The LIM/Homeodomain Protein Islet-1 Modulates Estrogen Receptor Functions

Frédérique Gay, Isabelle Anglade, Zhiyuan Gong and Gilles Salbert

Equipe Information et Programmation Cellulaire (F.G., G.S.) and Equipe Endocrinologie Moléculaire de la Reproduction (I.A.) UMR 6026 Centre Nationale de la Recherche Scientifique Université de Rennes I, Campus de Beaulieu 35042 Rennes cedex, France
Department of Biological Sciences (Z.G.) National University of Singapore Singapore 119260


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
LIM/Homeodomain (HD) proteins are classically considered as major transcriptional regulators which, in cooperation with other transcription factors, play critical roles in the developing nervous system. Among LIM/HD proteins, Islet-1 (ISL1) is the earliest known marker of motoneuron differentiation and has been extensively studied in this context. However, ISL1 expression is not restricted to developing motoneurons. In both embryonic and adult central nervous system of rodent and fish, ISL1 is found in discrete brain areas known to express the estrogen receptor (ER). These observations led us to postulate the possible involvement of ISL1 in the control of brain functions by steroid hormones. Dual immunohistochemistry for ISL1 and ER provided evidence for ISL1-ER coexpression by the same neuronal subpopulation within the rat hypothalamic arcuate nucleus. The relationship between ER and ISL1 was further analyzed at the molecular level and we could show that 1) ISL1 directly interacts in vivo and in vitro with the rat ER, as well as with various other nuclear receptors; 2) ISL1-ER interaction is mediated, at least in part, by the ligand binding domain of ER and is significantly strengthened by estradiol; 3) as a consequence, ISL1 prevents ER dimerization in solution, thus leading to a strong and specific inhibition of ER DNA binding activity; 4) ISL1, via its N-terminal LIM domains, specifically inhibits the ER-driven transcriptional activation in some promoter contexts, while ER can serve as a coactivator for ISL1 in other promoter contexts. Taken together, these data suggest that ISL1-ER cross-talk could differentially regulate the expression of ER and ISL1 target genes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The LIM/HD proteins are a subset of homeodomain (HD)-containing transcription factors defined on the basis of a common LIM domain, which consists of a conserved cysteine- and histidine-rich structure of two tandemly repeated zinc fingers. The acronym of LIM is derived from the first identified members of this family, namely Lin-11 from Caenorhabditis elegans (1), Islet-1 from rat (2), and Mec-3 from C. elegans (3). Up to date, LIM/HD transcription factors have been extensively studied for their critical roles during development in invertebrates and vertebrates. Most, if not all, the LIM/HD-encoding genes are expressed in the developing nervous system and have been implicated, in cooperation with other factors, in regulating crucial events during nervous system development such as neuronal survival, neuronal fate specification, axonal pathway and target selection, as well as neurotransmitter identity (for review, see Ref. 4).

Among the LIM/HD transcription factors, Islet-1 (ISL1) has been extensively studied for its regulatory role in motoneuron differentiation. Indeed, in chick embryo, ISL1 is expressed in motoneurons of the embryonic spinal cord soon after their final mitosis, even before the appearance of other differentiated properties (5, 6). Similarly, in zebrafish embryo, this LIM/HD protein expression is initiated in many primary neurons in the brain and spinal cord, including primary motoneurons, at the end of the gastrulation and precedes that of any other known neural marker (7, 8). These results therefore established ISL1 as the earliest marker of developing motoneurons. Subsequent studies of LIM/HD proteins in embryonic motoneurons have led to the identification of a combinatorial code of several LIM/HD proteins, including ISL1, that controls various aspects of motoneuron identity (6, 9, 10, 11, 12, 13).

Aside from its expression in embryonic motoneurons, ISL1 is broadly expressed in other cell types during embryogenesis, but also in adult. Consistent with its identification as an insulin enhancer binding protein (2), ISL1 is expressed in a variety of cell lines of pancreatic endocrine origin, as well as in normal adult rat islet cells (2). However, ISL1 expression is not restricted to pancreatic cells since this protein is widely distributed in endocrine cells of the thyroid and the anterior lobe of the pituitary (14, 15). In addition, ISL1 is also present in many neurons within the central nervous system of the embryonic and adult rat (15). In this last case, ISL1 is expressed in neurons of the caudate-putamen, the septal nuclei, and in the nuclei of the basal telencephalon. Finally, ISL1 is expressed in a subset of neurons of the diencephalon, a particularly high expression being detected in the pineal gland, in the thalamic reticular nucleus, and in neurons of the hypothalamus. The hypothalamic arcuate nucleus exhibits a particularly dense population of ISL1-expressing neurons (15). A similar expression pattern of ISL1 can be observed in salmonids showing that, in both rodent and fish central nervous systems, ISL1 expression maps brain nuclei involved in the control of the endocrine system (15, 16), the expression pattern observed in embryo being similar to that observed in adult. These data could suggest the involvement of ISL1 in both the development and/or the maintenance of the differentiated phenotypes of the ISL1-expressing neurons. In this respect, ISL1 may function in a similar way to Mec-3, a LIM/HD protein identified in C. elegans. This transcription factor is expressed in early embryo and throughout adulthood and controls both development of mechanoreceptor sensory cells and maintenance of their differentiated properties (3, 17). Previous studies have also reported the involvement of LIM/HD proteins in both acquisition and maintenance of appropriate neurotransmitter secretory phenotypes in distinct subsets of neurons. A role in neuronal identity specification has indeed been demonstrated for the Drosophila Islet protein, which is expressed in discrete subsets of interneurons including dopaminergic and serotoninergic neurons (18). Loss of Islet function causes dramatic defects in both axon pathfinding and targeting as well as loss of dopamine and serotonin synthesis, showing that in Drosophila embryo, Islet controls two distinct aspects of neuronal identity including secretion of the appropriate neurotransmitter (18). Similarly, the Drosophila Apterous LIM/HD protein is involved in a direct and cell-specific regulation of the Drosophila FMRFa neuropeptide gene and thereby contributes to both initiation and maintenance of FMRFa expression in a subset of neurons (19). Nevertheless, the ISL1-expressing neurons identified in the rat central nervous system do not share any obvious neuropeptide secretory phenotype (15). Thus, ISL1 is unlikely to control neurotransmitter identity of these neurons. However, particularly striking are the parallel expression patterns of ISL1 and of the estrogen receptor {alpha} (referred below as ER) in the brain of both rodent and fish (15, 16, 20, 21, 22, 23, 24, 25).

The evolutionary conservation of the highly parallel expression patterns of these two transcription factors in restricted brain areas led us to investigate the functional consequences of ISL1-ER coexpression. In this report, we give evidence for a complex and promoter-specific cross-talk between these two transcription factors. Such a transcriptional interference is likely to play a major role in the modulation of estrogen signaling in neurons.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Nuclear ER and the LIM/HD Protein ISL1 Colocalize in the Rat Hypothalamic Arcuate Nucleus
In the rat central nervous system, ISL1 has been previously shown to be expressed in brain nuclei controlling the endocrine system (15). Such a distribution is reminiscent of ER-containing neurons that are also found in nuclei involved in the control of the endocrine system (21). One of these, the arcuate nucleus, is densely populated by both ER and ISL1 expressing neurons (15, 21). We thus performed immunofluorescence studies to verify whether ER and ISL1 were effectively coexpressed in the same neurons of the arcuate nucleus (Fig. 1Go). We found that, in this nucleus, with very few exception (Fig. 1EGo, open arrowheads), all ER-positive (ER+) cells were also positive for ISL1 (Fig. 1Go, A and B), whereas some ISL1+ cells were not revealed by the ER-specific antibodies (Fig. 1Go, C and D, white arrowheads). This was also true for ER+ cells of the ventromedial hypothalamic nucleus since they also expressed ISL1 (data not shown). These results imply that, at least in the ventral hypothalamus, ER expression can be systematically associated with ISL1 expression. Such an extensive colocalization was not due to an interference between the detection systems since control sections that did not receive the biotinylated goat-antimouse immunoglobulins (see Materials and Methods) did not display any green fluorescence (data not shown).



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Figure 1. ISL1 and ER Colocalize in the Rat Hypothalamic Arcuate Nucleus

Dual immunofluorescence studies were run on rat brain cryostat sections. Both ER (red) and ISL1 (green) showed a strictly nuclear localization and were coexpressed in most cells of the arcuate nucleus. Panels A/B, C/D, and E/F show different regions of the arcuate nucleus (magnification: x180). The white arrowheads (D and F) point out ISL1-positive cells that do not express ER (C and E), while open arrowheads show ER-positive cells lacking ISL1 immunoreactivity (E).

 
ISL1 and ER Interact in Vivo
The functional relevance of the ISL1-ER colocalization in the rat central nervous system was assessed by in vivo coimmunoprecipitation studies, performed with homogenate from female rat hypothalamus, known to express both transcription factors. Animals were injected with 17ß-estradiol (E2) 1 h before the protein extract was prepared, half of the extract being incubated with a rabbit polyclonal antibody (ER715), directed against the hinge region of the rat ER. The specificity of the interaction was assessed by incubation of the remaining extract with rabbit preimmune serum. Immunoprecipitation was followed by immunoblotting with the mouse monoclonal anti-ISL1 antibody, and the results are presented in Fig. 2Go. As shown in lane 2, a single band of the expected molecular mass (39 kDa) was detected with the anti-ISL1 antibody when proteins extracted from rat hypothalamus had been incubated with the anti-ER antibody. This band was moreover specific since it was not present when the protein extract had been incubated with preimmune serum (lane 1). Thus, ER can specifically interact with ISL1 in vivo. Moreover, the fact that ISL1 can be immunoprecipitated with ER from rat hypothalamus extracts strongly suggests that the striking colocalization between ISL1 and ER observed in some rat hypothalamus nuclei could be physiologically relevant.



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Figure 2. ISL1 and ER Interact in Vivo

Protein extracts from female rat hypothalamus were incubated with rabbit preimmune serum (lane 1) or anti-ER antibodies (lane 2), as indicated at the top of the autoradiogram. Immunocomplexes were collected, washed, and resolved by SDS-PAGE analysis. After Western blotting, the membrane was probed with anti-ISL1 antibodies, and detection of the immunocomplexes was performed with a chemiluminescent detection kit. The molecular mass markers (in kilodaltons) are shown on the left.

 
ISL1 Interacts with a Subset of Nuclear Receptors in Vitro
We next wished to find out whether the ISL1-ER interaction observed in vivo could also occur in vitro. We therefore used the matrix-bound fusion protein of glutathione-S-transferase (GST) combined with ISL1 (GST-ISL1) for in vitro protein-protein interaction studies to test various nuclear proteins for their ability to interact with the zfISL1 protein in vitro. The majority of the 35S-methionine-labeled in vitro-translated proteins tested interacted with GST-ISL1 (Fig. 3Go), as shown by the bands observed when these 35S-methionine-labeled proteins were incubated with GST-ISL1. It was the case for the wild-type rat ER (rER; Fig. 3AGo, lane 9), the truncated ER protein, corresponding only to the ligand-binding domain (LBD) of the rER (TERP; Fig. 3AGo, lane 12), the rainbow trout ER (rtER; Fig. 3BGo, lane 3), the human nuclear orphan receptor, chicken ovalbumin upstream promoter (COUP)-TFI (hCOUP; Fig. 3BGo, lane 6), as well as for the zebrafish steroidogenic factor 1 (zfSF1; Fig. 3BGo, lane 9). In contrast, the Xenopus Cyclin E was not or very weakly retained on the GST-ISL1 bound-beads (xCyclin; Fig. 3BGo, lane 12). The GST-ISL1 fusion protein did not significantly retain the luciferase control protein (Luc; Fig. 3AGo, lane 6). Moreover, the protein-protein interactions involving ISL1 and the nuclear receptors were specific since the GST moiety of the fusion protein did not interact with any of the 35S-methionine-labeled in vitro-translated proteins. These gels were submitted to phosphoimage screen, and the relative amounts of 35S-methionine-labeled in vitro-translated proteins retained on the GST-ISL1 bound-beads were assessed by comparing the intensity of the corresponding band to that of the input lane. This analysis showed that 7% of the input was retained on the GST-ISL1-bound beads for the zfSF1-programmed lysate, while 5.71% of the input was retained for the in vitro-translated rER. The percentage of the input retained was quite similar for TERP, rtER, and hCOUP-TFI, ranging from 3% to 4%. Finally, only 0.8% and 0.4%, respectively, of the input was retained for the xCyclin E and luciferase proteins. Taking the specific activity of the labeled proteins into account, we thus could determine that, among the tested proteins, ISL1 presented a similar and maximal relative affinity for zfSF1 and hCOUP-TFI, its relative affinity for TERP, rER and rtER being 1.3- to 2.3-fold weaker. Finally, ISL1 showed only weak or negligible affinity for xCyclin E and luciferase. As ISL1 seemed to associate directly and specifically with various nuclear receptors including ER, we next studied the potential effect of the ligand on ISL1-rER interaction. In order to reach this goal, we performed GST pull-down assays with rER-programmed lysate in the absence (Fig. 3CGo, lanes 1–3) or in the presence of 17ß-estradiol (Fig. 3CGo, lanes 4–6). The amount of rER protein retained was significantly higher in the presence of ligand. As confirmed by phosphoimage screen, a 2-fold induction in the amount of 35S-methionine-labeled rER bound by GST-ISL1 in the presence of ligand was observed compared with that retained in the absence of ligand (data not shown). Thus, ISL1 binding to rER is affected by the presence of ligand in an E2-stimulated manner and is likely to involve the C-terminal LBD of the nuclear receptor since ISL1 also interacts in vitro with the natural truncated rER variant, TERP.



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Figure 3. ISL1 Interacts with Various Nuclear Receptors in Vitro

Panels A and B show SDS-PAGE analysis of the 35S-methionine-labeled in vitro-translated proteins retained by the GST-ISL1 fusion protein. Glutathione-agarose beads containing GST (panels A and B, lanes 2, 5, 8, and 11) or GST-ISL1 (panels A and B, lanes 3, 6, 9, and 12) proteins were incubated with various 35S-methionine-labeled in vitro-translated proteins indicated at the bottom of the gels. Panel C shows SDS-PAGE analysis of the 35S-methionine-labeled in vitro-translated rER retained by the GST-ISL1 fusion protein, in the absence or presence of ligand. The rER-programmed lysate was treated in the absence (panel C, lanes 1–3) or in the presence (panel C, lanes 4–6) of E2 before incubation with glutathione-agarose beads containing GST (panel C, lanes 2 and 5) or GST-ISL1 (panel C, lanes 3 and 6). For each lysate, the input lane corresponds to the amount of 35S-methionine-labeled in vitro-translated proteins used for the GST pull-down assay.

 
ISL1-ER Interaction Inhibits ER Dimerization in Vitro
Since ISL1 and ER interacted in vivo and in vitro, it was of interest to examine a potential effect of this protein-protein interaction on ER dimerization function. GST pull-down competition assays were set up to analyze rER binding to a GST-hER LBD fusion protein in the absence or in the presence of zfISL1 (Fig. 4Go). These competition assays were performed in the presence of limiting amounts of E2-treated rER-programmed lysate (1.5 µl) and GST-hER LBD fusion protein (500 ng), as determined in preliminary experiments. Increasing amounts (1.5–15 µl) of unlabeled zfISL1-programmed lysate were added, such that ISL1 was present from a 3- to a 30-fold molar excess, as assessed by in vitro translations of rER and zfISL1, performed in the presence of 35S-methionine and taking the specific activity of both 35S-methionine-labeled proteins into account. In the absence of competitor, rER was able to interact with the LBD of hER, as shown by the band observed when 35S-methionine-labeled rER was incubated with the GST-hER LBD fusion protein (Fig. 4Go, lane 3). The relatively low intensity of this band could probably reflect the difficulty for hER LBD to destabilize the dimers formed in solution by the E2-treated in vitro-translated rER. Even though a significant amount of rER interacted with the GST-hER LBD-bound beads in the absence of zfISL1, this protein-protein interaction was moreover specific since the GST moiety did not retain the 35S-methionine-labeled rER (Fig. 4Go, lane 2). Addition of increasing amounts of in vitro-translated zfISL1 led to a gradual decrease in the retention of 35S-methionine-labeled rER by GST-hER LBD (Fig. 4Go, lanes 4–7), reflecting a competition between rER and zfISL1 for binding to the GST-hER LBD fusion protein. Competition assays performed in the absence of E2 gave similar results (data not shown). Finally, in contrast to the marked inhibition of rER binding to the fusion protein observed in the presence of the highest dose of cold ISL1-programmed lysate, addition of an equivalent dose of unprogrammed lysate did not modify the amount of rER-programmed lysate retained on the GST-hER LBD-bound beads (Fig. 4Go, lane 8), showing that the inhibitory effect observed previously was specific for ISL1. These data suggest that ISL1 binding to the ER precludes ER dimer formation, thus giving evidence for a specific and dose-dependent inhibition of ER dimerization function by ISL1 in vitro.



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Figure 4. ISL1-ER Interaction Inhibits rER Dimerization

SDS-PAGE analysis of the 35S-methionine-labeled in vitro-translated rER retained by the GST-hER LBD fusion protein in the absence or presence of unlabeled ISL1-programmed competitor lysate. The rER-programmed lysate (1.5 µl) was incubated with limiting amounts (500 ng) of glutathione-agarose beads containing GST (lane 2) or GST-hER LBD (lanes 3–8) proteins, as indicated at the bottom of the gel. Both rER-programmed lysate and GST-hER LBD fusion protein were treated by E2 before incubation. The input lane (lane 1) corresponds to 20% of the total amount of 35S-methionine-labeled in vitro-translated rER used for the GST pull-down competition assay. The amount of labeled in vitro-translated rER retained by the GST-hER LBD protein in the absence of any competitor lysate is shown in lane 3. Increasing amounts (1.5–15 µl) of ISL1-programmed lysate were added, such that ISL1 was present in a molar excess ranging 3- to 30-fold, as indicated at the top of the gel (lanes 4–7). Unprogrammed competitor lysate (15 µl) was used as a control (lane 8).

 
ISL1-ER Interaction Specifically Decreases rER Binding to DNA
Since ISL1 destabilized ER dimers in solution, we next wished to determine whether this inhibitory effect could occur in the presence of ER DNA target sequence and affect the binding of the receptor to its recognition site. We performed electrophoretic mobility shift assay (EMSA) experiments to analyze the effect of addition of increasing amounts of GST-ISL1 fusion protein on the rER binding to a 32P-labeled estrogen responsive element (ERE) consensus sequence. Preliminary experiments using GST and GST-ISL1 proteins extracted from total insoluble bacterial lysates revealed that increasing amounts of unpurified GST-ISL1 fusion protein failed to significantly affect the DNA binding activity of the in vitro translated rER. Since the GST-ISL1 was highly unstable and only represented a small proportion of the total insoluble proteins, we thus repeated these EMSA experiments using increasing amounts of fusion proteins enriched from the insoluble fraction by affinity chromatography with glutathione-agarose beads. As shown in Fig. 5AGo, incubation of rER-programmed lysate with labeled double-stranded EREc in the absence of fusion protein (lanes 2 and 4) led to the formation of a preponderant shifted ER-ERE complex (indicated by an arrowhead). The major retarded complex reflected the specific binding of rER to the EREc probe since no similar shifted band was observed when unprogrammed lysate was incubated with the probe under the same conditions (Fig. 5AGo, lanes 1 and 3). The intensity of the shifted band corresponding to the specific ER-ERE complex was not affected when rER-programmed lysate was coincubated with 450 ng of purified GST before addition of the probe (Fig. 5AGo, lane 6), indicating that the GST moiety of the GST-ISL1 fusion protein did not affect the ER binding to its DNA recognition site. Neither the GST moiety (Fig. 5AGo, lane 5) nor the GST-ISL1 fusion protein (Fig. 5AGo, lanes 7, 9, and 11) alone interacted with the labeled double-stranded ERE oligonucleotide. Interestingly, addition of increasing amounts (150 ng, 300 ng, and 450 ng) of purified GST-ISL1 in the presence of receptor (Fig. 5AGo, lanes 8, 10, and 12, respectively) resulted in a progressive decrease in the ER-ERE complex intensity. In contrast, the intensity of the nonspecific bands was modified neither by GST nor by GST-ISL1. Similar gel shift experiments were performed with rER-programmed lysate treated with 17ß-estradiol, but no effect of the ligand was observed (data not shown). Thus, ISL1-ER interaction inhibits the specific ER-DNA interaction in a dose-dependent manner, both in the absence and in the presence of ligand. Requirement of relatively high amounts of GST-ISL1 protein to significantly inhibit rER binding to DNA could be related to a very weak stability of the fusion protein (data not shown) and/or to a partial renaturation of the GST-ISL1 protein during dialysis (see Materials and Methods). Since ISL1 also interacted with hCOUP-TFI in vitro, we next tested the potential effect of increasing amounts of purified GST-ISL1 on hCOUP-TFI binding to DNA. Previous work has shown that the orphan receptor hCOUP-TFI is able to bind a DR24-type element within the rtER gene promoter (26). EMSA experiments using a 32P-labeled double-stranded DR24 oligonucleotide as a probe were performed rigorously in the same conditions as those mentioned above (Fig. 5BGo). Incubation of hCOUP-TFI-programmed lysate with labeled probe in the absence of fusion protein (Fig. 5BGo, lanes 2 and 4) led to the formation of a major hCOUP-TFI-DR24 shifted complex (indicated by an arrowhead), giving evidence of the specific binding of hCOUP-TFI to the DR24 type element since no similar shifted band was observed when unprogrammed lysate was incubated with the probe under the same conditions (Fig. 5BGo, lanes 1 and 3). Coincubation of the hCOUP-TFI programmed lysate with 450 ng of purified GST protein did not modify the intensity of the shifted band (Fig. 5BGo, lane 6). Similarly, coincubation of hCOUP-TFI with increasing amounts of purified GST-ISL1 fusion proteins (Fig. 5BGo, lanes 8, 10, and 12) did not significantly modify the shifted band intensity. Thus, although ISL1 interacts in vitro with both the rER and the human nuclear orphan receptor COUP-TFI, these protein-protein interactions differentially modulate DNA binding by the nuclear receptors.



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Figure 5. ISL1-ER Interaction Specifically Decreases rER Binding to DNA

Effects of ISL1-ER interaction on the rER binding to an EREc probe (panel A) and effects of ISL1-COUP-TFI interaction on the hCOUP-TFI binding to a DR24 probe (panel B) were assessed by EMSA analysis. Unprogrammed (lanes 1, 3, 5, 7, 9, and 11), ER-programmed lysate (panel A, lanes 2, 4, 6, 8, 10, and 12) or hCOUP-TFI-programmed lysate (panel B, lanes 2, 4, 6, 8, 10, and 12) were coincubated with either 450 ng of purified GST (lanes 5–6) or 150 ng, 300 ng, and 450 ng of purified GST-ISL1 (lanes 7 and 8, 9 and 10, and 11 and 12, respectively) before addition of the 32P-labeled double-stranded oligonucleotide indicated at the bottom of the gel. Incubations of unprogrammed (lanes 1 and 3), ER-programmed lysate (panel A, lanes 2 and 4) or hCOUP-TFI-programmed lysate (panel B, lanes 2 and 4) alone with the labeled probe were performed either in binding buffer alone (lanes 1 and 2) or in the same buffer conditions (i.e. binding buffer plus dialysis buffer) as in lanes 5–12 (lanes 3 and 4). The position of the main ER-ERE and COUP-TFI-DR24 complexes is indicated by an arrowhead while nonspecific bands are indicated by asterisks.

 
ISL1 Inhibits rER Transactivation Function in a Dose-Dependent Manner
The functional relevance of the ISL1-ER interaction was tested in transient transfections of Chinese hamster ovary (CHO) cells, using an artificial estrogen-responsive reporter gene ERE-SV-Luciferase (Fig. 6Go, A and B). This reporter gene contains the FP1 sequence of the rtER gene (bp -85 to -56), which encompasses an estrogen-responsive element (27, 28). Cells were cotransfected with or without rER, zfISL1, and zfISL2 expression vectors and were either untreated or treated with E2 after transfection. Cotransfection of the ERE-SV-Luc reporter construct with rER expression vector alone only led to a slight increase in the basal luciferase activity but allowed induction in the luciferase activity when E2 was present (Fig. 6AGo). In contrast, cotransfection with zfISL1 expression vector alone affected neither the basal nor the E2-induced transcriptional activity of the ERE-SV-Luc reporter construct (data not shown). When cells were cotransfected with rER expression vector and increasing amounts of zfISL1 expression vector, no significant modification of the basal luciferase activity was observed, while ISL1 led to a gradual decrease in the E2-induced luciferase activity (Fig. 6AGo). Even the lowest dose (5 ng per well) of zfISL1 expression vector led to a 35% inhibition of the ligand-dependent activity of the reporter gene (Fig. 6Go, A and B). Finally, the transcriptional induction by E2 was almost completely abolished in the presence of 100 ng of zfISL1 expression vector (Fig. 6Go, A and B). These data are consistent with the previous EMSA results and show that ISL1 is able to repress the ligand-dependent transcriptional activation mediated by rER on an artificial estrogen-responsive promoter. In contrast, when cells were cotransfected with rER expression vector and increasing amounts (5–100 ng per well) of zfISL2 expression vector, no such inhibition of the estrogen-dependent reporter gene activation was observed (Fig. 6AGo). These results clearly demonstrate that inhibition of rER transactivation function is specific for ISL1 since the related LIM/HD protein ISL2 had no effect on the ligand responsiveness of the reporter gene. The specificity of ISL1 inhibitory effect for rER was also investigated using a COUP-TFI-dependent reporter construct (G. Salbert, unpublished results). While cotransfection of 50 ng of hCOUP-TFI expression vector alone allowed a strong activation of the reporter construct transcriptional activity, cotransfection of hCOUP-TFI with increasing amounts (5–100 ng per well) of zfISL1 expression vector had no effect on the induction mediated by hCOUP-TFI (data not shown). These data are consistent with our EMSA results showing that ISL1 does not affect hCOUP-TFI DNA binding and further attest to the specificity of ISL1-ER functional interaction. To map the functional(s) domain(s) of ISL1 required for the repression of rER transactivation function, the ERE-SV-Luc reporter plasmid was cotransfected with or without rER, zfISL1, and zfISL1{Delta}HD/AD expression vectors in CHO cells. The CMV-zfISL1{Delta}HD/AD plasmid encodes for a truncated protein corresponding only to the N-terminal LIM domains of zfISL1, thus lacking both the homeodomain and the C-terminal transactivation domain. Estradiol treatment of the cells cotransfected with rER alone led to a strong increase in the reporter gene transcriptional activity (Fig. 6BGo). As previously shown, cotransfection of both rER and increasing amounts (5–100 ng per well) of zfISL1 expression vectors resulted in a strong and progressive decrease in the induction mediated by E2 (Fig. 6BGo). Similarly, cotransfection of rER expression plasmid with increasing amounts of zfISL1{Delta}HD/AD expression vector strongly reduced the E2-induced luciferase activity but did not affect the basal luciferase activity (Fig. 6BGo). These results suggest the involvement of the N-terminal LIM domains of ISL1 in ISL1-ER interaction and in the subsequent modulation of rER functions: the LIM domains of ISL1 indeed appear to be sufficient to mediate the full inhibition of rER transactivation function, suggesting that the homeo- and transactivation domains are dispensable for ISL1 effect. We then examined whether ISL1 could inhibit ER function through exclusion of ER from the nucleus. This was done by cotransfection of GFP-rER with ISL1 in COS-7 cells. No difference in the subcellular distribution of ER was observed upon cotransfection with ISL1 (data not shown).



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Figure 6. ISL1 Inhibits rER Transactivation Function in a Dose-Dependent Manner

The ERE-SV-Luc reporter was cotransfected in CHO cells with a constant amount of CMV-rER expression vector and with increasing amounts of CMV-zfISL1, CMV-zfISL2, and CMV-zfISL1{Delta}HD/AD expression vectors. Cells were either treated or untreated with E2 (10-8 M) as indicated. Panel A shows relative luciferase activities corresponding to luciferase activities corrected for transfection efficiency by the ß-galactosidase values. Untreated cells are shown by gray bars while E2-treated cells are shown by black bars. Panel B shows "fold inductions" of the reporter gene activities (luciferase activities normalized with ß-galactosidase activities) in the presence of E2 compared with the activity in the absence of ligand for each expression vector combination. All results are expressed as the means ± SEM of three values obtained in a representative experiment.

 
ISL1 Modulates the Ligand-Dependent Transcriptional Activity of the rtER Gene Promoter
We next wished to confirm the previous results in a natural estrogen-responsive promoter context. With this goal in mind, we used the rtER gene promoter previously isolated and characterized in our laboratory since it contains an estrogen-responsive unit (27, 28), as well as three putative ISL1 DNA binding sites, I1, I2, and I3 (this study). Two reporter plasmids, namely the 0.2-Basic reporter gene and the 2.0-Basic reporter gene were used (26). These plasmids contain either bp -207 to +28 or bp -2,079 to +28, respectively, of the rtER gene inserted in front of the luciferase coding sequence. The 0.2-Basic reporter plasmid only contains the estrogen-responsive unit of the rtER gene promoter, composed of an imperfect functional ERE and a consensus half -binding site for nuclear receptors (27, 28). The consensus half-site together with the downstream ERE half-site form a DR24-type element, which is bound by hCOUP-TFI (26). The 2.0-Basic reporter construct contains, in addition to the estrogen-responsive unit, three distal putative ISL1-binding sites identified in the rtER gene promoter (I1, I2, and I3), which are absent in the 0.2-Basic reporter plasmid. CHO cells were cotransfected with either 0.2-Basic or 2.0-Basic reporter constructs with or without rER, hCOUP-TFI, and zfISL1 expression vectors (Fig. 7Go). When cells were cotransfected with the 0.2-Basic construct and rER expression vector, a 2-fold induction in the luciferase activity was observed in the presence of E2. When both rER and hCOUP-TFI expression vectors were cotransfected, a marked increase in the fold induction by E2 was observed (Fig. 7Go), showing that the orphan receptor hCOUP-TFI was able to enhance rER activation properties as it was the case for rtER (26). Transfection of zfISL1 expression vector alone did not significantly affect the 0.2-Basic reporter gene activity, suggesting that ISL1 had on its own neither activatory nor inhibitory effect on the transcriptional activity of the 0.2- kb rtER promoter fragment lacking the ISL1 potential target sequences (Fig. 7Go). In contrast, when both rER and zfISL1 expression vectors were cotransfected with the same reporter construct, the E2-induced luciferase activity decreased significantly, the induction by E2 being 55% of that observed with rER alone (Fig. 7Go). When rER, hCOUP-TFI, and zfISL1 expression plasmids were cotransfected, ISL1 was still able to inhibit the ligand-dependent transcriptional activation, the fold induction by E2 representing only 45% of the induction mediated by rER and hCOUP-TFI in the presence of ligand (Fig. 7Go). When cotransfected in CHO cells, the 2.0-Basic reporter construct had a quite similar responsiveness to rER, although the ligand-dependent induction in the transcriptional activity was slightly weaker than that observed with the shorter 0.2-Basic construct. As above, hCOUP-TFI was able to enhance the E2-stimulated luciferase activity, therefore leading to an increase in the fold induction by E2 when compared with the value obtained with rER alone (Fig. 7Go). Despite the presence of ISL1-binding sites in this reporter construct, ISL1, on its own, had no significant effect on the transcriptional activity of the rtER promoter (i.e. did not repress or activate the reporter gene). Moreover, in this promoter context, ISL1 did not affect the ligand-dependent transcriptional activation mediated by either rER alone or rER and hCOUP-TFI (Fig. 7Go). Thus, while ISL1, on its own, has no regulatory effect on the activity of the tested reporter genes, this transcription factor is able to modulate the ligand-dependent activation of the estrogen-responsive rtER gene proximal promoter. However, the ISL1 inhibitory effect occurs only in the 0.2-Basic reporter gene context, a construct that, in contrast to the 2.0-Basic reporter gene, lacks the ISL1 target DNA sequences identified in the rtER gene promoter.



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Figure 7. ISL1 Modulates the Ligand-Dependent rtER Transcriptional Activity of the 0.2-Basic Reporter Construct but Has No Effect on the 2.0-Basic Reporter Construct

The 2.0-Basic reporter plasmid contains the estrogen-responsive unit of the rtER gene promoter composed of a functional ERE (bp -80 to -65) and a half-binding site for nuclear receptor together forming a DR24 type element that is bound by hCOUP-TFI. This construct also contains the three putative ISL1-binding sites identified in the rtER gene promoter (I1, I2, and I3), which are absent in the 0.2-Basic reporter plasmid. The 0.2-Basic and the 2.0-Basic reporter genes were cotransfected in CHO cells with 50 ng of various expression vectors, CMV-rER, CMV-hCOUP-TFI, and CMV-zfISL1. Cells were either untreated or treated with E2 (10-8 M) after transfection. Results are shown as "fold inductions" of the reporter gene activities in the presence of E2 compared with the activity observed in the absence of ligand. Results are shown as the means ± SEM of 12 values obtained in five independent experiments.

 
The rtER Gene Promoter Contains ISL1-Binding Sites
We then performed EMSA experiments to investigate whether the zfISL1 protein could bind to the three putative ISL1-binding sites, I1, I2, and I3, identified in the rtER gene promoter. These sequences were defined by sequence analysis of the rtER gene promoter, based on their sequence identity with the DNA consensus sequences previously described for the chinook salmon Islet-2-related protein (29). As shown in Fig. 8Go, GST (lanes 1, 3, 5, 7, and 9) or GST-ISL1 (lanes 2, 4, 6, 8, and 10) fusion proteins were incubated with various 32P-labeled double-stranded oligonucleotides, namely P1 (lanes 1 and 2), I1 (lanes 3 and 4), I2 (lanes 5 and 6), I3 (lanes 7 and 8), or EREc (lanes 9 and 10), as indicated at the bottom of the gel. The P1 oligonucleotide was synthesized based on the P1 element of the rat insulin I gene enhancer that is bound by ISL1 (2). As expected, incubation of GST-ISL1 fusion protein with labeled double-stranded P1 oligonucleotide led to the formation of a major retarded complex (Fig. 8Go, lane 2), which corresponds to the specific binding of ISL1 to the P1 sequence. The shifted band was indeed not observed when GST alone was incubated under the same conditions with labeled P1 oligonucleotide (Fig. 8Go, lane 1). Incubation of GST-ISL1 fusion protein with labeled I1, I2, or I3 oligonucleotides resulted in the formation of a shifted complex (Fig. 8Go, lanes 4, 6, and 8, respectively) reflecting specific binding of the GST-ISL1 fusion protein. Finally, incubation of the fusion protein with a labeled double-stranded EREc oligonucleotide did not result in the formation of any retarded complex, demonstrating that, as expected, ISL1 does not bind to the EREc sequence (Fig. 8Go, lane 10). Competition EMSA carried out with labeled P1 oligonucleotide in the presence of increasing amounts of unlabeled P1, I1, I2, and I3 oligonucleotides confirmed the binding of ISL1 to the three rtER gene promoter sequences and showed that ISL1 has a quite similar affinity for both P1 and I1 sequences, its affinity for I2 or I3 being weaker (data not shown). According to these EMSA results, we can therefore conclude that, even though ISL1 has, on its own, no regulatory effect on the full-length rtER gene promoter, this transcription factor can bind the three ISL1-binding sites identified within the rtER gene promoter.



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Figure 8. The rtER Gene Promoter Contains Functional ISL1-Binding Sites

The binding of ISL1 to the I1, I2, and I3 potential target DNA sequences identified in the rtER gene promoter was assessed by EMSA. Fifty nanograms of unpurified GST (lanes 1, 3, 5, 7, and 9) or GST-ISL1 (lanes 2, 4, 6, 8, and 10) fusion proteins were incubated with 32P-labeled P1 (lanes 1 and 2), I1 (lanes 3 and 4), I2 (lanes 5 and 6), I3 (lanes 7 and 8), or EREc (lanes 9 and 10) double-stranded oligonucleotides. A black arrowhead indicates the position of the ISL1-DNA complex.

 
ISL1 Does Not Affect rER Transactivation Function in the Presence of ISL1-Binding Sites
A new set of transfection experiments was then performed to determine whether the presence of ISL1-binding sites could interfere with the ISL1-mediated inhibition of ER activity in an artificial promoter context. The ERE-SV-Luc artificial estrogen-responsive plasmid, which contains no ISL1-binding sites and whose ER-driven transcriptional activity was previously shown to be inhibited by ISL1 (Fig. 6Go), was used as a reporter construct. As the previous results suggested that the inhibitory effect of ISL1 would not occur in a ISL1 binding site-containing promoter context, we generated two recombinant reporter plasmids based on the ERE-SV-Luc construct. The ERE/I1-SV-Luc reporter gene contains one of the ISL1-binding sites identified in the rtER promoter, and the ERE/I2/I3/I1-SV-Luc reporter gene contains the three ISL1 target sequences downstream of the estrogen-responsive element. CHO cells were transfected with these reporter constructs, with or without rER and zfISL1 expression vectors, and cells were treated or not with E2. When cells were cotransfected with the ERE-SV-Luc reporter gene and rER expression vector (Fig. 9Go), an E2 treatment markedly increased the luciferase activity. Cotransfection of zfISL1 expression vector alone did not lead to any significant modification of the reporter gene activity (Fig. 9Go). In contrast, when rER and zfISL1 expression vectors were cotransfected with the ERE-SV-Luc reporter construct, the induction mediated by E2 decreased markedly compared with that observed with rER expression vector alone (Fig. 9Go). The two recombinant ERE/I1-SV-Luc and ERE/I2/I3/I1-SV-Luc reporter genes had a similar estrogen responsiveness than the parental ERE-SV-Luc plasmid. The transcriptional activation mediated by rER alone in the presence of E2 were about 2.5- and 2.3-fold, respectively, compared with the basal activities (Fig. 9Go). Despite the presence of ISL1-binding sites in these reporter constructs, cotransfection of zfISL1 expression vector alone did not affect (i.e. neither increased nor decreased) the reporter genes’ transcriptional activities (Fig. 9Go). In contrast to the inhibitory effect of ISL1 on the ER-driven transcriptional activation of the ERE-SV-Luc construct, no significant inhibition of the estrogen responsiveness of the two recombinant reporter genes was observed in the presence of both rER and ISL1. The transcriptional activation mediated by rER in the presence of E2 and of ISL1 were indeed about 2.6- and 2.4-fold, respectively, for the ERE/I1-SV-Luc and ERE/I2/I3/I1-SV-Luc reporter genes, compared with the basal activities (Fig. 9Go). We can therefore conclude that ISL1 is able to inhibit the ER-driven transcriptional activation in an artificial estrogen-responsive promoter context, this inhibitory effect occurring only in the absence of ISL1-binding sites. The presence of one or more ISL1-binding sites in the reporter gene seems to be sufficient to restore the full transcriptional activation of the estrogen-responsive promoter mediated by rER in the presence of E2 and ISL1.



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Figure 9. ISL1 Represses rER Transactivation Function in an Artificial Estrogen-Responsive Promoter Context Devoid of ISL1-Binding Sites

The ERE/I1-SV-Luc and ERE/I2I3/I1-SV-Luc reporter genes contain, respectively, one (I1) or a combination (I2/I3/I1) of the three ISL1 DNA target sequences identified in the rtER gene promoter downstream of the ERE. These reporter genes were cotransfected in CHO cells with 50 ng of the indicated expression vectors CMV-rER and CMV-zfISL1. Cells were either treated or untreated with E2 (10-8 M) after transfection. Results are expressed as "fold inductions" of the reporter gene activities in the presence of E2 compared with the activity in the absence of ligand for each expression vector combination. Results are shown as the means ± SEM of 12 values obtained in five independent experiments.

 
rER Can Act as a Coactivator for ISL1 in the Absence of an ERE
In regard to the previous data, distinct but not mutually exclusive hypotheses could be envisaged to explain the results obtained in natural and artificial promoter contexts containing both an ERE and ISL1-binding sites. First, it could be postulated that ISL1-binding sites and rER compete with each other for interaction with ISL1; binding of ISL1 to its DNA target sequences may decrease or prevent ISL1-rER interaction, thus abolishing ISL1 inhibitory effect on rER functions. Alternatively, rER may interact with the DNA-bound ISL1 and serve as a coactivator of the LIM/HD protein, the estrogen-dependent induction in the reporter constructs containing both an ERE and ISL1-binding sites being thus mediated via the ISL1-binding sites. To test this last hypothesis, we generated two artificial reporter constructs, I1-SV-Luc and I2/I3/I1-SV-Luc, containing, respectively, one or a combination of the three ISL1-binding sites identified in the rtER gene promoter but lacking an ERE. CHO cells were transfected with these reporter constructs as well as with the control SV-Luc plasmid, with or without zfISL1 and rER expression vectors, and were treated or not with E2 (Fig. 10Go). In the absence as well as in the presence of ISL1-binding sites, cotransfection of ISL1 expression vector alone did not modify the transcriptional activity of any of the reporter constructs, neither in untreated nor in estradiol-treated cells (Fig. 10Go). Similarly, when rER expression vector was cotransfected alone, we did not observe any activation or repression of the reporter construct activities, even in the presence of E2 (Fig. 10Go). Cotransfection of the I1-SV-Luc reporter construct with zfISL1 expression plasmid and increasing amounts (50–100 ng per well) of rER expression vector did not affect (neither increased nor decreased) the basal transcriptional activity, but gradually enhanced the reporter gene activity observed in the presence of E2. This resulted in a ligand- and rER dose-dependent induction in the I1-SV-Luc reporter construct activity (Fig. 10Go). Similar results were obtained with the I2/I1/I3-SV-Luc reporter plasmid, the E2-dependent induction observed in the presence of both ISL1 and increasing amounts of rER expression vectors being even more pronounced than that observed under the same conditions with the I1-SV-Luc reporter construct (Fig. 10Go). In contrast, when similar cotransfection experiments were performed using the zfISL1{Delta}HD/AD expression vector in spite of the full-length zfISL1 encoding vector, no such E2-dependent induction in the I1-SV-Luc and I2/I1/I3-SV-Luc transcriptional activities was observed in the presence of increasing amounts of rER expression vector (data not shown). These data strongly suggest that ER can serve as a coactivator for ISL1 in certain promoter contexts, since ER is able to confer estrogen dependency to artificial reporter constructs containing ISL1-binding sites but lacking ERE. This effect is dependent not only on the presence of ISL1-binding sites within the promoter but also on the number of these sites. The coactivation requires ISL1 binding to DNA through the homeodomain and may involve ER monomer tethering to the promoter by the LIM domains of ISL1, this binary complex allowing transactivation by E2.



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Figure 10. rER Acts as a Coactivator for ISL1 in an Artificial ISL1-Binding Sites-Containing Promoter Context Devoid of ERE

The I1-SV-Luc and I2I3/I1-SV-Luc reporter genes contain, respectively, one (I1) or a combination (I2/I3/I1) of the three ISL1 DNA target sequences identified in the rtER gene promoter inserted in the SV-Luc reporter construct. These reporter genes were cotransfected in CHO cells with the indicated amounts of expression vectors CMV-zfISL1 and CMV-rER. Cells were either treated or untreated with E2 (10-8 M) after transfection. Results are expressed as "fold inductions" of the reporter gene activities in the presence of E2 compared with the activity in the absence of ligand. Results are shown as the means ± SEM of three values obtained in a representative experiment.

 
ISL1 Binding to Its DNA Target Sequences Modulates ISL1-rER Interaction
In the presence of both the ISL1-binding sites and the ERE within a promoter, one would expect, according to the preceding results, a higher estrogen-dependent activation when ISL1 and rER are present than that mediated by rER alone. Furthermore, in these promoter contexts, a more pronounced E2-dependent transcriptional induction by ISL1 and rER would also be expected in the presence of three ISL1-binding sites than in the presence of a single ISL1-binding site. Nonetheless, Fig. 9Go shows that the estrogen-induced transcriptional activation was identical in the presence or absence of ISL1 and occurred irrespective of the number of ISL1-binding sites (from 1 to 3) present in the ERE-containing promoters. This observation could suggest that, once bound to DNA, ISL1 has a reduced affinity for ER, and that the estrogen response of promoters containing both type of recognition sites is mainly due to rER interaction with the ERE. To check this hypothesis, we set up GST pull-down assays using the matrix-bound GST-hER LBD fusion protein to study its ability to retain the 35S-methionine-labeled in vitro-translated zfISL1, either in the absence or in the presence of ISL1-binding sites. Consistent with our previous results, the in vitro-translated zfISL1 protein was able to interact with the LBD of ER in the absence of DNA, as revealed by the band observed when 35S-methionine-labeled zfISL1 lysate was incubated with the E2-treated GST-hER LBD fusion protein (Fig. 11AGo, lane 3). This protein-protein interaction was moreover specific, since no similar band was present when the zfISL1-programmed lysate was incubated with the GST fusion protein bound-beads (Fig. 11AGo, lane 2). The presence of increasing amounts of I1 double-stranded oligonucleotide, corresponding to the most distal ISL1-binding site characterized within the rtER gene promoter, led to a strong and dose-dependent decrease in the amount of 35S-methionine-labeled zfISL1 protein retained by the GST-hER LBD (Fig. 11AGo, lanes 4–7), showing that ISL1 displayed a weaker affinity for ER in the presence of ISL1-binding sites than that observed in the absence of DNA. In contrast, coincubation of the 35S-methionine-labeled zfISL1 lysate with the GST-hER LBD fusion protein in the presence of increasing amounts of double-stranded AP1 oligonucleotide did not influence ISL1-ER interaction (Fig. 11AGo, lanes 8–11), showing that the inhibitory effect of DNA on ISL1-ER interaction is specific for the I1 sequence. These data not only confirm that ISL1 can interact in vitro with ER via the LBD of the nuclear receptor, but also establish that the presence of ISL1-binding sites specifically interfere with the protein-protein interaction, binding of ISL1 to its DNA target sequences decreasing its affinity for ER.



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Figure 11. ISL1 Binding to DNA Decreases ISL1-rER Interaction

Panel A shows SDS-PAGE analysis of the 35S-methionine-labeled in vitro-translated zfISL1 retained by the GST-hER LBD fusion protein in GST pull-down assays, performed in the absence or in the presence of double-stranded I1 and AP1 oligonucleotides. The zfISL1-programmed lysate was incubated with limiting amounts of glutathione-agarose beads containing GST (lane 2) or GST-hER LBD (lanes 3–11) proteins, as indicated at the bottom of the gel. The input lane (lane 1) corresponds to 50% of the total amount of 35S-methionine-labeled in vitro-translated zfISL1 used for the GST pull-down assay. The amount of labeled in vitro-translated ISL1 retained by the GST-hER LBD protein in the absence of any oligonucleotide is shown in lane 3. Increasing amounts (25, 50, 100, or 200 ng) of double-stranded I1 and AP1 oligonucleotides (lanes 4–7 and lanes 8–11, respectively), were added as indicated at the top of the gel. Panel B shows transfection experiment results, expressed as relative luciferase activities, corresponding to luciferase activities corrected for transfection efficiency by the ß-galactosidase values. The ERE-SV-Luc reporter construct contains the FP1 proximal region of the rtER promoter overlapping the imperfect ERE. This reporter gene was cotransfected in CHO cells with the indicated amounts of expression vectors CMV-rER and CMV-zfISL1. Cells were either treated or untreated with E2 (10-8 M) after transfection. Each value corresponds to the mean ± SEM of three values obtained in a representative experiment. Untreated cells are shown by gray bars while E2-treated cells are shown by black bars. Note that in some wells, double-stranded (ds) oligonucleotides (I1, AP1, and EREc) were cotransfected with the different plasmids.

 
A last set of transfection experiments was then performed to confirm that the presence of ISL1-binding sites could interfere with the ISL1-mediated inhibition of ER activity in an artificial promoter context. The ERE-SV-Luc artificial estrogen-responsive plasmid, which contains no ISL1-binding sites and whose ER-driven transcriptional activity was previously shown to be inhibited by ISL1 (Fig. 6Go), was used as a reporter construct. Untreated or E2-treated CHO cells were transfected with this reporter construct, with or without rER and zfISL1 expression vectors, and in the absence or presence of various double-stranded oligonucleotides (I1, AP1, and EREc). As previously shown, in the absence of oligonucleotides, rER activation of the reporter gene was counteracted by ISL1 (Fig. 11BGo). Cotransfection of I1 oligonucleotide (50 ng or 100 ng) with rER expression vector alone did not lead to any significant modification of either the basal or the E2-stimulated luciferase activity of the ERE-SV-Luc construct (Fig. 11BGo). By contrast, when increasing amounts of I1 oligonucleotide were cotransfected in the presence of rER and ISL1, a gradual, dose-dependent reversion of the inhibitory effect of ISL1 on the estrogen responsiveness was observed. The highest dose of I1 oligonucleotide (100 ng) restored a fold induction by E2 similar to that observed in the presence of rER expression alone (Fig. 11BGo). Cotransfection of AP1 oligonucleotide with rER expression vector alone led to a slight decrease in both the basal and the E2-stimulated luciferase activity, the fold induction by E2, however, being unaffected. In cells cotransfected with both rER and zfISL1 expression vectors, the presence of AP1 oligonucleotide did not modify the inhibitory effect of ISL1 on the ER-driven transcriptional activation that was still observed (Fig. 11BGo). Finally, cotransfection of EREc oligonucleotide with rER expression vector alone led to a complete loss of induction by E2 of the ERE-SV-Luc reporter construct (Fig. 11BGo). We can therefore conclude that, while ISL1 is able to inhibit the ER-driven transcriptional activation in an artificial estrogen-responsive promoter context, this inhibitory effect is reversed when ISL1 binds to DNA. These data suggest that binding of ISL1 to DNA decreases its affinity for rER, thus allowing the restoration of a full ligand-dependent transcriptional activation of an ERE-containing promoter by rER.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Focusing on the adult rat hypothalamic arcuate nucleus, which has been previously shown to be densely populated with ISL1-expressing neurons (15), we report here that ISL1 and ER colocalize. ISL1 and ER showed a strictly nuclear localization and were coexpressed in most cells of this hypothalamic nucleus, very few neurons expressing only one of these proteins. This result raises the possibility that ISL1 could be involved in the maintenance of the differentiated phenotype of the ER-expressing neurons. To date, little is known about the molecular mechanisms of LIM/HD protein functions in specification and maintenance of neuronal properties since no or few LIM/HD target genes have been identified. Based on the colocalization of ISL1 and ER in the central nervous system, we hypothesized that ISL1 could functionally interact with ER in vivo and, subsequently, modulate estrogen-dependent target gene expression. Using in vivo coimmunoprecipitation, we first demonstrated the existence of a specific ISL1-ER interaction in vivo, suggesting that the striking colocalization between ISL1 and ER observed in rat hypothalamic nuclei could be physiologically relevant. GST pull-down assays confirmed that these two transcription factors are able to physically interact in vitro. Despite the fact that a number of interacting partners have already been identified for nuclear receptors, as well as for some LIM/HD transcription factors, this is, to our knowledge, the first evidence for direct protein-protein interaction between members of these two families of transcription factors in vitro and in vivo. We show here that ISL1 interacts in vitro with several nuclear receptors, including orphan receptors such as COUP-TFI and SF-1. In addition, ISL1 interacts with the rat and rainbow trout ERs, but also with the natural ER variant TERP, which consists only in the C-terminal LBD of rER (30). This last observation is particularly interesting since it shows that the ISL1-rER interaction is, at least in part, mediated by the LBD of rER. The fact that estradiol enhances this protein-protein interaction is in accordance with this idea. It can indeed be envisaged that ligand binding to ER induces a conformational change within the LBD that facilitates ISL1-ER interaction. Focusing on the LBD, we give in vitro evidence that ISL1 may interact with ER residues required for the generation of the LBD dimerization interface, ISL1 precluding ER dimer formation in solution. Moreover, ISL1 is able, probably through inhibition of ER dimerization, to specifically inhibit ER-ERE complex formation. No similar inhibition of COUP-TFI DNA binding activity was observed, although ISL1-COUP-TFI interaction was demonstrated in pull-down assays, suggesting that ISL1 does not negatively interfere with this orphan receptor. As a consequence, the LIM/HD protein ISL1 specifically represses ER transactivation function. The observation that ISL1 interacts with TERP may moreover suggest that TERP could relieve ISL1-mediated inhibition of ER functions. Previous works have shown that TERP exerts a biphasic action on ligand-activated ER-dependent transcription, depending on the relative amounts of both proteins (31). The inhibitory effect of TERP on ER transcriptional activity observed at TERP to ER ratios equal to or greater than 1:1 have been shown to occur by formation of inactive TERP-ER heterodimers and by competition for SRC-1 coactivator (32). Although little is known about the molecular mechanisms underlying the stimulation of ER transactivation function by lower TERP levels, one could envisage that, under these conditions, TERP might titer a repressor of ER activity, thus enhancing ER-dependent transcription. Such a repressor could be ISL1.

Several previous studies have already shown that ER-mediated transcriptional activation can be modulated by interaction with other transcription factors. Indeed, in addition to its ability to associate with other nuclear receptor superfamily members (33, 34, 35), ER has already been described as interacting with homeodomain-containing proteins, these interactions resulting in synergy between the two transcription factors. For example, Pit1 increases the estrogen responsiveness of the PRL promoter, the integrity of the DNA binding POU-specific domain being required (36). This effect is likely to be due to a cooperative binding of Pit1 and ER, the binding of Pit1 to its DNA target site promoting interaction of ER with the nonpalindromic ERE within the PRL promoter (36). Such cooperative interactions and subsequent modulation of target gene expression have also been reported between ER and the POU family member Brn3b (37). In this case, the POU domain protein directly interacts with ER in vitro and in vivo, and this interaction seems to occur independently of DNA and of estradiol. This physical association requires the POU domain of Brn3b and the DNA binding domain of the steroid receptor and significantly enhances the affinity of ER for DNA. As a consequence, this POU domain protein modulates the transcriptional activation of estrogen-dependent promoters mediated by ER (37). Our results provide the first evidence for a functional interaction between ER and a member of the LIM/HD protein family. We report here that inhibition of ER transactivation function by ISL1 is likely to be mediated by the N-terminal LIM domains of ISL1: a truncated ISL1 protein consisting only of the LIM motifs is indeed able to repress, in a dose-dependent manner, ER-mediated transcriptional activation in a similar extent to the full-length ISL1. A wide range of protein-protein interaction involving LIM/HD proteins have already been reported, most of which are mediated by the LIM domains (for review, see Ref. 38). The LIM interacting proteins include cytoskeletal components (39, 40, 41), bHLH proteins (42, 43, 44, 45), the insulin receptor (46, 47), as well as various POU-HD transcription factors (48, 49, 50, 51), and other members of the LIM/HD family (38, 40, 52, 53). In addition, LIM domains have recently been shown to interact with a family of cofactors, including NLI/LBD1 or its Drosophila homolog Chip (53, 54, 55, 56, 57, 58). As a result of their ability to homodimerize, these cofactors allow indirect association of several LIM/HD proteins, such as ISL1 and ISL2, which absolutely require NLI/LBD1 to homo- and heterodimerize (53). Several data suggest that LIM protein cofactors regulate LIM/HD proteins activity in vitro and in vivo, but the mechanism by which these cofactors act remains unclear (45, 54, 55, 56, 57, 58, 59, 60, 61, 62). Although NLI/LBD1/Chip have been suggested to act, at least in part, by relieving the interference exerted by LIM domains on LIM/HD proteins activity (54, 56, 59), recent data argue against such a mechanism (61, 63). Aside from their role in intermolecular interactions, LIM domains have indeed been proposed to mediate intramolecular inhibition of LIM/HD activity (49, 50, 51, 56, 60, 64, 65). This hypothesis was first proposed based on the inhibitory effect of the LIM domains of the rat ISL1 protein on its DNA binding activity (64). Consistent with this result, the full-length ISL1 is unable to activate transcription of a reporter gene containing the "TAAT" motif, and both DNA binding and transactivation activities are restored by deletion of the LIM domains (64). Another study reports that overexpression of Islet 3 (ISL3) LIM domains in zebrafish embryo causes dramatic cerebellar and ocular defects (60). These morphological abnormalities can be rescued by coexpression of full-length ISL3, suggesting that the LIM domains act as a specific dominant-negative variant of ISL3 (60). Binding of coactivator proteins to the LIM domains would be required to relieve the intramolecular inhibition they exert and to allow activation of ISL3 (60). Consistent with this hypothesis, mutation or deletion of the LIM domains activates xLIM1 in a Xenopus axis induction assay (65), while binding of the LBD1 cofactor to the LIM domains of the intact xLIM1 activates the LIM/HD protein in vivo (54). Likewise, NLI/LBD1 enhances transactivation properties of P-Lim in a similar manner to that subsequent to the deletion of LIM domains (56). However, this model of intramolecular inhibition is still controversial. Indeed, in both the proglucagon and the somatostatin gene promoter context, no inhibitory effect of the LIM domains was observed for the rat ISL1 protein (66, 67). Consistent with these last results, the LIM domains of the chinook salmon ISL2 protein affect neither the specificity nor the affinity of DNA binding (29). Furthermore, recent data show that deletion of the LIM domains of Apterous results in a loss of biological activity of this LIM/HD protein (63), which argue against a putative repressor role for the LIM domains. Moreover, overexpression of the Drosophila cofactor Chip results in a decrease in Apterous activity in vivo, instead of the enhancement of the LIM/HD protein function that would be expected according to the intramolecular inhibition model (61).

In the present study, we give evidence that the full-length zebrafish ISL1 protein specifically binds the P1 high-affinity target sequence identified in the rat insulin enhancer (2), as well as the three ISL1-binding sites, defined in the rtER gene promoter in regard to their sequence identity with the consensus DNA targets characterized in fish (29). Although ISL1 efficiently binds to these DNA sequences in vitro, this transcription factor has, on its own, neither any positive nor any negative transcriptional regulatory effect. However, in artificial promoter contexts containing one or more ISL1-binding sites but lacking an ERE, ER is able to serve as a coactivator for ISL1, leading to a strong ligand-induced transcriptional activation. This effect depends both on the presence of ISL1-binding sites within the promoter and on the number of these sites and requires the homeo- and/or transactivation domains of ISL1. Coactivation is likely to involve ISL1 binding to DNA through the homeodomain, and interaction of ER with the LIM domains, the DNA-bound ISL1 thus tethering an ER monomer to the promoter. Ligand-induced recruitment of coactivators by ER may potentiate transcriptional activation. However, our results strongly suggest that coactivation is unlikely to occur in an ERE-containing promoter context. Indeed, in an artificial promoter context containing both the ISL1-binding sites and the ERE, a similar ligand-induced transcriptional activation was observed in the presence of ER and ISL1 than that mediated by ER alone. If ER acted as a coactivator for ISL1 in this promoter context, one would expect a higher estrogen-dependent activation when ISL1 and ER are present than that mediated by ER alone. Moreover, in an artificial ERE-containing promoter, a more pronounced E2-dependent transcriptional induction by ISL1 and ER would also be expected in the presence of three ISL1-binding sites than in the presence of a single ISL1-binding site, which was not the case. Thus, it seems more likely that the estrogen response of promoters containing both type of recognition sites is mainly due to ER interaction with the ERE, inhibition of ER functions by ISL1 being alleviated by binding of the LIM/HD protein to its DNA target sequences. In accordance with this hypothesis, we demonstrate that, once bound to DNA, ISL1 has a reduced affinity for ER and does no longer inhibit ER transactivation function. We thus propose a model for the regulation of ER functions by ISL1 in which, in the context of estrogen-responsive promoters lacking ISL1-binding sites, ISL1 inhibits ER-driven transcriptional activation (Fig. 12AGo). This specific repressor effect of ISL1 on ER function is likely to be mediated through direct protein-protein interaction involving the LIM domains of ISL1 and the LBD of the nuclear receptor. Ligand-induced ISL1-ER interaction would inhibit ER dimerization and subsequent DNA binding, decreasing estrogen-dependent target gene expression. In promoter contexts containing both types of recognition element, binding of ISL1 to its DNA binding sites would weaken ISL-ER interaction and relieve interference with ER transactivation function (Fig. 12BGo). Finally, in the case of promoters containing ISL1-binding sites but lacking EREs, monomeric ER may acts as a coactivator for ISL1. Interaction of ER with the DNA-bound ISL1 may recruit coactivators and promote transcription initiation, thus leading to the estrogen-dependent activation of genes lacking EREs (Fig. 12CGo).



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Figure 12. A Model for the Functional Cross-Talk between ISL1 and ER

Panels A and B show the putative underlying molecular mechanisms of the modulation of ER functions by ISL1 in a promoter context containing an ERE, in the absence (panel A) or presence (panel B) of ISL1-binding sites (IBS). Panel C shows a putative molecular basis for the enhancement of ISL1-driven transcriptional regulation by ER in a promoter context containing only ISL1-binding sites (IBS).

 
Based on the striking colocalization of ISL1 and ER in some hypothalamus nuclei, and on the in vivo ISL1-ER interaction, we postulate the existence of a physiologically relevant cross-talk between these proteins: ISL1 could modulate the expression of estrogen-dependent genes in a promoter-specific manner. Targeted mutation of the mouse ISL1 gene has been shown to perturb the differentiation of many cell types (68). However, ISL1-/- embryo are arrested in their development (E9.5) and die too early (E11.5) to allow the analysis of a putative role of ISL1 function in modulating the neuronal response to estrogen (68). Thus, the in vivo functional relevance of ISL1-ER interaction and the regulatory mechanisms modulating this cross-talk remain to be elucidated. Since in any given cell nucleus both ISL1- and ER-binding sites are likely to be present, under physiological conditions in which both transcription factors bind DNA, one can hypothesize that these proteins are unlikely to interfere with each other. Nevertheless, there might be particular situations in which the inhibitory cross-talk occurs. Mechanisms regulating the DNA binding activity of ISL1 would be particularly relevant to the inhibitory activity of ISL1 on ER functions, since ISL1 binding to DNA modulates ISL1-ER interaction. Phosphorylation events have been extensively described to modulate transcription factors activity, affecting the structure, the dimerization, DNA binding or transactivation activities, as well as the interaction with coactivators/corepressors or with components of the transcriptional machinery of various transcription factors. The target proteins include basic-helix-loop-helix proteins, members of the basic-leucine zipper family, and protooncogene products, as well as a vast variety of nuclear receptor superfamily members (for review, see Refs. 69, 70, 71, 72). Although no data are to date available concerning LIM-homeodomain proteins, a putative role for phosphorylation events in the regulation of ISL1 biological activity cannot be excluded. Such a regulatory role on protein-protein interaction involving the Antennapedia homeodomain protein have already been described (73). Phosphorylation has also been reported to affect the DNA binding ability of POU- and HD-containing transcription factors including Pit-1, Oct-1, Oct-2, Engrailed, and Cut (74, 75, 76, 77, 78, 79). Other regulatory mechanisms could be involved in the modulation of ISL1 functions and, more particularly, in the regulation of the DNA binding activity of ISL1. Although the hypothesis of intramolecular inhibition of the HD-mediated DNA binding activity is highly controversial, one cannot exclude that such an inhibition would occur in some particular cases, still increasing the complexity of the mechanisms underlying ISL1 inhibition of ER functions. Although the precise function of the LIM domains is not completely elucidated, these domains have been clearly demonstrated to bind zinc and iron (29, 80, 81, 82, 83, 84, 85). The fact that the modulation of the redox status of ISL1 abrogates DNA binding by the ISL1 homeodomain suggests an additional mechanism for regulating ISL1 DNA binding activity (86). Finally, the activity of various LIM/HD proteins has been shown to be regulated by the NLI/LBD/Chip cofactors both in vitro and in vivo. Recent data demonstrate that NLI is found in the nuclei of developing embryonic neuronal cells and is coexpressed with ISL1 early in motoneuron differentiation (55). The synchronous expression of both proteins in such cells during the initial stages of differentiation strongly suggests a role for this cofactor in the ISL1-dependent development of motoneurons (55). Coexpression and modulation of ISL1 biological activity by NLI in other neuronal populations during development and in the adult could provide an additional mechanism for the regulation of ER function by the LIM/HD protein ISL1.

Altogether, these data and our results give insight into the highly complex mechanisms directing cell type- and promoter-specific gene expression, which are due not only to the number and diversity of transcription factors binding to regulatory sequences, but also to a complex cross-talk between these transcription factors and to the variety of regulatory events that modulate their biological activities.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Chemicals and Materials
The affinity-purified rabbit anti-ER antibody (ER{alpha} MC-20), Texas Red (TR)-labeled streptavidin, and protein G-agarose beads were from TEBU (Le Perray-en-Yvelines, France). The anti-Islet1 monoclonal antibody (39.4D5) and the rabbit anti-ER antibody (ER715) were provided, respectively, by the Developmental Studies Hybrydoma Bank (Iowa City, IA) and by the National Hormone and Pituitary Program (Baltimore, MD). Biotinylated goat antirabbit secondary antibodies, goat antimouse secondary immunoglobulins, and fluorescein isothiocyanate (FITC)-conjugated streptavidin were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Klenow fragment of E. coli DNA polymerase I and T4 polynucleotide kinase were from Boehringer (Mannheim, Germany). The TNT-coupled reticulocyte lysate was from Promega Corp. (Madison, WI). Glutathione-agarose beads, reduced glutathione, protease inhibitors, E2, charcoal-dextran, and culture media were from Sigma (St Louis, MO). FCS and antibiotics were from Life Technologies, Inc. (Eragny, France). The luciferase assay system was from Promega Corp., and luciferase activity was measured on a Dynatech Corp. luminometer (Guyancourt, France). The chemiluminescent substrate protein detection kit (ECL) was from Amersham Pharmacia Biotech (Orsay, France).

In Vivo Coimmunoprecipitation
In vivo coimmunoprecipitation was carried out to assess the interaction between ER and ISL1 in vivo. Animal investigations were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Eight adult Wistar strain female rats, weighing 250–300 g, were intraperitoneally injected with 5 µg of E2 dissolved in 20% ethanol/80% saline (0.9% NaCl) solution. After 1 h, animals were killed, and the hypothalamus was dissected and immersed in NaCl 0.9% at 4 C. The tissue was homogenized in buffer 1 [0.2 M sucrose; 3 mM MgCl2; 10 mM Tris-HCl, pH 7.4; 1 mM phenylmethylsulfonyl fluoride (PMSF); 50 mM NaF; 10 µg/ml aprotinin; 10 µg/ml leupeptin; 10 µg/ml pepstatin] in a Dounce homogenizer at 4 C. The homogenate was centrifuged at 1,000 x g for 20 min at 4 C to separate nuclei from cytosol. The pellet was then resuspended in buffer 2 [0.1 M KCl; 50 mM Tris, pH 7.9; 3 mM MgCl2; Nonidet P-40 0.1%; 20% glycerol; 1 mM dithiothreitol (DTT) and protease inhibitors]. After three cycles of freezing (-80 C) and thawing (37 C), the lysate was centrifuged at 12,000 rpm for 10 min at 4 C to pellet debris. For immunoprecipitation, the protein extract was precleared by incubation at 4 C for 4 h with 2 µg of rabbit preimmune serum and 50 µl of protein G-agarose slurry (TEBU). After centrifugation the supernatant was separated into two equal fractions, which were incubated for 16 h at 4 C with either 2 µg of the rabbit anti-ER antibody (ER715; National Hormone and Pituitary Program) or 2 µg of rabbit preimmune serum. The immunocomplexes were then collected by incubation with 50 µl of protein G-agarose slurry for 4 h at 4 C. The protein beads were washed twice in buffer 2, boiled in 2x SDS loading buffer for 5 min and resolved in a SDS-10% polyacrylamide gel. After electrophoresis, the proteins were electrotransferred from the gel to a nitrocellulose membrane (Hybond C; Amersham Pharmacia Biotech) and stained with Ponceau S to localize the mol wt markers. After 2 h saturation with PBS containing 0.1% Tween 20, 1% BSA, and 5% milk powder, the membrane was probed with a mouse monoclonal antibody directed against the rat ISL1 protein (39.4D5; Developmental Studies Hybrydoma Bank) diluted 1:25. After overnight incubation, the chemiluminescent substrate protein detection kit (ECL; Amersham Pharmacia Biotech) was used according to the manufacturer’s protocol for immunodetection of ISL1.

Double Nuclear Staining Immunohistochemistry
Adult Wistar strain female rats, weighing 250–300 g at the time of perfusion, were deeply anesthetized with sodium pentobarbital. Animals were then perfused with physiological saline (0.9% NaCl) followed by fixative containing 4% paraformaldehyde in a 0.1 M sodium phosphate buffer (pH 7.2). After overnight immersion in a 20% sucrose solution, the brain was frozen in isopentane cooled with liquid nitrogen. Coronal sections (12 µm) were obtained with a cryostat and collected on gelatin-coated slides. A veronal buffer (diethylmalonylurea 0.1 M; pH 7.4) containing 0.75% NaCl was used for immunohistochemistry. For all rinses, 0.2% Triton X-100 was added. Dilutions of all primary and secondary antibodies as well as dilutions of the streptavidin conjugates were performed in veronal buffer containing 0.5% milk powder, and all incubations were carried out at room temperature. Sections were first saturated in veronal buffer containing both 0.5% milk powder and 0.2% Triton X-100 for 45 min at room temperature. The sections were then exposed overnight to the affinity-purified rabbit anti-ER antibody (ER{alpha} MC-20; TEBU) diluted 1:250. Slides were submitted to three rinses and incubated for 90 min with biotinylated goat antirabbit secondary immunoglobulins diluted 1:2500 (Jackson ImmunoResearch Laboratories, Inc.). After three rinses, sections were exposed for 90 min to Texas Red (TR)-labeled streptavidin (TEBU) diluted 1:1500. The sections were rinsed three times and incubated with the second primary antibody, raised against a bacterially expressed peptide corresponding to the C-terminal portion of the rat Islet-1 protein (39.4D5; Developmental Studies Hybrydoma Bank). After overnight incubation with the mouse monoclonal antibody diluted 1:25, the sections were rinsed three times. Biotinylated goat antimouse secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.) diluted 1:2500 were then added for 90 min. After three more rinses, the sections were finally exposed for 90 min to FITC-conjugated streptavidin (Jackson ImmunoResearch Laboratories, Inc.) diluted 1:1500. Specificity of the staining was assessed with control sections treated exactly as previously described, except that the biotinylated goat antimouse secondary antibody was omitted. In this case, no green fluorescence was observed. These control sections allowed us to verify that the FITC-labeled streptavidin did not interact with the biotinylated goat antirabbit secondary antibodies already saturated with TR-labeled streptavidin. After three final rinses, the slides were mounted with phosphate buffer/glycerol (1:1) and examined using a Provis photomicroscope (Olympus Corp., Tokyo, Japan).

Oligonucleotides
The following oligonucleotides (Eurogentec, Seraing, Belgium) were used double stranded for EMSA or for transient transfection experiments. The positions relative to the transcription start point of the rainbow trout ER (rtER) gene are indicated in brackets for the FP1, I1, I2, and I3 sequences. EREc, 5'-CCAGGTCACAGTGACCTGAGCTAAAATAACACATTCAG-3'; P1, 5'-TCGACGCCCTTAATGGGCCAAACGGCAG-3'; AP1, 5'-TCGACGCTTGATGACTCAGCCGGAA-3'; FP1 (bp -85 to -56), 5'-TTGCTGTGTCATGTTGACCTGCTCTAGAGA-3'; I1 (bp -1,859 to -1,841), 5'-GATCCTGATCTTAAGTGACTTTT-3'; I2 (bp -1,341 to -1,323), 5'-GATCACCTGTCTAATGATGTATT-3'; I3 (bp -861 to -843), 5'-GATCTAGAGGTTAATGAACCATC-3'.

Plasmids
The GST-ISL1 fusion construct was made by in-frame ligation of the zebrafish Islet-1 coding sequence to the GST fusion protein vector pGEX-2T (Amersham Pharmacia Biotech). The pGEX-2TK-ER vector allowed expression of the GST-hER LBD fusion protein and contained the human ER spanning amino acids 282 to 595 (87). Recombinant plasmids used for in vitro translation were generated by inserting the coding sequences of the rER [wild-type ER and truncated protein TERP (30)], zebrafish SF-1 (Steroidogenic Factor-1), and human COUP-TFI (Chicken Ovalbumin Upstream Promoter-Transcription Factor) into pcDNA3 vector (Invitrogen, San Diego, CA). The rainbow trout ER (rtER) expression vector has already been described (28), and the Pepex recombinant vector contained the coding sequence of the Xenopus Cyclin E (88). The ERE/I1-SV-Luciferase and ERE/I2/I3/I1-SV-Luciferase reporter plasmids were generated by subcloning the ISL1-binding sites defined in the rtER gene promoter in the ERE-SV-Luciferase reporter construct (27). The Isl1-binding sites were inserted between the FP1 sequence of the rtER gene promoter, which encompasses the ERE (27, 28), and the SV40 promoter. The I1-SV-Luciferase and I2/I3/I1-SV-Luciferase reporter constructs were generated by removing the FP1 sequence, respectively, from the ERE/I1-SV-Luciferase and the ERE/I2/I3/I1-SV-Luciferase plasmids. The two reporter plasmids named 2.0-Basic and 0.2-Basic used in transient transfection experiments have been described previously (26). The CMV-rER and CMV-hCOUP-TFI expression vectors were the same constructs as those used for in vitro translation. The CMV-zfISL1 expression vector was obtained by cloning the zebrafish Islet-1 coding sequence into pcDNA3 plasmid. All experiments in this study were thus performed using the zebrafish Islet-1 (zfISL1) protein. Amino acid sequences deduced from the cloned rat (2) and zebrafish (7) ISL1 cDNAs showed that these proteins share 98% identity. To obtain the CMV-zfISL1{Delta}HD/AD expression vector, the first 388 bp of the zfISL1 coding sequence were subcloned in Bluescript, before being reinserted into pcDNA3 plasmid. The resulting expression vector encodes a truncated (129 amino acids) ISL1 protein corresponding to the N-terminal LIM domains of zfISL1 and to a short additional sequence (13 amino acids), but lacking both the homeodomain and the transactivation domain. The CMV-zfISL2 expression vector was obtained by cloning the zebrafish Islet-2 coding sequence into pcDNA3 plasmid.

Generation of Proteins
The TNT-coupled reticulocyte lysate (Promega Corp.) was used with cold or 35S-methionine (ICN Pharmaceuticals, Inc., Cleveland, OH) according to the manufacturer’s protocol to produce various proteins. Translation efficiencies of the 35S-labeled proteins were checked by SDS-10% polyacrylamide gel (SDS-PAGE) analysis of 2 µl of translation products. To study the potential effects of the ligand, 25 µl of in vitro-translated wild-type rER were incubated for 1 h at room temperature with an equal volume of TEG buffer (50 mM Tris-HCl, pH 7.4; 50 mM NaCl; 1.5 mM EDTA; 10% glycerol; 5 mM MgCl2; 1 mM DTT) in the absence or presence of 10-5 M E2. The GST and GST-ISL1 fusion proteins were generated using the pGEX-2T vector, which allows bacterial expression under isopropyl-1-thio-ß-D-galactopyranoside (IPTG) induction. Similarly, the GST-hER LBD fusion protein was generated using the pGEX-2TK recombinant vector. After IPTG induction, cells were collected by centrifugation at 4,000 rpm for 20 min at 4 C. The supernatant was discarded, and the pellet was resuspended in NETN buffer (20 mM Tris, pH 8.0; 100 mM NaCl; 1 mM EDTA; 0.5% Nonidet P-40; 1 mM PMSF; 5 µg/ml aprotinin; 5 µg/ml leupeptin; 5 µg/ml pepstatin), and sonicated twice for 30 sec. The suspension was then centrifuged for 15 min at 12,000 rpm and the protein concentration of the supernatant (soluble fraction), containing the GST and GST-hER LBD fusion proteins, was assessed by Bradford assay. To study the potential effects of the ligand, the GST-hER LBD fusion protein was treated in the absence or presence of 10-5 M E2. Since SDS-PAGE analysis showed that the fusion protein GST-ISL1 occurred predominantly as insoluble inclusion bodies, the insoluble fraction was washed with 4 M urea in 50 mM Tris, pH 8.5, and solubilized in 8 M urea in 50 mM Tris, pH 8.5, in the presence of 10 mM DTT. Inclusion body proteins were then refolded by overnight dialysis at 4 C against 1,000 volumes of dialysis buffer (50 mM Tris, pH 8.5; 10 mM DTT), and the protein concentration in the supernatant was assessed by Bradford assay. Before the GST pull-down assay, a fraction of each sample was loaded onto a SDS-10% polyacrylamide gel and analyzed by Coomassie blue staining.

GST Pull-Down Assay
For in vitro protein-protein interactions, 10 µg of GST or refolded GST-ISL1 were incubated overnight at 4 C with 10 µl of glutathione-agarose beads (Sigma) prepared and stored in NETN buffer. The beads were washed twice with NETN buffer and resuspended into 5 volumes of binding buffer (50 mM Tris, pH 8.0; 50 mM NaCl; 0.02% Tween 20; 0.02% BSA; 1 mM PMSF; 10 µg/ml aprotinin; 10 µg/ml leupeptin; 10 µg/ml pepstatin). Equivalent amounts of 35S-methionine-labeled in vitro-translated proteins (1–5 µl) were then incubated with 10 µg of GST fusion protein-bound beads in 250 µl of binding buffer for 2 h at 4 C. For the GST pull-down competition assay, limiting amounts of GST and GST-hER LBD fusion proteins-bound beads (500 ng) and of 35S-methionine-labeled in vitro-translated proteins (1.5 µl) were used, as determined in preliminary experiments. Increasing amounts (1.5–15 µl) of unlabeled competitor lysate were added to the reaction mixture. To study the influence of DNA binding on in vitro protein-protein interaction, similar amounts of GST and GST-hER LBD fusion protein-bound beads and of 35S-methionine-labeled in vitro-translated proteins were incubated in the presence of increasing amounts (25–200 ng) of double-stranded I1 and AP1 oligonucleotides. In all cases, beads were then washed with washing buffer (50 mM Tris, pH 8.0; 150 mM NaCl; 0.02% Tween 20; 1 mM PMSF; 10 µg/ml aprotinin; 10 µg/ml leupeptin; 10 µg/ml pepstatin) and the proteins were solubilized in 1x SDS loading buffer, heated at 100 C for 5 min, and resolved in a SDS-10% polyacrylamide gel. After electrophoresis, the gel was dried and exposed to radiographic film and the amounts of 35S-methionine-labeled in vitro translated protein retained on the beads were assessed with a PhosphoImager.

EMSA
To characterize the ISL1-binding sites identified in the rtER gene promoter, EMSA was performed using 50 ng of unpurified bacterially expressed GST and GST-ISL1 fusion proteins, obtained directly after the dialysis step. The proteins were incubated with 1 µg polydIdC in 20 µl binding buffer (20 mM HEPES, pH 7.9; 1 mM DTT; 50 mM KCl; 10% glycerol; 2.5 mM MgCl2). After 20 min at room temperature, samples were incubated with 0.2 ng of double-stranded oligonucleotide probe labeled with {alpha}-32P dCTP (20,000 cpm) for another 20 min. The effect of ISL1-ER interactions on the DNA binding activity of the nuclear receptor was assessed by coincubation of 4 µl of unprogrammed or rER-programmed lysate with increasing amounts of purified fusion proteins (0–500 ng of GST or GST-ISL1 proteins). In this case, the fusion proteins were enriched by affinity chromatography with glutathione-agarose beads (Sigma) according to the manufacturer’s protocol after the dialysis step. Specifically bound GST and GST-ISL1 proteins were eluted with 20 mM reduced glutathione in 100 mM Tris-HCl, pH 8.0/120 mM NaCl and coincubated with the in vitro-translated products for 1 h at room temperature. The samples were then treated as previously, and the protein-DNA complexes were separated from the free probe by nondenaturating electrophoresis on 4% polyacrylamide gel in 0.5x TBE (45 mM Tris base; 45 mM boric acid; 1 mM EDTA). The gel was dried before autoradiography.

Cell Cultures and Transient Transfection
CHO cells were routinely grown at 37 C in DMEM-F12 (Sigma) containing 10% FCS (Life Technologies, Inc.). The medium was replaced by DMEM-F12 supplemented with 10% charcoal/dextran-treated FCS 1 h before transfection. Both media contained 100 U/ml penicillin, 100 µg/ml streptomycin, and 25 µg/ml Amphotericin (Sigma). Cells were transfected in 24-well plates by using a calcium phosphate/DNA precipitation method as previously described (26). Briefly, 820 ng of each 0.2-Basic or 2.0-Basic reporter plasmid were cotransfected with 30 ng of control plasmid CMV-ßGAL and 50 ng of various pcDNA3 expression plasmids (CMV-rER, CMV-zfISL1, and CMV-hCOUP-TFI). In a similar manner, 200 ng of each SV-Luciferase or ERE-SV-Luciferase reporter constructs were cotransfected with 30 ng of CMV-ßGAL, 50–100 ng of CMV-rER plasmid and increasing amounts (5–100 ng per well) of CMV-zfISL1, CMV-zfISL1{Delta}HD/AD, and CMV-zfISL2 expression vectors. In some wells, increasing amounts (50–100 ng per well) of double-stranded I1, AP1, or EREc oligonucleotides were added. In all cases, the total amount of CMV promoter was maintained constant in all wells through addition of empty pcDNA vector when necessary. Similarly, the total amount of DNA was maintained constant (1 µg per well) through addition of Bluescript plasmid. After 18 h, cells were washed once with PBS and fresh medium was added. Hormone was also added (10-8 M E2) and cells were harvested 36 h later. For luciferase assay, 10% of the cellular extract was used to measure the luciferase activity. Half of the remaining extract was taken to perform ß-galactosidase assay. Luciferase activities were normalized for transfection efficiency with the ß-galactosidase activities.


    ACKNOWLEDGMENTS
 
We are extremely grateful to M. L. Thieulant for the gift of ER{alpha} MC-20 and ER715 antibodies. We thank C. Tiffoche for the gift of the rER and TERP expression vectors, K. Le Guellec for providing us with the Cyclin E expression vector, and Y. Le Dréan for the gift of SF1 expression vector. We also thank B. Katzenellenbogen for the gift of the pGEX-2TK-ER plasmid. We are grateful to E. Pellegrini for technical assistance during the immunohistochemistry protocol and help with data analysis. We also thank R. Métivier for helpful advice on the GST pull-down protocol. Finally, we thank O. Kah and D. Mazurais who did us the favor of reading and correcting this manuscript.


    FOOTNOTES
 
Address requests for reprints to: Dr. Gilles Salbert, Equipe Information et Programmation Cellulaire, UMR 6026 Centre National de la Recherche Scientifique, Université de Rennes I, Campus de Beaulieu, 35042 Rennes cedex, France. E-mail: gilles.salbert{at}univ-rennes1.fr

This work was supported by grants from the Ministère de la Recherche et de l’Enseignement and the Centre National de la Recherche Scientifique.

Received for publication October 25, 1999. Revision received June 6, 2000. Accepted for publication July 11, 2000.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Freyd G, Kim SK, Horvitz HR 1990 Novel cysteine-rich motif and homeodomain in the product of the Caenorhabditis elegans cell lineage gene Lin-11. Nature 344:876–879[CrossRef][Medline]
  2. Karlsson O, Thor S, Norberg T, Ohlsson H, Edlund T 1990 Insulin gene enhancer binding protein Isl-1 is a member of a novel class of proteins containing both a homeo- and a Cys-His domain. Nature 344:879–882[CrossRef][Medline]
  3. Way J, Chalfie CM 1988 Mec-3, a homeobox-containing gene that specifies differentiation of the touch receptor neurons in C. elegans. Cell 54:5–16[Medline]
  4. Curtiss J, Heilig JS 1998 DeLIMiting development. Bioessays 20:58–69[CrossRef][Medline]
  5. Ericson J, Thor S, Edlund T, Jessell TM, Yamada T 1992 Early stages of motor neuron differentiation revealed by expression of homeobox gene Islet-1. Science 256:1555–1560[Medline]
  6. Tsuchida T, Ensini M, Morton SB, Baldassare M, Edlund T, Jessell TM, Pfaff SL 1994 Topographic organization of embryonic motor neurons defined by expression of LIM homeobox genes. Cell 79:957–970[Medline]
  7. Inoue A, Takahashi M, Hatta K, Hotta Y, Okamoto H 1994 Developmental regulation of Islet-1 mRNA expression during neuronal differentiation in embryonic zebrafish. Dev Dynam 199:1–11[Medline]
  8. Korzh V, Edlund T, Thor S 1993 Zebrafish primary neurons initiate expression of the LIM homeodomain protein Isl-1 at the end of gastrulation. Development 118:417–425[Abstract/Free Full Text]
  9. Appel B, Korzh V, Glasgow E, Thor S, Edlung T, Dawid IB, Eisen JS 1995 Motoneuron fate specification revealed by patterned LIM homeobox gene expression in embryonic zebrafish. Development 121:4117–4125[Abstract/Free Full Text]
  10. Lumsden A 1995 A "LIM code" for motor neurons? Curr Biol 5:491–495[Medline]
  11. Pfaff S, Kintner C 1998 Neuronal diversification: development of motor neuron subtypes. Curr Opin Neurobiol 8:27–36[CrossRef][Medline]
  12. Thor S, Andersson SGE, Tomlinson A, Thomas JB 1999 A LIM-homeodomain combinatorial code for motor neuron pathway selection. Nature 397:76–80[CrossRef][Medline]
  13. Varela-Echavarria A, Pfaff SL, Guthrie S 1996 Differential expression of LIM homeobox genes among motor neuron subpopulations in the developing chick brain stem. Mol Cell Neurosci 8:242–257[CrossRef][Medline]
  14. Dong J, Asa SL, Drucker DJ 1991 Islet cell and extrapancreatic expression of the LIM domain homeobox gene Isl-1. Mol Endocrinol 5:1633–1641[Abstract]
  15. Thor S, Ericson J, Brännstrsm T, Edlund T 1991 The homeodomain LIM protein Isl-1 is expressed in subsets of neurons and endocrine cells in the adult rat. Neuron 7:881–889[Medline]
  16. Gong Z, Hui CC, Hew CL 1995 Presence of Isl-1-related LIM domain homeobox genes in teleost and their similar patterns of expression in brain and spinal cord. J Biol Chem 270:3335–3345[Abstract/Free Full Text]
  17. Way JC, Chalfie M 1989 The mec-3 gene of Caenorhabditis elegans requires its own product for maintained expression and is expressed in three neuronal cell types. Genes Dev 3:1823–1833[Abstract]
  18. Thor S, Thomas JB 1997 The Drosophila islet gene governs axon pathfinding and neurotransmitter identity. Neuron 18:397–409[CrossRef][Medline]
  19. Benveniste RJ, Thor S, Thomas JB, Taghert PH 1998 Cell type-specific regulation of the Drosophila FMRF-NH2 neuropeptide gene by Apterous, a LIM homeodomain transcription factor. Development 125:4757–4765[Abstract/Free Full Text]
  20. Anglade I, Pakdel F, Bailhache T, Petit F, Salbert G, Jego P, Valotaire Y, Kah O 1994 Distribution of estrogen receptor-immunoreactive cells in the brain of the rainbow trout (Oncorhynchus mykiss). J Neuroendocrinol 6:573–583[Medline]
  21. Cintra A, Fuxe K, Harfstrand A, Agnati LF, Miller LS, Greene JL, Gustafsson JA 1986 On the cellular localization and distribution of estrogen receptors in the rat tel- and diencephalon using monoclonal antibodies to human estrogen receptor. Neurochem Int 8:587–595[CrossRef]
  22. Pfaff D, Keiner M 1973 Atlas of estradiol-concentrating cells in the central nervous system of the female rat. J Comp Neurol 151:121–158[Medline]
  23. Salbert G, Bonnec G, Le Goff P, Boujard D, Valotaire Y, Jego P 1991 Localisation of estradiol receptor mRNA in the forebrain of rainbow trout. Mol Cell Endocrinol 76:173–180[CrossRef][Medline]
  24. Salbert G, Atteke C, Bonnec G, Jego P 1993 Differential regulation of the estrogen receptor mRNA by estradiol in the trout hypothalamus and pituitary. Mol Cell Endocrinol 96:177–182[CrossRef][Medline]
  25. Stumpf WE 1970 Estrogen-neurons and estrogen-neuron systems in the periventricular brain. Am J Anat 129:207–218[Medline]
  26. Lazennec G, Kern L, Valotaire Y, Salbert G 1997 The nuclear orphan receptors COUP-TF and ARP-1 positively regulate the trout estrogen receptor gene through enhancing autoregulation. Mol Cell Biol 17:5053–5066[Abstract]
  27. Lazennec G, Kern L, Salbert G, Saligaut D, Valotaire Y 1996 Cooperation between the human estrogen receptor (ER) and MCF-7 cell-specific transcription factors elicits high activity of an estrogen inductible enhancer from the trout ER gene promoter. Mol Endocrinol 10:1116–1126[Abstract]
  28. Le Dréan Y, Lazennec G, Kern L, Saligaut D, Pakdel F, Valotaire Y 1995 Characterization of an estrogen-responsive element implicated in regulation of the rainbow trout estrogen receptor gene. J Mol Endocrinol 15:37–47[Abstract]
  29. Gong Z, Hew CL 1994 Zinc and DNA binding properties of a novel LIM homeodomain protein Isl-2. Biochemistry 33:15149–15158[Medline]
  30. Friend KE, Ang LW, Shupnik MA 1995 Estrogen regulates the expression of several different estrogen receptor mRNA isoforms in rat pituitary. Proc Natl Acad Sci USA 92:4367–4371[Abstract]
  31. Schreihofer DA, Resnick EM, Soh AY, Shupnik MA 1999 Transcriptional regulation by a naturally occuring truncated rat estrogen receptor (ER), truncated ER product-1 (TERP-1). Mol Endocrinol 13:320–329[Abstract/Free Full Text]
  32. Resnick EM, Schreihofer DA, Periasamy A, Shupnik MA 2000 Truncated estrogen receptor product-1 supresses estrogen receptor transactivation by dimerization with estrogen receptors {alpha} and ß. J Biol Chem 275:7158–7166[Abstract/Free Full Text]
  33. Le Dréan Y, Liu D, Wong OL, Xiong F, Hew CL 1996 Steroidogenic factor 1 and estradiol receptor act in synergism to regulate the expression of the salmon gonadotropin IIß subunit gene. Mol Endocrinol 10:217–229[Abstract]
  34. Klinge CM, Silver BF, Driscoll MD, Sathya G, Bambara RA, Hilf R 1997 Chicken ovalbumin upstream promoter-transcription factor interacts with estrogen receptor, binds to estrogen response elements and half-sites, and inhibits estrogen-induced gene expression. J Biol Chem 272:31465–31474[Abstract/Free Full Text]
  35. Lee SK, Choi HS, Song MR, Lee MO, Lee JW 1998 Estrogen receptor, a common interaction partner for a subset of nuclear receptors. Mol Endocrinol 12:1184–1192[Abstract/Free Full Text]
  36. Day RN, Koike S, Sakai M, Muramatsu M, Maurer RA 1990 Both Pit-1 and the estrogen receptor are required for estrogen responsiveness of the rat prolactin gene. Mol Endocrinol 4:1964–1971[Abstract]
  37. Budhram-Mahadeo V, Parker M, Latchman DS 1998 POU transcription factors Brn-3a and Brn-3b interact with the estrogen receptor and differentially regulate transcriptional activity via an estrogen response element. Mol Cell Biol 18:1029–1041[Abstract/Free Full Text]
  38. Dawid IB, Breen JJ, Toyama R 1998 LIM domains: multiple roles as adapters and functional modifiers in protein interactions. Trends Genet 14:156–162[CrossRef][Medline]
  39. Sadler I, Crawford AW, Michelsen JW, Beckerle MC 1992 Zyxin and cCRP: two interactive LIM domain proteins associate with cytoskeleton. J Cell Biol 119:1573–1587[Abstract]
  40. Schmeichel KL, Beckerle MC 1994 The LIM domain is a modular protein-binding interface. Cell 79:211–219[Medline]
  41. Arber S, Caroni P 1996 Specificity of single LIM motifs in targeting and LIM/LIM interactions in situ. Genes Dev 10:289–300[Abstract]
  42. German MS, Wang JH, Chadwick RB, Rutter WJ 1992 Synergistic activation of the insulin gene by a LIM-homeodomain protein and a basic helix-loop-helix protein: building a functional insulin minienhancer complex. Genes Dev 6:2165–2176[Abstract]
  43. Johnson JD, Zhang W, Rudnick A, Rutter WJ, German MS 1997 Transcriptional synergy between LIM-homeodomain proteins and basic helix-loop-helix proteins: the LIM2 domain determines specificity. Mol Cell Biol 17:3488–3496[Abstract]
  44. Wadman IA, Li J, Bash RO, Forster A, Osada H, Rabbitts TH, Baer R 1994 Specific in vivo association between the bHLH and LIM proteins implicated in human T cell leukemia. EMBO J 13:4831–4839[Abstract]
  45. Wadman IA, Osada H, Grütz GG, Agulnick AD, Westphal H, Forster A, Rabbitts TH 1997 The LIM-only protein Lmo2 is a bridging molecule assembling an erythroid, DNA-binding complex which includes the TAL1, E47, GATA-1 and Ldb1/NLI proteins. EMBO J 16:3145–3157[Abstract/Free Full Text]
  46. Wu RY, Gill GN 1994 LIM domain recognition of a tyrosine-containing tight turn. J Biol Chem 269:25085–25090[Abstract/Free Full Text]
  47. Wu RY, Durick K, Songyang Z, Cantley LC, Taylor SS, Gill GN 1996 Specificity of LIM domain interactions with receptor tyrosine kinases. J Biol Chem 271:15934–15941[Abstract/Free Full Text]
  48. Xue D, Tu Y, Chalfie M 1993 Cooperative interactions between the Caenorhabditis elegans homeoproteins UNC-86 and MEC-3. Science 261:1324–1328[Medline]
  49. Bach I, Rhodes SJ, Pearse RV, Heinzel T, Gloss B, Scully KM, Sawchenko PE, Rosenfeld MG 1995 P-Lim, a LIM homeodomain factor, is expressed during pituitary organ and cell commitment and synergizes with Pit-1. Proc Natl Acad Sci USA 92:2720–2724[Abstract]
  50. Lichtsteiner S, Tjian R 1995 Synergistic activation of transcription by UNC-86 and MEC-3 in Caenorhabditis elegans embryo extracts. EMBO J 14:3937–3945[Abstract]
  51. Xue D, Finney M, Ruvkun G, Chalfie M 1992 Regulation of the mec-3 gene by the C. elegans homeoproteins UNC-86 and MEC-3. EMBO J 11:4969–4979[Abstract]
  52. Feuerstein R, Wang X, Song D, Cooke NE, Liebhaber SA 1994 The LIM double zinc-finger motif functions as a protein dimerization domain. Proc Natl Acad Sci USA 91:10655–10659[Abstract/Free Full Text]
  53. Jurata LW, Pfaff SL, Gill GN 1998 The nuclear LIM domain interactor NLI mediates homo- and heterodimerization of LIM domain transcription factors. J Biol Chem 273:3152–3157[Abstract/Free Full Text]
  54. Agulnick AD, Taira M, Breen JJ, Tanaka T, Dawid IB, Westphal H 1996 Interactions of the LIM-domain-binding Lbd1 with LIM homeodomain proteins. Nature 384:270–272[CrossRef][Medline]
  55. Jurata LW, Kenny DA, Gill GN 1996 Nuclear LIM interactor, a rhombotin and LIM homeodomain interacting protein, is expressed early during neuronal development. Proc Natl Acad Sci USA 93:11693–11698[Abstract/Free Full Text]
  56. Bach I, Carrière C, Ostendorff HP, Andersen B, Rosenfeld MG 1997 A family of LIM domain-associated cofactors confer transcriptional synergism between LIM and Otx homeodomain proteins. Genes Dev 11:1370–1380[Abstract]
  57. Jurata LW, Gill GN 1997 Functional analysis of the nuclear LIM domain interactor NLI. Mol Cell Biol 17:5688–5698[Abstract]
  58. Morcillo P, Rosen C, Baylies MK, Dorsett D 1997 Chip, a widely expressed chromosomal protein required for segmentation and activity of a remote wing margin enhancer in Drosophila. Genes Dev 11:2729–2740[Abstract/Free Full Text]
  59. Visvader JE, Mao X, Fujiwara Y, Hahm K, Orkin SH 1997 The LIM-domain binding protein Lbd1 and its partner LOM2 act as negative regulator of erythroid differentiation. Proc Natl Acad Sci USA 94:13707–13712[Abstract/Free Full Text]
  60. Kikuchi Y, Segawa H, Tokumoto M, Tsubokawa T, Hotta Y, Uyemura K, Okamoto H 1997 Ocular and cerebellar defects in zebrafish induced by overexpression of the LIM domains of Islet-3 LIM/homeodomain protein. Neuron 18:369–382[CrossRef][Medline]
  61. Fernandez-Funez P, Lu C, Rincon-Limas DE, Garcia-Bellido A, Botas J 1998 The relative expression amounts of apterous and its cofactor dLbd/Chip are critical for dorso-ventral compartmentalization in the drosophila wing. EMBO J 17:6846–6853[Abstract/Free Full Text]
  62. Milan M, Cohen SM 1999 Regulation of LIM homeodomain activity in vivo: a tetramer of dLBD and Apterous confers activity and capacity for regulation by dLMO. Mol Cell 4:267–273[Medline]
  63. O’Keefe DD, Thor S, Thomas JB 1998 Function and specificity of LIM domains in Drosophila nervous system and wing development. Development 125:3915–3923[Abstract/Free Full Text]
  64. Sanchez-Garcia I, Osada H, Forster A, Rabbitts TH 1993 The cysteine-rich LIM domains inhibit DNA binding by the associated homeodomain in Isl-1. EMBO J 12:4243–4250[Abstract]
  65. Taira M, Otani H, Saint-Jeannet JP, Dawid IB 1994 Role of the LIM class homeodomain protein Xlim-1 in neural and muscle induction by the Spemann organizer in Xenopus. Nature 372:677–679[CrossRef][Medline]
  66. Wang M, Drucker DJ 1995 The LIM domain homeobox gene Isl-1 is a positive regulator of islet cell-specific proglucagon gene transcription. J Biol Chem 270:12646–12652[Abstract/Free Full Text]
  67. Vallejo M, Penchuk L, Habener JF 1992 Somatostatin gene upstream enhancer element activated by a protein complex consisting of CREB, Isl-1-like, and {alpha}-CBF-like transcription factors. J Biol Chem 267:12876–12884[Abstract/Free Full Text]
  68. Pfaff SL, Mendelsohn M, Stewart CL, Edlung T, Jessell TM 1996 Requirement for LIM homeobox gene Isl1 in motor neuron generation reveals a motor neuron-dependent step in interneuron differentiation. Cell 84:309–320[Medline]
  69. Jackson SP 1992 Regulating transcription factor activity by phosphorylation. Trends Cell Biol 2:104–108[CrossRef]
  70. Hunter T, Karin M 1992 The regulation of transcription by phosphorylation. Cell 70:375–387[Medline]
  71. Karin M 1994 Signal transduction from the cell surface to the nucleus through phosphorylation of transcription factors. Curr Opin Cell Biol 6:415–424[Medline]
  72. Karin M, Hunter T 1995 Transcriptional control by protein phosphorylation: signal transmission from the cell surface to the nucleus. Curr Biol 5:747–757[Medline]
  73. Jaffe L, Ryoo HD, Mann RS 1997 A role for phosphorylation by casein kinase II in modulating Antennapedia activity in Drosophila. Genes Dev 11:1327–1340[Abstract]
  74. Tanaka M, Herr W 1990 Differential transcriptional activation by Oct-1 and Oct-2: interdependent activation domains induce Oct-2 phosphorylation. Cell 60:375–386[Medline]
  75. Kapiloff MS, Farkash Y, Wegner M, Rosenfeld MG 1991 Variable effects of phosphorylation of Pit-1 dictated by the DNA response elements. Science 253:786–789[Medline]
  76. Segil N, Roberts SB, Heintz T 1991 Mitotic phosphorylation of the Oct-1 homeodomain and regulation of Oct-1 DNA binding activity. Science 254:1814–1816[Medline]
  77. Bourbon HM, Martin-Blanco E, Rosen D, Kornberg TB 1995 Phosphorylation of the drosophila engrailed protein at a site outside its homeodomain enhances DNA binding. J Biol Chem 270:11130–11139[Abstract/Free Full Text]
  78. Coqueret O, Berube G, Nepveu A 1996 DNA binding by cut homeodomain proteins is down-regulated by protein kinase C. J Biol Chem 271:24862–24868[Abstract/Free Full Text]
  79. Coqueret O, Martin N, Berube G, Rabbat M, Lichtfield DW, Nepveu A 1998 DNA binding by cut homeodomain proteins is down-regulated by casein kinase II. J Biol Chem 273:2561–2566[Abstract/Free Full Text]
  80. Hempe JM, Cousins RJ 1991 Cystein-rich intestinal protein binds zinc during transmucosal zinc transport. Proc Natl Acad Sci USA 88:9671–9674[Abstract]
  81. Li PM, Reichert J, Freyd G, Horvitz HR, Walsh CT 1991 The LIM region of a presumptive Caenorhabditis elegans transcription factor is an iron-sulfur- and zinc-containing metallodomain. 88:9210–9213
  82. Michelsen JW, Schmeichel KL, Beckerle MC, Winge DR 1993 The LIM motif defines a specific zinc-binding protein domain. Proc Natl Acad Sci USA 90:4404–4408[Abstract/Free Full Text]
  83. Archer VE, Breton J, Sanchez-Garcia I, Osada H, Forster A, Thomson AJ, Rabbitts TH 1994 Cystein-rich LIM domains of LIM-homeodomain and LIM-only proteins contain zinc but not iron. Proc Natl Acad Sci USA 91:316–320[Abstract]
  84. Kosa JL, Michelsen JW, Louis HA, Olsen JI, Davis DR, Beckerle MC, Winge DR 1994 Common metal ion coordination in LIM domain proteins. Biochemistry 33:468–477[Medline]
  85. Nishiya N, Sabe H, Nose K, Shibanuma M 1998 The LIM domains of Hic-5 protein recognize specific DNA fragments in a zinc-dependent manner in vitro. Nucleic Acids Res 26:4267–4273[Abstract/Free Full Text]
  86. Sanchez-Garcia I, Rabbitts TH 1993 Redox regulation of in vitro DNA-binding activity by the homeodomain of the Isl-1 protein. J Mol Biol 231:945–949[CrossRef][Medline]
  87. Lazennec G, Ediger TR, Petz LN, Nardulli AM, Katzenellenbogen BS 1997 Mechanistic aspects of estrogen receptor activation probed with constitutively active estrogen receptors: correlations with DNA and coregulator interactions and receptor conformational changes. Mol Endocrinol 11:1375–1386[Abstract/Free Full Text]
  88. Chevalier S, Couturier A, Chartrain I, Le Guellec R, Beckhelling C, Le Guellec K, Philippe M, Ford CC 1996 Xenopus cyclin E, a nuclear phosphoprotein, accumulates when oocytes gain the ability to initiate DNA replication. J Cell Sci 109:1173–1184[Abstract/Free Full Text]