored as COUP-TF1 expression declines. In the PCP-2 gene, however, the regions that confer COUP-TF1-dependent T3 suppression is mediated by promoter sequences distinct from the TRE (12, 13). The CaMKIV element confers both T3 induction and COUP-TF1 repression in overlapping sequences.

COUP-TF1 is required for normal nervous system development including neuronal arborization, and the COUP-TF1 gene knockout in mice is a perinatal lethal mutation (58, 59). COUP-TF1 silencing function involves interactions with both N-CoR (nuclear receptor corepressor) and SMRT (silencing mediator of retinoid and thyroid receptors) (60). A recent study (61) showed that TR favors interactions with N-CoR and RAR favors recruitment of SMRT to DNA response elements. Preferential recruitment of SMRT by COUP-TF1, for example, could favor RAR activation. Although such a preference of COUP-TF1 for one corepressor has not been shown using two-hybrid and coimmunoprecipitation assays (60), the DNA response element may influence COUP-TF1 corepressor recruitment. SRC1 (steroid receptor coactivator) and N-CoR are differentially expressed during cerebellar development but are not regulated by thyroid hormone (62). COUP-TF1 interact with a number of other transcription factors, including ARP1 and Ear2 (63), and the newly identified zinc finger proteins, COUP-TF-interacting proteins 1 and 2 (64).

Several other candidates for genes that interact with TR/RAR or are regulated by T3 in the brain have been identified. The staggerer (sg) mouse, which does not express the orphan receptor ROR{alpha}, has cerebellar defects similar to those seen in hypothyroidism. Expression of TR in the developing cerebellum colocalizes with ROR{alpha} (65), and in vitro studies show that ROR{alpha} augments TR-mediated transcriptional activation (66). A recent study has shown that CaMKIV potentiates ROR{alpha} transcription 20- to 30-fold (39). Additional T3-regulated genes in the brain identified include hairless (hr), a zinc finger protein that binds to TR and inhibits thyroid hormone action (67), and thyroid hormone-responsive protein, a substrate for c-Abl tyrosine kinase activity (68).

The binding studies indicate that COUP-TF1 binds predominantly to the upstream AB domain of the CaMKIV TRE and TR/RXR and RAR/RXR to the BC domain. Although mutations of the BC domain completely eliminated TR and RAR binding and T3 and RA induction, it is possible that other sequences participate in the endogenous hormone response. The mechanism of COUP-TF1 repression of T3 action is not established from our studies, although several possibilities are suggested. COUP-TF1 bound in close proximity to the TR/RXR complexes that may allow it to effectively compete with TR/RXR for limiting coactivators or links to the basal transcriptional machinery. COUP-TF1 modestly augmented RAR/RXR binding and did not show a band reflecting independent binding to the TRE as was seen for TR/RXR. This configuration may promote augmentation of function for RA induction.

A wide range actions of CaMKIV have been recognized, especially in regard to transcriptional regulation (14, 15). The CaMKIV response element that we report binds TR and COUP-TF1 and confers T3 induction and COUP-TF1 repression to a heterologous reporter. The identification of the T3 response element should allow for further studies to determine the interaction of tissue-specific expression of the CaMKIV gene and T3 regulation. Finally, this element should provide a model to study the mechanism of COUP-TF1 repression of T3 induction and augmentation of the RA response. It has become increasingly recognized that the DNA response element configuration can influence nuclear receptor-cofactor interactions.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture and Differentiation Conditions
ES cells (J1 type) were cultured in DMEM with 20% FBS, 0.1 mM nonessential amino acids, and 0.1 mM ß-mecaptomethanol on a mouse primary embryonic fibroblast feeder cell layer in a 0.2% gelatin-coated tissue culture dish (69). ES cell neural differentiation was modified from a previously described technique (43, 44). ES cells were plated without feeder cells or gelatin coating to form embryoid bodies. To initiate neural differentiation, embryoid bodies were plated and grown in DMEM/F-12 medium supplemented with insulin, transferrin, sodium selenite, and with addition of hormones to a final concentration of 1 nM T3, 1 µM RA, or combined RA/T3. The hormonal treatment was continued for 5 d, followed by RNA isolation.

RNase Protection Assay
The RNase protection assay (RPAIII Kit, Ambion, Inc., Austin, TX), used a 259-bp PCR-amplified DNA probe from the CaMKIV cDNA, in vitro transcribed with 32P-uridine triphosphate labeling (Maxiscript Kit, Ambion, Inc.). The probe was gel purified and hybridized with total RNA isolated from ES cells with various hormone treatments. The nonhybridized RNA fragments and probe were digested with RNase, and the protected band recovered by precipitation and analyzed on a 5% polyacrylamide gel. The CaMKIV primers were: sense primer 5'-GGACAGCACAGATCTTCTGGG-3' and antisense primer with attached T7 promoter 5'-GGATCCTAATACGACTCACTATAGGGCCTCCACACTCTTCAGCTTC-3' (70). The bands of interest from the RNase protection assay were quantified by PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and normalized to the corresponding mouse ß-actin mRNA levels (also determined by RNase protection assay).

Plasmid Construction
The 5'-deletion constructs of the rat CaMKIV promoter were a gift of Drs. Christopher Kane and Anthony Means (Duke University Medical Center, Durham, NC). The oligonucleotide wild-type TRE (-750 to -700) and its mutants were synthesized and inserted into the pGL-3-promoter vector (Promega Corp., Madison, WI) at BamHI-SamI sites located upstream of a luciferase reporter gene driven by the Simian virus (SV) 40 promoter. The cDNAs of COUP-TF1 were a gift of Dr. Ming Tsai (Baylor College of Medicine, Houston, TX). TR{alpha}1 and RARß cDNAs were cloned into the expression vector pCDNA3.0 (Promega Corp.), which utilizes the SV40 promoter.

Transient Transfection
ES cells were plated in 24-well dishes at a density of 5 x 104 cells/well and grown 24 h in serum-free medium. Cells were either transfected with rat CaMKIV promoter-CAT-reporter constructs (0.4 µg) alone or cotransfected with constructs expressing mouse TR{alpha}1 (0.4 µg) and/or mouse COUP-TF1 (0.4 µg). In a similar manner, 0.4 µg of luciferase reporter constructs containing oligonucleotide response elements upstream of the SV40 promoter were either transfected alone or cotransfected with TR{alpha}1 (0.4 µg) and mouse COUP-TF1 (0.4 µg) expressing vectors. The empty pCDNA expression vector was added to the transfection as necessary to maintain constant DNA concentration in all transfections. Expression of the reporter constructs was compared with luciferase activity of the empty pGL-3-promoter vector. After transfection, cells were grown in serum-free medium supplemented with hormones, 1 µM RA, 1 nM T3, or combined RA and T3. CAT activity was analyzed 48 h after transfection. The Dual Luciferase Reporter Assay System (Promega Corp.) was used with the transfection control, pRL-SV40 vector, in each assay to control for transfection efficiency. Expression of the reporter and control luciferase activity was quantified by luminometer 48 h after transfection.

Gel Mobility Shift Assay
TR{alpha}, RARß, RXRß, and COUP-TF1 protein were prepared by in vitro transcription/translation (Promega Corp. TNT kit) and tested for binding to 32P-labeled oligonucleotide DNA TRE. Briefly, for the protein-DNA binding assay, 4–6 µl of in vitro translated protein (depending on translation efficiency) were incubated with the 32P-labeled oligonucleotide-TRE (10,000 cpm) in a 20-µl reaction mixture containing 10 mM HEPES (pH 7.9), 50 mM KCl, 1 mM dithiothreitol, 2.5 mM MgCl2, 10% glycerol, and 1.5 µg of poly(deoxyinosine-deoxycytidine) at room temperature for 15 min. The reactions were then loaded on a 5% nondenaturing polyacrylamide gel (29:1) made in 1x TBE buffer and electrophoresed at constant 150 V for 2 h. For supershift, after an initial 15-min incubation, 1 µl of TR{alpha} (Affinity BioReagents, Inc., Golden, CO) or COUP-TF1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added and incubated for an additional 10 min. The reaction was electrophoresed in a nondenaturing gel (19:1) in 1x Tris-glycine buffer (0.025 M Tris, 0.19 M glycine, 1 mM EDTA). The gel was dried before autoradiography. The protein/DNA complex intensity was quantified by PhosphorImager.

Statistical Analysis
Studies were performed in triplicate, unless otherwise noted, and the data are presented as mean ± SD. Significant differences in mRNA expression and promoter activity between two different treatment were compared using two-tailed Student’s t test with significance considered at P < 0.05. Retardation gels shown are representative of at least three separate experiments.


    ACKNOWLEDGMENTS
 
We are grateful to Drs. Christopher Kane and Anthony Means (Duke University Medical Center, Durham, NC) for providing the CaMKIV promoter constructs and Dr. Ming Tsai (Baylor College of Medicine, Houston, TX) for COUP-TF1 constructs used for our studies. Dr. Jimin Xu (VA Greater Los Angeles Healthcare System and UCLA School of Medicine, Los Angeles, CA) provided helpful suggestions in this work


    FOOTNOTES
 
These studies were supported by research funds from the Department of Veterans Affairs, NIH Grant RO1-CA-89364, and the American Thyroid Association.

Abbreviations: AB, Direct repeat; ABC, wild-type element; BC, overlapping inverted repeat; CaMKIV, Ca2+calmodulin-dependent protein kinase IV; CAT, chloramphenicol acetyltransferase; COUP-TF1, chicken ovalbumin upstream promoter-transcription factor 1; CREB, cAMP-response element binding protein; ES, embryonic stem; N-COR, nuclear receptor corepressor; PCP-2, Purkinje cell protein-2; RA, retinoic acid; RAR, retinoic acid receptor; RNase, ribonuclease; ROR, RAR-related orphan receptor; RXR, retinoid X receptor; SMRT, silencing mediator of retinoid and thyroid receptors; SV, Simian virus; TR, thyroid hormone receptor; TRE, thyroid hormone response element.

Received for publication November 30, 2001. Accepted for publication July 23, 2002.


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