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Endocrinology Vol. 144, No. 7 3148-3158
Copyright © 2003 by The Endocrine Society

Ini, a Small Nuclear Protein that Enhances the Response of the Connexin43 Gene to Estrogen

Elisa Oltra, Ingrid Pfeifer and Rudolf Werner

Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101

Address all correspondence and requests for reprints to: Rudolf Werner, Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, P.O. Box 016129, Miami, Florida 33101.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This article describes the structural and functional characterization of Ini (AF495522), a novel highly conserved zinc-finger protein that had been identified by screening an estrogen-induced rat myometrial expression library. Ini localizes to the nucleus of HeLa cells and binds to the proximal connexin43 (cx43) promoter, as demonstrated by EMSA. In addition, transient transfection experiments performed with estrogen receptor {alpha} (ER{alpha}) cDNA show that overexpression of Ini enhances, in a dose-dependent fashion, the up-regulation of the cx43 gene by estrogen. On binding to the cx43 promoter, Ini stimulates the transcriptional activating function (AF)-1, but not the AF-2, of the ER{alpha}. This makes Ini one of the few known coactivators specific for AF-1. Because estrogen up-regulates Ini mRNA in the myometrium, it is likely that Ini’s physiological role in this tissue is to modulate the response of the cx43 gene to estrogen. Transfection studies with an Ini antisense construct seem to indicate that Ini plays an additional role in the cellular response to estrogen affecting both AF-1 and AF-2 activities of the ER{alpha}. This broader effect may be associated with cell cycle progression that in yeast has been shown to require Ini.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
WHILE STUDYING THE regulation of expression of the connexin43 (cx43) gene, we isolated a clone coding for a novel small protein, Ini, which binds to the proximal promoter of the cx43 gene (1). Here we report on the characterization of Ini and its participation in the up-regulation of Cx43 by estrogen.

Connexin43 is a member of a family of transmembrane proteins, which on oligomerization form cell-to-cell channels. Assemblies of such channels in the plasma membrane are called gap junctions. Cx43 is expressed early in the developing embryo. In the adult it is found in many different tissues. The highest levels of Cx43 are found in heart muscle in which expression of the cx43 gene is constitutive. The myocardial intercellular channels are thought to be responsible for the propagation of action potentials during the beating of the heart. Cx43 is also expressed in the uterus in which gap junctions are required to coordinate synchronous contraction of the muscle at the end of pregnancy. In contrast to the heart, however, the cx43 gene in the myometrium is under the control of steroid hormones, being up-regulated by estrogen and down-regulated by progesterone (2, 3, 4, 5, 6). There is convincing evidence that both tissue-specific and hormone-induced regulation of the cx43 gene occur at the transcriptional level (6, 7, 8, 9). Little is known about the mechanisms governing this differential regulation of the cx43 gene, even though its promoter had been identified and sequenced over a decade ago.

Ligand-activated estrogen receptors (ERs) have been shown to activate transcription by binding to estrogen response elements (EREs) in the promoter of estrogen-responsive genes. The rat cx43 promoter contains several half-EREs, which in other genes have been shown to mediate the response to estrogen (7, 10, 11). Recent experiments in this laboratory, however, have shown that these half-EREs are not required for the induction of the cx43 gene; a promoter containing only 145 nucleotides and lacking all half-EREs was shown to be sufficient to achieve full estrogen response (12).

To explain how the estrogen induction of the cx43 gene is achieved in the myometrium, Chen et al. (13) proposed that the activator protein (AP)-1 site, located 12 bp upstream from the TATA box in the murine cx43 promoter, was indirectly involved through the binding of the c-jun and c-fos family of transcription activators, whose up-regulation by estrogen temporally preceded that of Cx43 (14, 15). It was also proposed that a switch in the estrogen receptor subtype (from {alpha} to ß) at the end of pregnancy is required for this up-regulation of Cx43 (16). However, both receptor types seem to bind identical sequences, the EREs, which are not present in the first 145 bp of the cx43 promoter.

Recent experiments in this laboratory have shown that mutating the AP-1 site, to disrupt c-fos and c-jun binding, did not abolish the response of the cx43 gene to estrogen (12). Instead, another site located just upstream of the AP-1 site, was found to be, at least in part, responsible for the induction of Cx43 by estrogen. The results presented here show that Ini actively participates in the estrogen response by binding to this proximal region of the cx43 promoter. Ini appears to belong to a new class of small transcription factors or cofactors that are highly conserved throughout evolution. Its expression has been shown to be essential in yeast (17, 18) and more recently in Caenorhabditis elegans (19).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
HeLa (human cervix epithelioid carcinoma cells), COS-7 (African green monkey SV40 transformed kidney cells), and HepG2 (human hepatocellular carcinoma cells) were cultured in phenol-red free DMEM F-12, high glucose, DMEM low glucose, and Eagle’s MEM{alpha} (Life Technologies, Inc., Grand Island, NY), respectively. All media were supplemented with either 10% heat-inactivated fetal bovine serum (FBS) (Life Technologies, Inc.) or charcoal-stripped serum (HyClone Laboratories, Inc., Logan, UT) for estrogen response assays, 100 U/ml of penicillin, and 0.1 mg/ml streptomycin (Sigma, St. Louis, MO). Cells were grown under an atmosphere of 5% CO2 and 95% water-saturated air at 37 C.

Plasmids and bacterial strains
For bacterial expression of Ini, we used pBluescript-FLAG-Ini, a construct that contains the Ini coding sequence carrying the FLAG epitope (DYKDDDDK) at its amino terminus. Ini expression in this plasmid is driven by an isopropyl-1-thio-ß-D-galactopyranoside (IPTG)-inducible lac promoter.

For experiments designed to study the estrogen response, we used pE0 (14), a construct containing 145 bp of the cx43 promoter and its entire 5' UTR (exon 1 and 13 bases of exon 2) linked to the coding sequence for the firefly luciferase inserted into the pGL-Basic vector (Promega Corp., Madison, WI); pE0-IBSdel (Ini-binding site deletion), a construct similar to pE0 but lacking the sequence of the cx43 promoter between nucleotides -71 and -34; HEO, a pSG5 expression plasmid encoding the full-length human ER{alpha}; HE15 and HE19, which encode truncated forms corresponding to the amino-terminal or the carboxy-terminal portion of the human ER{alpha}, respectively (20). For the expression of Ini in mammalian cells, we used either pcDNA-Ini or pcDNA-FLAG-Ini, coding for an N-terminal FLAG-tagged version of Ini. These constructs were made with the pcDNA3.1(+) vector (Invitrogen, Carlsbad, CA) by insertion of the Ini coding region including its 3' UTR between the KpnI and XhoI sites of its multicloning site. The pcDNA-Inirev contained the Ini coding sequence and its 3'UTR in reverse orientation. The pcDNA-Ini{Delta}3 contained only the coding sequence for the first 57 amino acids (aas) of Ini. The pcDNA-Ini{Delta}1,2 contained the sequence coding for aas 58–110 of Ini. To determine transfection efficiency, we used the pRL-TK vector (Promega Corp.), encoding the Renilla luciferase gene under the control of the thymidine kinase promoter.

For subcellular localization of Ini, we used pEGFP-C1-Ini a construct in which the Ini cDNA is linked in frame to the carboxy-terminal end of the enhanced green fluorescent protein (EGFP) coding sequence in the pEGFP-C1 vector (CLONTECH Laboratories, Inc., Palo Alto, CA).

Bacterial strains
Escherichia coli JM109 was used for all plasmid growth and XL1-Blue MRF’ (Stratagene, La Jolla, CA) for protein expression.

Oligonucleotide synthesis and DNA sequencing analysis
Oligonucleotides were synthesized in the DNA Core Facility of the University of Miami (Miami, FL, Department of Biochemistry and Molecular Biology) in a 394 DNA/RNA synthesizer (Perkin-Elmer, Wellesley, MA). DNA was sequenced in the same facility in an automated 377 DNA sequencer (Perkin-Elmer).

DNA EMSA
EMSA was performed with the Lightshift Chemiluminescent EMSA kit (Pierce Chemical Co., Rockford, IL) according to manufacturer’s recommendations. The 5' biotin-labeled double-stranded oligonucleotides containing the sequence of the rat cx43 promoter between nucleotides -71 and -34 were used: 5'biotin-CTTTCTCCTGGCCCCTCCTTCCAGTTGAGTCAGTGGCT-3'; 5'-AGCCACTGACTCAACTGGAAGGAGGGGCCAGGAGAAAG-3' (annealed).

Five to 10 µg bacterial extracts containing FLAG-Ini recombinant protein per reaction were used. Controls contained 5–10 µg bacterial extracts lacking FLAG-Ini recombinant protein. For the supershift assays, 2 µg monoclonal anti-FLAG M2 antibody (Sigma) were added to the binding reaction. This was followed by additional 15-min incubation at room temperature. DNA-protein complexes were subjected to a 6% native PAGE and transferred to a 0.45-µm Immobilon-P polyvinyl difluoride membrane (Millipore Corp., Bedford, MA) in a mini-PROTEAN II transfer cell (Bio-Rad Laboratories, Inc., Hercules, CA) set at a constant voltage of 50 V for 2 h. A chemiluminescent detection method using a luminal/enhancer solution and stable peroxide solution (Pierce Chemical Co.) was used as described by the manufacturer, and membranes were exposed to Biomax MR x-ray films (Kodak, Rochester, NY) for 30 sec to 5 min before development.

Database search and sequence alignment
Homology search was done at the protein and nucleotide level in GenBank’s nonredundant and expressed sequence tag databases, using the basic local alignment search tool (BLAST) developed by NCBI (National Center for Biotechnology Information) (21). Protein alignment was done using the CLUSTALW program for multiple sequence alignments (22).

Expression of recombinant protein
In bacteria FLAG-tagged Ini protein was produced by adding IPTG (final concentration 1 mM) to a culture of exponentially growing (A600 = 0.4–0.6) E. coli XL-1 Blue MRF’ cells containing the pBluescript-FLAG-Ini construct and continuing the incubation at 37 C for 2–3 h with constant shaking. Bacteria cells were harvested by centrifugation at 8000 rpm (Sorvall SLA-1500 rotor) at 4 C for 15 min and stored at 80 C until used. Soluble FLAG-Ini protein was recovered from these cells after lysis in bacterial protein extraction reagent (B-PER, Pierce Chemical Co.) according to the manufacturer’s midiscale protocol. In mammalian cells HeLa cells transiently transfected with pcDNA-FLAG-Ini were lysed 48 h post transfection in M-PER (Pierce Chemical Co.) according to manufacturer’s recommendations. Protein concentrations were determined with Bradford protein assay reagent (Bio-Rad Laboratories, Inc.).

Western blot analysis
Five to 20 µg protein extracts were denatured with SDS sample buffer followed by 5 min boiling and then separated by 12–16.5% SDS-PAGE (23). Following electrophoresis the proteins were transferred in 1 x transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol, pH ~8.4) to a 0.45-µm Immobilon-P polyvinyl difluoride membrane (Millipore Corp.) in a mini-PROTEAN II transfer cell (Bio-Rad Laboratories, Inc.) set at a constant voltage of 50 V for 2 h. Membranes were then blocked in a 5% nonfat dry milk PBS solution for at least 1 h at room temperature. Incubation with the primary antibody (monoclonal anti-FLAG M2, or monoclonal anti-{alpha}-tubulin, 1 µg/ml, Sigma) proceeded for at least 3 h. Membranes were washed four times with PBS, incubated with horseradish peroxidase-linked goat antimouse antibodies (Pierce Chemical Co.) (1/10,000 dilution) for 1 h, and then washed four times with PBS. Signal detection was performed with a chemiluminescence kit (Super Signal, Pierce Chemical Co.) and exposure to Biomax film (Kodak).

Northern blot analysis
Total RNA was obtained from female Sprague Dawley rats (175–225 g) by homogenization of 50–100 mg tissue per milliliter of TRI reagent (Molecular Research Center, Inc., Cincinnati, OH) with an Ultra-Turrax homogenizer (Tekmar, Cincinnati, OH). Extraction of RNA was done according to the manufacturer’s protocol. Ten micrograms total RNA, quantitated by OD260, were subjected to electrophoresis in a 1.2–1.4% agarose/formaldehyde gel at 5 V/cm for 3 h. The gel was blotted onto a GeneScreen Plus nylon membrane (Biotechnology Systems, NEN Life Science Products, Boston, MA), and UV cross-linked. Hybridization and autoradiography were done as described elsewhere (24). 32P-labeled hybridization probes were generated by PCR in the presence of {alpha}32P-dCTP. The Ini-specific probe consisted of a 246-bp fragment containing the coding sequence for aas 1–84. The 18S ribosomal probe was made from pGEM100D9 (United Technologies International Inc., Calgary, Canada) by PCR using T7 and SP6 primers.

Transient transfections
Fifty to 80% confluent HeLa cells were transfected with Lipofectamine reagent (Life Technologies, Inc.) according to the manufacturer’s protocol. Approximately 2–3 µg total plasmid DNA and 6 µl Lipofectamine reagent in 200 µl Opti-MEM I (Life Technologies, Inc.) were used per 35-mm dish. After 6 h the transfection mixture was replaced by regular medium, and incubation continued at 37 C for an additional 42 h. For luciferase assays the cells were lysed with passive lysis buffer provided in the luciferase reporter assay system (Promega Corp.). Firefly and Renilla luciferase activities were assayed in a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA) set to dual mode with a 2-sec delay and 10-sec integration time. Equimolar ratios of cx43 promoter reporter plasmid (pE0 or pE0-IBSdel), ER (HEO, HE19, or HE15), and Ini (pcDNA-Ini, pcDNA-Inirev, pcDNA-Ini{Delta}3, or pcDNA-Ini{Delta}1,2) expression plasmids were used. Corresponding empty plasmids, pSG5 (the vector used in the cloning of the estrogen receptor), and pcDNA3.1(+) (the vector used in the cloning of Ini) were used to normalize DNA concentration in control transfection mixtures. Every culture was also cotransfected with 10–50 ng of a Renilla luciferase expression vector (pRL-TK, Promega Corp.) as an internal control for efficiency of transfection. Estrogen treatments consisted of the addition of 17ß-estradiol (Sigma) to a final concentration of 25 nM at 6 and 24 h post transfection. Control cells received an identical volume of ethanol vehicle.

For cellular localization of Ini, HeLa cells were seeded on 12-mm diameter glass slides (catalog no. 12–545-80 Fisher), grown in complete medium [10% FBS (DMEM)] and transfected as described above with 200 ng of either pEGFP-C1 (CLONTECH Laboratories, Inc.) or pEGFP-C1-Ini and 2–4 µl Lipofectamine reagent in 50 µl Opti-MEM I (Life Technologies, Inc.). Twenty-four to 48 h after transfection, the cells were washed twice with 1 ml PBS solution and either fixed in freshly prepared 4% paraformaldehyde for 30 min at room temperature or directly observed. Fixed cells were stained with the fluorescent DNA intercalating dye 4,6-diamidino-2-phenylindole (DAPI) with VECTASHIELD mounting medium (H-1200, Vector Laboratories, Burlingame, CA) and observed in a Axiovert 200 inverted fluorescence microscope (Carl Zeiss, Thornwood, NY) with a x32 objective and a Chroma DAPI/Hoechst/AMC D360/40X, 400DCLP, D460/50m, or Chroma narrow-band green fluorescent protein (GFP) HQ480/20x, Q495LP, HQ510/20m filter. Confocal images were collected after exciting the HeLa cells at 488 nm with an argon laser NY attached to a Fluoview scanning confocal microscope (Olympus Corp., Lake Success, NY) with a x60 oil objective. GFP emission was viewed through a 496- to 505-nm band pass filter. Stacks of images were produced digitally with Fluoview software.

Statistical analysis
Relative luciferase (firefly/Renilla) units (RLUs) were expressed as the mean ± SE of triplicate sets of at least three individual experiments. Statistical comparisons were made by the two-tailed t test, and probability values of 0.05 or less were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ini highly conserved during evolution
The rat Ini protein consists of 110 aas with three zinc-finger motifs, a leucine zipper, and a nuclear translocation signal sequence at the carboxy terminus (Fig. 1AGo). Its sequence is found in many different species ranging from yeast to human, but no homologous sequence was identified in a BLAST search of the prokaryotic database. Comparison of the mouse and human Ini coding sequences to our rat clone shows 94% and 91% identity, respectively. At the protein level, however, the Ini sequence is 100% identical in all vertebrates tested. If the comparison is limited to multicellular organisms, which includes rat, mouse, human, zebra fish, the fruit fly Drosophila melanogaster, the mosquito Anopheles gambiae, the nematode C. elegans, and the plants Arabidopsis thaliana and Oryza sativa, the degree of sequence identity is over 80%. Even the most distant Ini ortholog, found in the budding yeast Saccharomyces cerevisiae, is 55% identical to the vertebrate Ini. Such a high degree of conservation through evolution suggests an important function for the Ini protein. Figure 1BGo shows the alignment of Ini’s vertebrate sequence with the orthologous sequences from several species.



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  		Figure 1. Analysis of the Ini sequence. A, Schematic structure of Ini. Positions of the cysteine and histidine residues, possibly involved in zinc binding, and the residues forming the leucine zipper motif are indicated. The sequence of the NLS is shown. B, Alignment of Ini ortholog sequences by the ClustalW interactive multiple sequence alignment program at EBI (European Bioinformatics Institute, http://www.ebi.ac.uk). Identical residues are boxed in black. Conserved residues are boxed in gray.

 
Ini binds to the cx43 promoter
To determine whether Ini binds to the cx43 promoter, we created a construct that contained the complete Ini coding sequence linked to the carboxy terminus of the FLAG sequence under the control of the lac promoter (pBluescript-FLAG-Ini). The expression of the FLAG-tagged Ini protein in E. coli XL1-Blue MRF’ was induced with IPTG, as shown by Western blot analysis of bacterial extracts (Fig. 2AGo). These extracts were then used in a DNA mobility shift experiment with biotin-labeled double-stranded DNA corresponding to nucleotides -71 to -34 of the cx43 promoter (Fig. 2BGo). The formation of a specific complex (SC) was observed with extracts from IPTG-induced bacteria but not with extracts from uninduced cells (Fig. 2BGo, lanes 1 and 3). The specific complex was partially masked by the presence of a strong nonspecific (NS) complex that appeared in all lanes. Shorter exposures were necessary to clearly discern between SCs and NS complexes (Fig. 2BGo, lower panel, lanes 1 and 3). The addition of anti-FLAG M2 monoclonal antibodies (Sigma) produced a supershift (Fig. 2BGo, lane 2) confirming the presence of Ini in the protein-DNA complex. This slower migrating complex was not observed in the uninduced control reaction (Fig. 2BGo, lane 4).



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  		Figure 2. Ini-DNA binding assays. A, Western blot analysis of E. coli. XL-Blue MRF’ lysates with an anti-FLAG M2 monoclonal antibody (1:1000, Sigma). The cells had been previously transformed with the pBluescript-FLAG-Ini plasmid and grown in the absence (-) or presence (+) of IPTG (final concentration 1 mM). The molecular weights of the prestained markers M (in kilodaltons) (catalog no. 7702, New England Biolabs, Inc.) are indicated. B, EMSA of bacterial extracts, uninduced or induced with IPTG, using a 5' biotin-labeled double-stranded DNA probe corresponding to the sequence between nucleotides -71 to -34 of the cx43 promoter. The lower panel shows a shorter exposure of the bracketed portion of the upper panel. With this shorter exposure, a specific FLAG-Ini-cx43 probe complex can be observed (SC). At longer exposure times, its presence is partially masked by the presence of an NS complex. The FLAG-Ini-cx43 probe complex is supershifted to a slower migrating complex when 2 µg anti-FLAG M2 monoclonal antibodies (Sigma) are included in the binding reaction (lane 2).

 
To assay for the specificity of the binding of Ini to the probe, the same assay was repeated with another labeled double-stranded DNA probe containing a nonrelated sequence of the cx43 promoter of similar length (nucleotides -386 to -420). No SC was observed between this probe and the proteins in our bacterial extracts (data not shown), indicating that Ini binds specifically to the sequence of the proximal cx43 promoter between nucleotides -71 and -34.

Ini localized to the nucleus
Ini carries at its carboxy terminus the sequence RKKygfKKR, which has the characteristics of a nuclear localization signal (NLS) (25) (Fig. 1AGo). To study Ini’s subcellular distribution, we attached the coding sequence of the EGFP to the amino-terminal end of Ini’s coding sequence cloned in a mammalian expression vector. In this construct, pEGFP-C1-Ini, expression of the EGFP-Ini fusion protein is driven by the cytomegalovirus promoter. Fluorescence emission from transfected HeLa cells grown in 10% FBS (DMEM) F-12 medium was visualized 24–48 h after transfection. HeLa cells transfected with pEGFP-C1-Ini showed fluorescence that is restricted to the nucleus (Fig. 3AGo, upper left panel), as subsequently confirmed by DAPI staining (Fig. 3AGo, upper middle panel) and phase contrast imaging (Fig. 3AGo, upper right panel). In contrast, control cells transfected with an EGFP-C1 expression plasmid exhibited a cytoplasmic disperse fluorescent signal (Fig. 3AGo, lower panels). The nuclear fluorescence appears in a speckled pattern (Fig. 3BGo, left panel), likely associated with regions of Ini activity on the chromatin. It can also be seen that the nucleolus lacks fluorescent signal.



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  		Figure 3. Subcellular localization of Ini. Representative microscopic images of HeLa cells grown in 10% FBS (DMEM) F-12 medium. The cells had been previously transfected with either a plasmid encoding an EGFP-Ini fusion protein (pEGFP-C1-Ini) or an EGFP expression plasmid (pEGFP-C1), as indicated. A, 24–48 h post transfection, cells were fixed with 4% paraformaldehyde, DAPI stained, and observed in an Axiovert 200 inverted fluorescence microscope (Carl Zeiss) with a x32 objective and a Chroma narrow-band GFP HQ480/20x, Q495LP, HQ510/20m (left and right panels), or a Chroma DAPI/Hoechst/AMC D360/40X, 400DCLP, D460/50m (middle panels) filter. Right panel images include phase contrast overlay. B, 24–48 h post transfection, cells were directly observed in a confocal microscope (Olympus Corp.). A stack of 8–10 images produced digitally with Fluoview software (Olympus Corp.) is shown. The color palette shows the intensity mapping (range 0–4095; 12-bit data).

 
Ini expression is ubiquitous and in the uterus is up-regulated by estrogen
Northern blot analysis of total RNA from different tissues indicates that there is a single Ini transcript, approximately 1.2 kb in length, that is found is all tissues tested including brain, heart, ovary, uterus, skeletal muscle, and testis (data not shown). In addition, a BLAST search of the expressed sequence tag database identified the presence of Ini mRNA in numerous tissues as well as in many different cancer cell types including HeLa cells. This suggests that Ini is a ubiquitous protein possibly involved in the transcription of many different genes. Comparison of Ini mRNA levels in myometrium of normal and estrogen-treated rats by Northern blot analysis shows a 2-fold up-regulation of Ini mRNA by estrogen (Fig. 4Go).



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  		Figure 4. Northern blot analysis of Ini mRNA levels in the myometrium in the presence and absence of estrogen. Northern blot of total RNA (10 µg/lane) from rat myometrium uninduced (U) or induced with estrogen (UI). The 32P-labeled probe corresponded to either the Ini sequence coding for amino acids 1–84 (upper panel) or a 205 bp-long fragment of the18S rRNA sequence (lower panel). Integrated density values (IDVs) were obtained by quantitation of the intensity of the signals after background subtraction in the autoradiogram by the Spotdenso Program with {alpha} Ease software (Alpha Innotech, San Leandro, CA).

 
Ini enhances the response of the cx43 gene to estrogen
Because of the presence of three zinc-finger motifs, the speckled appearance of Ini within the nucleus, and its ability to bind to a specific region of the cx43 promoter, it seemed reasonable to suspect that Ini participates as a transcription factor or cofactor in the expression of the cx43 gene. Also, the up-regulation of Ini mRNA by estrogen in the myometrium suggested a role for Ini in the regulation of the cx43 gene by estrogen. To study this possibility, a reporter construct (pE0) containing 145 bp of the cx43 promoter linked to the firefly luciferase reporter gene together with a plasmid encoding a constitutively active form of ER{alpha} (HE15) was introduced into HeLa cells. If these cells contained pcDNA-Ini (a plasmid driving Ini’s expression from a cytomegalovirus promoter), luciferase levels increased about 2-fold over levels observed with ER{alpha} alone (Fig. 5AGo), indicating that Ini enhances ER{alpha} activity. Transfected cells lacking active ER{alpha}, however, showed no significant increase in luciferase (Fig. 5Go, A, B, and C), suggesting that Ini probably is not involved in the basal activity of this promoter. Please note the difference in scales between Fig. 5Go, A and B.



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  		Figure 5. Effect of Ini on the response of the cx43 promoter to active ER (HE15). A–C, Relative luciferase activities (RLUs) of HeLa extracts 48 h post transfection, calculated by dividing the firefly luciferase by the Renilla luciferase activities to account for differences in transfection efficiency. HeLa cells were transfected with either the pE0 plasmid (a luciferase expression construct containing 145 bp of cx43 promoter sequence) (A and C) or the pE0-IBSdel plasmid (a luciferase expression vector similar to pE0 but lacking the sequence of the cx43 promoter between nucleotides -71 to -34 to which Ini binds) (B). Other plasmids included in some transfections were HE15 (encoding a constitutively active form of the ER{alpha}) (+HE15), pcDNA-Ini (Ini expression plasmid), pcDNA-Ini{Delta}1,2 (encoding a truncated Ini lacking aas 1–52), pcDNA-Ini{Delta}3 (encoding a truncated Ini lacking aa 53–110), and pcDNA-Inirev (a plasmid encoding an antisense Ini mRNA). The presence or absence of each plasmid is indicated by + or - signs below the bar graphs. To keep DNA concentrations constant in all transfection mixtures, appropriate amounts of pcDNA3.1 (Invitrogen, empty vector) or pSG5 (the vector used in the cloning of the ER) (-ER) were included in some mixtures. Every culture was also cotransfected with 10–50 ng of a Renilla luciferase expression vector (pRL-TK, Promega Corp.) as an internal control for efficiency of transfection. Relative luciferase values significantly higher (P < 0.01) (*) or lower (P < 0.05) (**) than the control are indicated. Values are expressed as the means ± SE of at least three replicate experiments. D, Increment of the estrogen response of pE0 as a function of the amount of pcDNA-Ini used for transfection. E, Decrease of intracellular FLAG-tagged Ini protein in HeLa cells transiently transfected with 0.5 µg pcDNA-FLAG-Ini and increasing amounts of pcDNA-Inirev antisense construct by Western blot analysis. NT corresponds to nontransfected cell lysates. Twenty micrograms of cell extracts were probed with either anti-FLAG M2 monoclonal antibody (1:1000, Sigma, F3165) or anti-{alpha}-tubulin (control) (1:2000, Sigma, T5168) as indicated.

 
In addition, Ini-dependent activation of ER{alpha} activity could be abolished when the sequence of the cx43 promoter to which Ini binds in vitro was deleted (pE0-IBSdel, Fig. 5BGo). Thus, the mechanism by which Ini enhances the response of the cx43 gene to active ER{alpha} must involve binding of Ini to that specific sequence of the promoter (nucleotides -71 to -34).

Identical results were obtained in similar experiments that used the pcDNA-FLAG-Ini construct (data not shown), showing that the presence of the acidic FLAG domain at the amino-terminal end of the Ini protein does not interfere with its ability to enhance the estrogen response. This result was expected because FLAG-tagged Ini binds to the cx43 promoter in vitro (Fig. 2BGo).

A truncated form of Ini lacking its amino-terminal zinc-fingers (Ini{Delta}1,2) but retaining its DNA binding carboxy-terminal zinc-finger domain was not capable of stimulating the estrogen response (Fig. 5CGo). Similarly, a truncated form of Ini lacking the carboxy-terminal zinc-finger motif, but retaining both amino-terminal zinc-finger motifs (Ini{Delta}3), did not enhance the response of pE0 to active ER{alpha} either. This indicates that Ini requires all three zinc-finger motifs for activation of ER{alpha}.

The effect of Ini on the pE0 response to active ER{alpha} is proportional to the amounts of Ini expression plasmid used in the cotransfections and therefore probably to the cellular levels of Ini (Fig. 5DGo). At this time no conclusions can be made about the stoichiometry of the interaction between Ini and estrogen receptor.

If Ini is required for the full estrogen response of pE0, then a reduction of the endogenous levels of Ini would be expected to lower the estrogen effect. To decrease endogenous levels of Ini, HeLa cells were transfected with pcDNA-Inirev, a construct that is transcribed into anti-Ini RNA. Western blot analysis of extracts from cells cotransfected with pcDNA-FLAG-Ini (a plasmid encoding FLAG-tagged Ini) and various amounts of pcDNA-Inirev showed a drastic reduction in the levels of FLAG-Ini protein by the antisense construct (Fig. 5EGo). This indicates that the presence of this antisense Ini RNA does indeed interfere with the translation of Ini. The presence of pcDNA-Inirev also reduced the response of the cx43 pE0 construct to ER{alpha}, suggesting that Ini may be required for the cells to respond to active ER{alpha}. Unexpectedly, however, this negative effect of the antisense Ini mRNA on the estrogen induction of the pE0 construct was not abolished when the sequence of the cx43 promoter to which Ini binds was removed from the construct (Fig. 5BGo). Thus, Ini must play an additional role in the estrogen induction of pE0 that is independent of its binding to the cx43 promoter.

Ini specifically stimulates the activating function (AF)-1 domain of ER{alpha}
In all the experiments presented above to evaluate Ini’s role on the estrogen response of the cx43 gene, we used the HE15 plasmid for cotransfection of HeLa cells. This plasmid codes for a constitutively active truncated form of ER{alpha} containing only its ligand-independent amino-terminal AF-1 domain (20). It was of physiological relevance to determine whether Ini could also enhance the response of the cx43 gene by full-length ER{alpha} activated by estrogen. To answer this question, we repeated the transfection experiments with a plasmid expressing the complete ER{alpha}, HEO, containing both activating functions AF-1 and AF-2, and studied the response of the transfected cells to estrogen. As shown in Fig. 6AGo, cells that contained the Ini expression plasmid almost doubled the levels of luciferase produced in response to estrogen, similar to what had been observed with the HE15 truncated form of ER{alpha}.



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  		Figure 6. Effect of Ini on the estrogen response of pE0 in the presence of HEO or HE19. RLUs of HeLa extracts were determined 48 h post transfection. HeLa cells were transfected with the cx43 promoter pE0 reporter construct and either the full-length ER expression plasmid (HEO) (A) or the plasmid producing a truncated form of ER{alpha} lacking its amino-terminal AF-1 activity (HE19) (B). In addition, cells contained pcDNA-Ini or pcDNA-Inirev as indicated (+/-). To keep DNA concentrations constant in all transfection mixtures, appropriate amounts of pcDNA3.1 (empty vector) were included in some mixtures. Every culture was also cotransfected with 10–50 ng of a Renilla luciferase expression vector (pRL-TK, Promega Corp.) as an internal control for efficiency of transfection. Transfected cells were treated with either estradiol (E2) 25 nM final (+) or vehicle (-) at 6 and 24 h post transfection. RLUs significantly higher (P < 0.001) or lower (P < 0.01) than the control are indicated by (*) or (**), respectively. Values are expressed as the means ± SE of at least three replicate experiments.

 
To complete these studies, we used a third form of ER{alpha}, HE19, containing only the AF-2 domain. This truncated form of ER{alpha} becomes active on binding of estrogen (20). However, cells that overexpressed Ini did not respond any better to estrogen than cells containing only endogenous Ini (Fig. 6BGo). This result suggests that Ini’s action on the cx43 gene requires the ER{alpha} AF-1 domain.

In addition, cells cotransfected with pcDNA-Inirev did not achieve maximal estrogen induction either with HEO or with HE19. This indicates that Ini participates in the estrogen response mediated by both the AF-1 and the AF-2 domains of ER{alpha}. Because HeLa cells had been reported to be nonpermissive for AF-1 activity (26, 27), it was possible that Ini would further enhance the estrogen response in cells permissive to AF-1 activity. To test this possibility, we chose two AF-1 permissive cell lines, COS-7 (African green monkey kidney fibroblasts) and HepG2 (human hepatocellular carcinoma epithelial cells). It was surprising to find that Ini behaves differently in these two cell lines (Fig. 7Go). Whereas Ini increased the response of pE0 to active ER{alpha} by at least 2-fold in COS-7 cells, similar to HeLa cells, it had no effect in HepG2 cells. This indicates that Ini’s role in the activation of the estrogen response is cell type specific.



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  		Figure 7. Effect of Ini on the response of pE0 to active ER{alpha} (HE15) in COS-7 and HepG2 cells. RLUs of either COS-7 extracts (A) or HepG2 extracts (B) were determined 48 h post transfection. Cells were transfected with pE0, HE15, and either pcDNA-Ini (+) or pcDNA3.1vector (-), as indicated. To keep DNA concentrations constant, pSG5 plasmid was used in the transfection of control cells lacking ER (-ER). Every culture was also cotransfected with 10–50 ng of a Renilla luciferase expression vector (pRL-TK, Promega Corp.) as an internal control for efficiency of transfection. RLUs significantly higher (P ≤ 0.05) than the control are marked (*). Values are expressed as the means ± SE of at least three replicate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Since the discovery in 1998 of Ini as a novel small RING-finger like protein, potentially involved in the transcriptional regulation of the rat cx43 gene, other laboratories have described this protein in other species (19, 28).

Ini’s sequence is rich in metal ligand-binding residues (cysteines and histidines). These residues are spaced in a way to form three distinct zinc-finger motifs (29). The two most amino-terminal fingers almost match the consensus sequence for a RING-finger motif (30, 31, 32, 33, 34, 35). Alternatively, the two most carboxy-terminal zinc-fingers could fold into a plant homeodomain (PHD) finger-like structure (36, 37), as recently proposed by Trappe et al. (28) for the mouse Ini ortholog PHF5. The fact that a truncated form of Ini, containing only its carboxy-terminal zinc-finger domain, is capable of binding to the cx43 promoter (17) argues against the latter possibility. However, the final answer to the question of whether Ini folds into a RING or PHD finger-like structure will have to wait for the determination of its three-dimensional structure.

Both RING- and PHD-finger motif-containing proteins have been found in many different species from plants to humans and have been highly conserved throughout evolution, as is the case for Ini. So far, Ini appears to be one of the most highly conserved proteins known, being 100% identical in all tested vertebrates. This suggests that there existed a very high selective pressure to conserve Ini’s sequence without even a single aa change for a period of more than 530 million years, the date of the oldest fish fossil found (38).

Because no ortholog of Ini can be found in the genomes of prokaryotic organisms, it is possible that Ini’s presence and function is restricted to the eukaryotic world. This suggests that Ini appeared as long as 2.7 billion of years ago and may constitute one of the eukaryotic signature proteins that have been proposed to be required for the transition from the chronocyte cell to the first eukaryote (39, 40).

It is noteworthy to point out that aas at positions 77, 84, 91, and 98 of the vertebrate Ini proteins, which are located downstream of Ini’s third zinc-finger domain, consist of leucine, isoleucine, or glycine and could therefore fold into a leucine zipper structure (41). Like zinc-finger motifs, leucine zippers are found in many transcription factors. Not all Ini orthologs show this leucine zipper motif, indicating perhaps a species-specific role for this domain.

What is the function of this highly conserved protein? Our images of transfected HeLa cells grown in full serum clearly show that Ini localizes to the nucleus of the cell exhibiting a speckled appearance (Fig. 3Go). This same nuclear distribution has recently been shown for the Ini’s mouse ortholog in mouse NIH3T3 cells (28). The speckled distribution matches the localization pattern previously described for some steroid receptors (42, 43, 44, 45) and other transcription and splicing factors (45, 46, 47, 48). The presence of a putative NLS, which includes aas 102–110 of the mammalian Ini sequence, is consistent with these results.

The ability of Ini to bind to the cx43 promoter and the increase of its mRNA levels in the rat myometrium on estrogen treatment suggested that Ini participates in the up-regulation of the cx43 gene by estrogen. Indeed, when constitutively active ER{alpha} is present in the cell, overexpression of Ini increases the level of luciferase about 2-fold over the level observed without overexpression of Ini. Thus, it appears that Ini enhances the activity of ER{alpha} on the cx43 promoter.

At this time, we do not know if Ini is an essential part of the cx43 transcription machinery that responds to estrogen because HeLa cells contain Ini mRNA and therefore are likely to express endogenous Ini protein. Cotransfection of cells with pcDNA-Inirev reduced the level of luciferase induction by active ER{alpha} but did not block the response completely. This is expected, even if Ini was absolutely required for estrogen response because the antisense RNA probably does not suppress Ini synthesis entirely, and the cells probably contain a pool of already synthesized Ini that would not be affected by the antisense RNA.

Removal of the Ini-binding sequence from the cx43 promoter (pE0-IBSdel) abolishes the Ini effect on the estrogen induction of the cx43 gene, indicating that the binding of Ini to this specific DNA segment of the cx43 promoter is required for its action. It should be pointed out that the levels of luciferase activity obtained with the pE0-IBSdel construct were about an order of magnitude lower than those obtained with the pE0 construct. This result was expected because the deletion of the Ini-binding site also eliminates two known transcription activator sequences, the previously reported cx43 cis-activator (13) and the consensus AP-1 site.

It was somewhat surprising, however, to find that removal of the Ini-binding sequence from the reporter construct (pE0-IBSdel) did not prevent the negative effect of the Ini-antisense RNA on the response of the cx43 gene to active ER{alpha} (Fig. 5BGo). This suggests that Ini plays an additional role in the estrogen response, which is independent of its binding to the cx43 promoter. Because Ini is expressed in many different tissues, some of which are not responsive to estrogen, it is possible that Ini functions as a more general transcription cofactor. This pleiotropic effect of Ini is supported by the finding that the ini gene is essential in budding and fission yeast (17, 18) as well as in the worm C. elegans (19). Perhaps it is through participation in the known cross-talk between the estrogen receptor and other mitogenic signaling pathways that Ini affects estrogen responsiveness of the cx43 gene. In fact, we have recently shown that Schizosaccharomyces pombe cells lacking a functional ini gene arrest at the G2 phase of the cell cycle (Oltra, E., F. Verde, R. Werner, and G. D’Urso, manuscript in preparation). The requirement of Ini for cell cycle progression explains the presence of highly conserved orthologs of Ini in organisms lacking connexins. Furthermore, because the human ER{alpha} has been shown to function in yeast (49, 50), the required cofactors that mediate this hormonal response are expected to be similar from yeast to man.

With respect to the mechanism used by Ini to enhance the estrogen response, we can conclude from our data that both binding of Ini to the cx43 promoter (compare Fig. 5Go, A and B) and the presence of its amino-terminal zinc-finger motifs (Fig. 5CGo), possibly to allow protein-protein interactions, are required to achieve maximal activation of pE0 by estrogen. Therefore, for our understanding of Ini’s mode of action, it would be important to identify additional proteins that interact with Ini. Our experiments also show that Ini specifically affects the amino-terminal AF-1 domain of ER{alpha} and in the full-length receptor this trans-activation is dependent on the presence of estrogen (Figs 6Go, A and B). Thus, Ini is one of the very few known examples of proteins that activate the AF-1 domain. The recent discovery of a variant of the ER{alpha} lacking the ligand-binding domain as result of alternative splicing at the 3' end of exon 4 (51) opens new possibilities for the cellular function of the AF-1 activators.

Although most known nuclear receptor cofactors are far larger in size than Ini, at least two other small coactivators of nuclear receptors, PNRC2 and SNURF, have been described (52, 53). Like Ini, both proteins are small and ubiquitously expressed. SNURF contains a RING-finger motif and acts as a coregulator in steroid receptor-mediated gene transcription (54, 55). Even though these two proteins are not sequence related to Ini, their existence supports the notion that Ini belongs to a new class of relatively small transcription cofactors involved in the steroid response. Whether Ini’s activation of ER{alpha} is mediated through direct interaction of Ini with the receptor itself or requires additional factors remains to be determined.

In addition, we observed that the activation of ER{alpha} by Ini is dose dependent over a broad range of Ini concentrations. Thus, variations in the level of Ini could serve to fine-tune the extent of the response of the cx43 gene to estrogen in term myometrium in which maximal levels of Cx43 are required. The fact that Ini mRNA levels are increased on estrogen treatment in this same tissue (Fig. 4Go) agrees with this possibility. To better understand Ini’s physiological role in the myometrium, we need answers to the following questions: Does Ini up-regulation precede that of Cx43 in the pregnant myometrium? Is it possible to prevent preterm labor by reducing Ini expression? It would also be interesting to find out whether Ini affects exclusively the activity of ER{alpha} or whether it affects other ER isoform activities as well. Does Ini play a role in the response to estrogen of other genes and does it participate in the response of the cx43 gene to other hormones? In particular it seems important to investigate whether Ini affects the response of the cx43 promoter to PTH whose response element has been mapped to the region between nucleotides -31 to +1 just downstream of Ini’s binding site (56).

It was surprising to find that in COS-7 cells, known to be AF-1 permissive, up-regulation of pE0 by HE15 is significantly lower than in the AF-1 nonpermissive HeLa cells. However, we have to point out that the classification of this cell-line as AF-1 permissive was based on studies using either a consensus ERE or the trout ER promoter (50, 57). It is known that estrogen response is not only cell line but also promoter context dependent. Therefore, it is possible that this classification does not apply to all estrogen-responsive genes. Furthermore, Ini’s ability to enhance the estrogen response of the cx43 gene is not observed in HepG2 cells. This indicates that even though Ini is ubiquitously expressed, its function appears to be tissue specific. There are several possibilities that could explain Ini’s cell-specific activity despite its ubiquitous expression: differences in levels of expression, subcellular localization, posttranslational modifications, and/or association with other cell-specific proteins.

In conclusion, we have shown that Ini, a highly conserved small nuclear protein with interesting structural characteristics, enhances the estrogen response of the cx43 gene in a dose-dependent and tissue-specific fashion. It is through binding to the promoter region that Ini enhances the response of the cx43 gene to estrogen specifically stimulating the AF-1 activity of the ER{alpha}. For this activation Ini’s amino-terminal domain is also required, possibly to mediate interactions with other proteins, perhaps the ER itself. At the same time, Ini seems to play a role in the estrogen response that does not require its binding to the cx43 promoter and is not restricted to the AF-1 domain. This broader effect of Ini on the estrogen response could be a consequence of the requirement of Ini for cell cycle progression.


   Acknowledgments
 
The authors wish to thank Dr. Pierre Chambon, Centre Nationale de la Recherche Scientifique, Strasbourg, France, for the generous provision of the various estrogen receptor constructs. We also thank Dr. Alejandro Caicedo, Department of Ophthalmology, and Dr. Juan Dominguez-Bendala, Diabetes Research Institute, University of Miami, for their help in the capture of microscopic images.


   Footnotes
 
This work was supported by a grant from the NIH (HD-34152). DNA synthesis and sequence analysis was subsidized by the Sylvester Comprehensive Cancer Center through their DNA Core Lab.

Abbreviations: aa, Amino acid; AF, activating function; AP-1, activator protein 1; BLAST, basic local alignment search tool; cx43, connexin43; DAPI, 4,6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; ER estrogen receptor; ERE, estrogen response element; EST, expressed sequence tag; FBS, fetal bovine serum; GFP, green fluorescent protein; IBS, Ini-binding site; IPTG, isopropyl-1-thio-ß-D-galactopyranoside; NLS, nuclear localization signal; NS, nonspecific; PHD, plant homeodomain; RING, really interesting new gene; RLU, relative luciferase units; SC, specific complex.

Received December 19, 2002.

Accepted for publication March 27, 2003.


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