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Biology of Reproduction 60, 23-31 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Repression of Major Histocompatibility Complex Genes by a Human Trophoblast Ribonucleic Acid

John A. Peyman1,a

a Section of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The suppression of polymorphic major histocompatibility complex antigen expression in human trophoblasts is critical for the avoidance of a cell-mediated immune response by maternal lymphocytes against cells expressing paternal antigens. In this study, a repressor of major histocompatibility complex gene expression was cloned by negative immunoselection using a trophoblast cDNA expression library in interferon-{gamma}-responsive human cells. The sequence of this regulatory gene was analyzed, and the functions of the transfected cDNA or microinjected gene product were examined in interferon-{gamma}-responsive cells by immunocytochemical methods. The repressor, called TSU— trophoblast STAT (signal transducers and activators of transcription) utron (untranslated region of an mRNA)—reduced STAT1 nuclear translocation and suppressed major histocompatibility complex class II antigen expression at high doses of interferon-{gamma} and class I expression at low doses of interferon-{gamma}. TSU encoded a small, untranslated poly-A+-RNA that appeared to bind STAT1 through pairs of motifs analogous to STAT-binding promoter sequences. These promoter-like motifs, but no open reading frame, were conserved in a TSU-related gene in goats. Northern blot analysis demonstrated that TSU was expressed as a 0.5-kilobase (kb) RNA in placenta and as an ubiquitous 4.4-kb RNA. TSU expression may protect trophoblasts from immune attack and promote the survival of the placenta and fetus.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human trophoblasts of the chorionic villus form a transport surface and a nonimmunogenic barrier that excludes maternal blood cells from the fetal circulation [1–3]. Trophoblasts lack constitutive expression of polymorphic major histocompatibility complex (MHC) class I molecules and interferon-{gamma} (IFN-{gamma})-inducible expression of MHC class I or II genes. IFN-{gamma}-responsive MHC class II gene expression is blocked at the RNA level by an intracellular mechanism in first-trimester placenta and in the trophoblast-derived choriocarcinoma cell line Jar, although trophoblasts express the IFN-{gamma} receptor [4].

The mechanism of IFN-{gamma}-induced MHC gene expression in other cell types involves the subunits of the IFN-{gamma} receptor [5–7], Janus kinases (Jaks) and the STAT (signal transducers and activators of transcription) transcription factors [8–10], the interferon type I ({alpha}/ß)-stimulated response elements (ISRE) conserved in the promoters of MHC class I [11] and other genes, and the gamma-interferon activation site (GAS) elements conserved in the promoters of genes responsive to IFN-{gamma} and other cytokines [8–10]. Cytokine-activated, receptor-associated Jaks phosphorylate STATs, which dimerize and are translocated to the cell nucleus, where they stimulate transcription. MHC class I gene promoters contain ISRE motifs but not GAS motifs. Constitutive expression of MHC class I genes occurs through binding of transcription factors to the ISRE, {kappa}B, and site {alpha} promoter elements [12–17]. The IFN-{gamma}-stimulated expression of MHC class I genes requires STAT1 [18] and may involve binding of interferon-regulated factor (IRF)-1, STAT1-containing factors, and the class II transactivator gene (CIITA) [19–22]. Induction of MHC class II gene expression by IFN-{gamma} is initiated by Jak-STAT activation but also requires the de novo production of CIITA [23–25], which interacts with constitutively expressed DNA-binding proteins on conserved promoter sequences in MHC class II genes [26]. In summary, the expression of MHC class I and II genes is mediated by STAT1, CIITA, nuclear factor (NF)-{kappa}B, IRF-1, and other transcription factors, and, therefore, the trophoblast-related mechanism(s) of MHC repression may require inhibition of a number of intracellular signaling pathways.

Mammalian expression cloning was used to test the hypothesis that trophoblasts actively repress IFN-{gamma}-induced expression of the MHC class II antigen HLA-DR. In this study, a trophoblast cDNA was identified, and its effects on the expression of IFN-{gamma}-stimulated MHC class II genes, constitutive and inducible MHC class I genes, and intercellular adhesion molecule (ICAM)-1 gene, and on the IFN-{gamma}-stimulated translocation of STAT1 were studied.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Expression Cloning by Negative Immunoselection

Poly(A)+-RNA was prepared with the PolyATTract System (Promega, Madison, WI) from the trophoblast-derived choriocarcinoma cell line Jar (American Type Culture Collection [ATCC], Rockville, MD). Jar cell cDNAs were prepared with oligo-dT primers, size-selected, and cloned by standard methods [27] with directional adapters into the mammalian expression vector pSH4-hphm, which provides an SV40 promoter, splice site upstream of cloning site, and poly-A addition signal, as well as hygromycin resistance [28]. The cDNA library was grown in DH10B Electromax Escherichia coli (Gibco BRL, Gaithersburg, MD), and the 3-dimensional amplification procedure in 50-ml tubes was carried out to ensure reasonable representation by slow-growing bacteria [29]. Large plasmid preparations with two CsCl bandings produced stock solutions for transfection [27]. Size-selected sublibraries were prepared with 0.5- to 4-kilobase (kb) and 4- to 23-kb inserts. The Jar large-insert expression library consisted of 1.7 x 105 independent clones, and it was used in the experiments described here. A clone of the cervical carcinoma cell line HeLa (ATCC), clone 6, was isolated by limiting dilution culture that gave expression of HLA-DR antigen after stimulation by recombinant human IFN-{gamma} (Boehringer-Mannheim, Indianapolis, IN), and this clone was expanded for further use. The cDNA expression library was transfected by the calcium phosphate method [29, 30] into HeLa clone 6 cells. A total of approximately 2 x 104 stable transfectants resistant to 150 µg/ml hygromycin B (Boehringer-Mannheim) were screened from 4 transfections over several months. Three rounds of selection were performed by IFN-{gamma} challenge (200 U/ml for 2 days), and then sterile sorting by flow cytometry of live, lightly trypsinized cells stained in suspension at 4°C that were gated on the lowest 5–10% of the range of HLA-DR antigen staining. The HLA-DR monoclonal antibody (mAb) L243 (IgG2a isotype, ATCC) was used against nonimmune mouse IgG2a (Sigma, St. Louis, MO) as a negative control. Untransformed, IFN-{gamma}-treated HeLa cells served as positive control cells. mAb binding was detected with R-phycoerythrin-goat anti-mouse IgG secondary Ab (Molecular Probes, Eugene, OR) on a FACS IV instrument (Becton-Dickinson, Bedford, MA). The isolation of antigen-negative cells was completed by cloning through limiting dilution and screening subcultures grown in chamber slides (Nunc, Rochester, NY) by means of immunocytochemistry with L243 mAb as described [4]. Polymerase chain reaction (PCR) primers were prepared that amplified sequences between the promoter and the poly-A signal of the expression vector, pSH4–1 (5'-GATG-TTGCCTTTACTTCTAGGCCT-3') and pSH4–2 (5'-AAC-TCATCAATGTATCTTATCATG-3'). Amplification was performed over 30 cycles of 1 min at 94°C, 2 min at 55°C, and 3 min at 72°C in a thermal cycler (Perkin Elmer, Norwalk, CT) with 1 U of Taq DNA polymerase (Gibco BRL) [31]. PCR products were cloned into the pCR3 vector and grown in TOP10F' E. coli according to the instructions of the manufacturer (Invitrogen, Carlsbad, CA). The CMV promoter in pCR3 drives mammalian expression, and the vector provides neomycin resistance. Plasmids were purified on Qiagen tips (Qiagen Corp., Valencia, CA). Supercoiled pCR3 plasmid was prepared by ligation with no cDNA insert in the TA-cloning site. Restriction endonuclease mapping of 9 TSU (trophoblast STAT utron [untranslated region of an mRNA]) cDNA clones was performed to establish the orientation of the cDNA. Constructs were introduced into HeLa clone 6 cells by lipofection [32] or by calcium phosphate-mediated transfection. Stable secondary transfectants were selected with G418-containing medium at 300–600 µg/ml G418 (Gibco BRL).

DNA Sequencing and Sequence Analysis

Double-stranded plasmid DNA was sequenced by the fluorescent cycle sequencing method with an Applied Biosystems 373A DNA Sequencer. Primers used for sequencing were pSH4–1, pSH4–2, T7 promoter primer (5'-TAATACGACTCACTATAGGG-3'), and internal primers TSU-1 (5'-GTGTGATCTGAAAACCCTGCTTGG-3'), TSU-2 (5'-AGACTACTTCCCCATACATGCG-3'), and TSU-3 (5'-CCATACAGAGCAACATACCAGTAC-3'). Data were analyzed with the Genetics Computer Group (GCG, Madison, WI) programs [33]; searches of the sequence databases at the National Center for Biotechnology Information (Bethesda, MD) were performed with the BLAST program [34]; and searches of the eukaryotic transcription factor database [35] were carried out with the FindPatterns (GCG) program. The dot plot was generated by the Compare and Dotplot (GCG) programs with a window of 21 bases and a stringency of 14. The calculations of sequence identity between TSU and the goat expressed sequence tag (EST) were based on alignment by the Gap (GCG) program. The following nucleotides were ignored in the calculations: single-base gaps in the goat EST (considered sequencing errors), 4 other gaps in the goat EST required to align the sequences, 6 nonsequence bases (N) in the goat cDNA, the 2 single-base gaps in the TSU cDNA opposite possible insertions or sequence errors in the goat EST, and the poly-A tails. Total sequence compared was 419 bases. The Genbank accession numbers for the DNA sequence reported in this paper are AF080092, AF080093, and AF080094.

Northern Blot Analysis of Human Tissues

Poly(A)+ RNA (2 µg) was isolated from 16 normal human tissues and displayed on formaldehyde-agarose gels, and Northern blots were prepared (Clontech, Palo Alto, CA). 32P-Labeled TSU antisense RNA probe was synthesized by in vitro transcription with NotI-linearized TSU-pCR3 reversed construct and T7 RNA polymerase. Hybridization with a 106-cpm/ml probe at 55°C for 40 h in hybridization buffer [27] containing 50% formamide was followed by a final wash with 0.1-strength SSC (single-strength SSC is 0.15 M sodium chloride, 0.015 M sodium citrate)-0.5% SDS at 55°C for 1 h as described [4]. Sequential autoradiographic exposures were produced with an intensifying screen. Blots were reprobed with 32P-labeled ß-actin probe according to the instructions of the manufacturer (Clontech). Results represent two independent preparations and analyses.

Flow Cytometry

Flow cytometric analysis was performed on stably transfected pools of cells that were treated in culture with IFN-{gamma} (0, 20, 66, 200, and 1000 U/ml) for 42 h and suspended for analysis by light trypsinization. Cells were stained at 4°C with 10-µg/ml solutions of mAb CR3/43 (Dako, Carpinteria, CA), specific for MHC class II antigens HLA-DP, HLA-DQ, and HLA-DR; mAb G46–2.6 (PharMingen, San Diego, CA), specific for MHC class I antigens HLA-A, HLA-B, and HLA-C heavy chains; mAb HA58 (PharMingen), specific for ICAM-1 antigen; and either nonimmune mouse IgG1 or IgG2a as negative control. Antibody binding was detected with fluorescein isothiocyanate-goat anti-mouse antibody (Biomeda, Foster City, CA), and 20 000 cells were analyzed with a FACScan (Becton-Dickinson). The percentages of high-level staining cells in three independent experiments were averaged and compared in the t-test to determine levels of significance between these means.

Morphometric Analysis of Immunocytochemical Localization of STAT1 Antigen

HeLa cells stably transfected with the TSU expression construct or the pCR3 empty vector were treated with IFN-{gamma} continuously for 0, 0.4, 4, or 40 h. The cells were then stained for STAT1 antigen localization with G16920 mAb, specific for STAT1 (Transduction Laboratories, Lexington, KY), or mouse IgG1 isotype control to show background. The Ni2+-enhanced detection of antibodies bound to avidin-biotin-peroxidase complexes was performed with commercial reagents (Vector Laboratories, Burlingame, CA) [4]. Slides were read in a blind manner, and cellular localization of STAT1 antigen was scored according to the number of cells with a clearly discernible cytoplasm and nucleus in one of three categories: 1) gray cells (cytoplasmic STAT1 localization), 2) cells with gray nuclei (weak STAT1 nuclear localization), and 3) cells with black nuclei (strong STAT1 nuclear localization). The objects disregarded were free nuclei from dead cells in the culture. Twelve random fields from each sample were photographed by video capture to disk, and images were numbered consecutively. The first ten cells in the top left of each field were counted, and scores were recorded for each field. Confirmation was obtained of questionable counts noted by the blinded observer for 62 of the 720 fields. The counts for each sample were averaged and plotted as the percentage of cells exhibiting one of the three localizations as a function of the time of treatment with IFN-{gamma}. The statistical significance of the differences observed in strong nuclear localization of STAT1 antigen between the TSU and control transfectants at the physiologically relevant 0.4-h point was ascertained by use of the t-test.

Microinjection of RNA

The TSU-pCR3 expression construct was linearized with NotI (New England Biolabs, Beverly, MA) in the polylinker downstream of the insert, and recombinant RNA was produced by in vitro transcription with T7 RNA polymerase (New England Biolabs) as described [4]. Plasmid DNA was then removed by 15-min incubation with 1 U of RNase-free DNase I (Promega). TSU RNA was purified by two phenol-chloroform extractions, one chloroform extraction, and ethanol precipitation [27]. Yeast tRNA (Boehringer-Mannheim) stock solution (10 mg/ml) was sheared by sonication and extracted with phenol-chloroform three times, after which residual phenol was removed by chloroform extraction. RNA concentrations were determined by A260 measurement. The RNA in aqueous solution was denatured at 95°C for 1 min and renatured by cooling in a thermal cycler to 35°C over 30 min. Dulbecco's Modified Eagle's medium (DMEM) was replaced with {alpha}-minimum essential medium Eagle ({alpha}-MEM) containing 20 mM HEPES-NaOH, pH 7.4, 20% fetal bovine serum, and penicillin-streptomycin-antimycin A for microinjection and IFN-{gamma} treatment. Solutions of 1 µg/µl TSU RNA, 1 µg/µl yeast tRNA, or 120 mM KCl-10 mM Tris-HCl, pH 7.5 buffer were loaded into sterile femtotips (Eppendorf, Hamburg, Germany) and microinjected into the juxtanuclear cytoplasm of HeLa cells grown on coverslips at low density and individually identified by position in relation to a manually etched grid. Microinjection was performed on a Zeiss (Oberkochen, Germany) IM inverted microscope with Nomarski optics, with a Zeiss micromanipulator and an Eppendorf 5242 microinjector as described [36]. Successful microinjections were indicated by cell swelling of ~10% upon delivery of the bolus of ~15–20 pl of RNA or buffer and by the absence of blebs on the cell membrane. Microinjected cells were recorded on the schematic cell identification map for each coverslip, and after subsequent treatments each cell was reidentified. IFN-{gamma} was added to the medium after microinjection, and the cells were incubated for 30 min at 37°C, after which acetone fixation and immunocytochemical determination of STAT1 localization were carried out as described above.

Electrophoretic Mobility Shift Assay

Cotransfection of the pSG91 SV40 promoter-STAT1 expression construct (a kind gift from X.-Y. Fu, Yale University) with pSV2-Neo (obtained from D. DiMaio, Yale University) and selection with 600 µg/ml G418 (Gibco Life Sciences, Paisley, UK) yielded cell lines 3A3 and 3B5, which were chosen for further use because of their abundant STAT1 antigen and IFN-{gamma}-responsive STAT1 nuclear localization by immunocytochemical staining with the ISGF3 mAb as described above. The STAT1- cell line U3A was generously provided by G.R. Stark (Cleveland Clinic Foundation, Cleveland, IN). Cell cultures were treated with 200 U/ml IFN-{gamma} for 30 min, and cell extracts were prepared as described [37]. Small aliquots were frozen at -70°C for single use. Protein concentration in cell extracts was determined with the Bio-Rad (Richmond, CA) protein assay with BSA standards. Activated STAT1 DNA-binding in the extracts was confirmed by published methods [37]. The buffer for RNA-protein binding consisted of 150 mM KCl, 50 mM HEPES (pH 7.9), 2 mM MgCl2, 0.5 mM EDTA, 1 mM dithiothreitol, 200 µg/ml tRNA, and 0.4 U/µl RNasin (Promega). RNA probes and nonradioactive competitor RNAs were prepared and refolded as described above. The {gamma}-actin cDNA template was prepared by subcloning a 1.1-kb BamHI-XhoI fragment into pGEM-3Z plasmid, and linearization at the 3'-end with XhoI. The templates for preparation of probes from the GAS loop-loop construct and the mutant GAS loop-loop construct (see below) were linearized with HindIII. Inclusion of 5 mM MgCl2 in the denaturation-renaturation step was avoided since it led to the formation of aggregates of TSU and actin probes. RNA probe (20 ng) was mixed in binding buffer with 300, 900, or 3000 ng of cell extract protein, incubated at 25°C for 15 min, and loaded onto 1% agarose gels that were run with 50 mM Tris-glycine buffer, pH 8.9, at 50 V. The samples were blotted onto Gene Screen Plus membranes (NEN-DuPont, Boston, MA) with a PosiBlotter (Stratagene, La Jolla, CA), and autoradiographic exposures were prepared with x-ray film.

Preparation of Model Loop-Loop RNA Structures

Overlapping 46-base oligodeoxynucleotides were annealed and filled in with Klenow enzyme (New England Biolabs) to give blunt-ended DNA that was extended with dATP and Taq polymerase. The products were ligated to pCR3 vector DNA and used to transform TOP10F' E. coli by standard methods [27]. The oligonucleotides for preparation of the GAS loop-loop expression construct were 5'-GCAACGATTGAATTGGGTAAAGTAATACCAATCTGACAGGTGTACC-3' and 5'-GCAACTAATTATGTACATGACGTAACAGAATTATGACAGGTACACC-3', and for the mutant GAS loop-loop expression construct 5'-GCAACGATTGAATTGGCGAAAATCCTACCAATCTGACAGGTGTACC-3' and 5'-GCAACTAATTCTGG-GATGTCCGGTACATAATTATGACAGGTACACC-3'. Plasmids were purified from bacterial cultures by Qiagen columns, restriction-mapped, and sequenced across the inserts in both directions with T7 promoter primer and pCRII-SP6 promoter primer (5'-ATTTAGGTGACACTATA-3').


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning and Analysis of TSU Sequence and Expression

A cDNA expression library was prepared from the trophoblast cell line Jar in the pSH4-hphm vector, and stable transfectants of the cervical carcinoma cell line HeLa were screened for dominant suppressors. Those cells with low or negative expression of IFN-{gamma}-stimulated HLA-DR antigen were selected by three rounds of flow cytometry and cloned by limiting dilution culture. A total of 20 clones derived from 4 transfections were expanded for further analysis because after IFN-{gamma} treatment they expressed low HLA-DR antigen levels on the cell membrane or in intracellular compartments when analyzed by avidin-biotin-peroxidase immunocytochemistry. PCR was carried out with flanking vector primers to rescue cDNAs from genomic DNA prepared from 19 of the transfectant clones. Only 5 of the 19 cell clones gave rise to detectable PCR products, and two of these clones yielded a ~0.6-kb amplimer. The nature of the cDNA insert in the other clones is under investigation. The 0.6-kb PCR products were cloned into the pCR3 expression vector, and plasmid stocks were prepared for mapping, sequencing, and re-testing of the regulatory functions of the cDNAs. The DNA sequence of TSU is shown in Figure 1A. The name trophoblast STAT utron (TSU) denotes a factor isolated from trophoblasts that interacts with STAT transcription factor(s) and that consists of an independently functioning untranslated region of an mRNA or "utron." Analysis of the sequence with the GCG programs [33] indicated that TSU was encoded by a 481-base pair (bp) cDNA with no significant similarities to known sequences in the database, except that TSU was identical (except for random sequencing errors) to more than 100 previously cloned ESTs from many human tissues and cell lines, and it was also similar to an EST from the goat. The EST were identified in the sequence databases with BLAST [34]; nothing had been reported about their functional characteristics. Both the human TSU cDNA and the goat EST included a poly-A addition signal upstream of a 3-base and a 30-base poly-A tail, respectively.



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  		FIG. 1. A) Sequence of TSU cDNA. Gene promoter-like motifs and the poly-A addition signal are shown in bold letters. B) Dot-plot analysis shows short regions conserved in TSU cDNA and the related goat EST. Numbers 1–12 refer to the location of promoter motifs and poly-A signal. C) Gene promoter-like motifs in TSU and the goat EST. Bases are underlined to indicate the position of the consensus sequence. Those bases conserved between human and goat are shown in bold lettering.

Surprisingly, the TSU sequence did not encompass a long open reading frame, and the human and goat cDNAs contained no conserved open reading frames. This suggested that no protein is encoded. The longest open reading frame in TSU could encode a 21-amino acid polypeptide. Conserved in these sequences, however, were a total of 11 sequences that encoded the RNA equivalent of gene promoter motifs related to cytokine signaling: 5 GAS, 4 ISRE, and 2 interleukin-4 (IL-4) response elements (IL-4RE, TTNCNNNNAA). The GAS and ISRE were observed as either coding strand (GAS, TTNCNNNAA; ISRE, AGTTTCNNTTYYYY, Y=C/T) or complementary strand (GAS complement, TTNNNGNAA; ISRE complement, RRRRAANNGAAACT, R=A/G) versions of the DNA consensus sequences. The TSU cDNA was 73% identical to the goat EST, 58% in the 5'-220 bp and 80% in the 3'-261 bp. The 11 motifs were 70% identical in the 5'-220 bp and 94% identical in the 3'-261 bp, if one excludes the variable bases in the consensus sequences. The homology between the TSU cDNA and the goat EST is shown graphically by dot plot analysis (Fig. 1B), with the diagonal line indicating sequence identity [33]. The promoter-like RNA motifs were located within the conserved regions, except for the ISRE motif 3, and each conserved region contained at least one motif, except for the region around bp 197, which contained a 16-base palindrome conserved in the goat EST. The GAS, ISRE, and IL-4RE sequences are shown in Figure 1C. Inverted repeats were found surrounding motifs 1, 7, 8, and 10, which in the folded RNA gene product could each theoretically form a stem-loop with the promoter-like motif in the single-stranded loop. While GAS variant sequences occur frequently in DNA sequences, the chance of finding two pairs of complementary GAS motifs and one pair of complementary ISRE motifs together in one RNA is exceedingly small.

Northern blot analysis of 16 human tissues showed that placenta was the only tissue that expressed predominantly a ~0.5-kb poly(A)+-TSU RNA (Fig. 2). Larger TSU-related mRNAs of 4.4 kb were observed in all tissues tested, and a 2.4-kb mRNA was also observed in placenta, heart, and pancreas. Placenta also expressed a 6.0-kb TSU mRNA. The identification of these related mRNAs will be pursued in further work. Southern blot analysis gave a single 3.5-kb EcoRI fragment that hybridized in human genomic DNA with a TSU probe, indicating that TSU is encoded by a single-copy gene in the human genome. The production of the TSU RNA in placenta, therefore, may involve alternative splicing. Cloning and sequencing of the genomic TSU gene and the related mRNAs should resolve this question. A search of the Human Gene Map database indicated that the TSU gene is located on the proximal long arm of human chromosome 11.



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  		FIG. 2. Northern blot analysis of expression of TSU in human tissues. A radiolabeled antisense TSU probe and a random-primer-labeled ß-actin probe were used sequentially to probe the blots. The positions of RNA size standards of 9.5, 7.5, 4.4, 2.4, and 1.35 kb are indicated on the right side.

Suppression of MHC and ICAM-1 Antigens by Transfected TSU cDNA

Pools of cells stably transfected with the TSU-pCR3 expression construct that were resistant to 400 µg/ml G418 were tested to determine the effects of TSU expression on the constitutive and IFN-{gamma}-inducible expression of several endogenous genes. Immunocytochemical staining of TSU-expressing cells from two independent transfections showed reduced induction of intracellular MHC class II expression, and reduced constitutive and inducible MHC class I expression, but no effect on Na+,K+-ATPase expression.

The effect of TSU on cell surface expression of MHC class II antigens (including HLA-DR), MHC class I antigens (including HLA-B), and ICAM-1 antigen was studied by flow cytometry of cells stained with specific mAbs (Fig. 3, A and B). Cells stably transfected with the TSU-pCR3 expression construct or pCR3 vector were treated with 0, 20, 66, or 200 U/ml IFN-{gamma} for 42 h. Results of a representative experiment that shows differences in expression on cells treated with 0 or 200 U/ml IFN-{gamma} are depicted in Figure 3A. Induction of high-level expression (hi) of MHC class II antigens was reduced by TSU expression. Constitutive expression of MHC class I antigens and ICAM-1 antigen was reduced in TSU-pCR3 transfectants compared to pCR3 vector-transfected cells. IFN-{gamma} treatment of pCR3-transfected control cells resulted in expression of high levels of MHC class I and ICAM-1 on most cells, while the presence of TSU partially blocked this stimulated expression.



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  		FIG. 3. Function of TSU RNA in stably transfected HeLa cells. A) Flow cytometric analysis of suppression of constitutive and IFN-{gamma} (200 U/ml)-inducible cell surface expression of MHC class II, MHC class I, and ICAM-1 antigens. Binding of mAbs CR3/43 (MHC class II), G46–2.6 (MHC class I), HA58 (ICAM-1), or nonimmune IgG1 or IgG2a was detected by use of fluorescein-conjugated anti-mouse antibody. FACS profiles produced by negative control IgG1 are shown as filled-in curves. B) Quantitation of suppression of constitutive expression and inducible responses of stably transfected cell pools treated with 20, 66, 200, and 1000 U/ml IFN-{gamma} for 42 h. Cells were gated (as shown in A) into negative, low, or high groups in three independent FACS analyses. Results are expressed as frequency (% of total cells in each category) of TSU-pCR3-transfected cells relative to frequency of pCR3-transfected cells. The calculated values shown are the mean with standard deviation.

The effects of several doses of IFN-{gamma} on the frequency of cells expressing high or low levels of MHC class II, MHC class I, and ICAM-1 in TSU-pCR3 transfectants relative to pCR3 transfectants are shown in Figure 3B. Compared to controls, MHC class IIhi cells were reduced to 22%, 22%, and 29% when treated with 66, 200, and 1000 U/ml IFN-{gamma}, respectively. The differences between the mean percentages over three experiments of TSU-pCR3-transfected cells and vector-transfected control cells were significant, with p < 0.001 under these three IFN-{gamma} treatment conditions. There was essentially no cell surface expression of MHC class II antigens at 0 U/ml IFN-{gamma} (Fig. 3A; compare top panels to negative control staining in bottom panels), and little expression at 20 U/ml for control or TSU-transfected cells. Identical results were observed with IgG1 and IgG2a nonimmune antibodies. The numbers of cells constitutively expressing high levels of MHC class I and ICAM-1 were reduced to 30% and 24%, respectively, relative to pCR3 vector transfectants, but with increasing doses of IFN-{gamma} the TSU-mediated suppression of MHC class I and ICAM-1 surface expression was overcome by the increasing signals from the IFN-{gamma} receptor. At 1000 U/ml IFN-{gamma}, the levels of MHC class Ihi and ICAM-1hi reached 73% and 70% of control, respectively (Fig. 3B). The differences between the mean percentages over three experiments for MHC class I and ICAM-1 were significant, with p < 0.01 for the 0 U/ml IFN-{gamma} condition. Southern blot analysis of EcoRI-cut genomic DNA from TSU-transfected and control transfected cells with a TSU probe indicated that copy numbers of integrated plasmids were similar.

Inhibition of STAT1 Nuclear Translocation by Transfected TSU cDNA and Microinjected TSU RNA

The suppression of IFN-{gamma}-inducible MHC class II expression and the partial suppression of constitutive and inducible MHC class I and ICAM-1 expression by the TSU cDNA suggested the possible inhibition of STAT1. The phosphorylation, dimerization, and rapid transport to the nucleus of STAT1 can be followed by immunocytochemical localization of the STAT1 antigen in IFN-{gamma}-treated cells [38, 39]. To determine whether the function of STAT1 was impaired by TSU, pools of cells stably transfected with TSU-pCR3 or empty pCR3 vector were treated with one of several concentrations of IFN-{gamma}, and STAT1 antigen was localized with a specific mAb. Cells were scored in a blind manner for exhibiting one of three STAT1 antigen localization patterns in digitized, randomly collected photomicrographs (Fig. 4). In the absence of IFN-{gamma} (0 h), more than 99% of cells contained exclusively cytoplasmic STAT1 antigen. In the control pCR3 vector-transfected cells, IFN-{gamma} treatment at 200 U/ml for 0.4 h caused the transport of STAT1 antigen into the nucleus and strong nuclear localization in about half of the cells, and weak nuclear localization in the remainder. A one-half log lower IFN-{gamma} dose (66 U/ml) resulted in 29% strong and 71% weak nuclear localization, and one log lower (20 U/ml) gave a slightly lower frequency of strong and weak nuclear localization and some cytoplasmic localization at 0.4 h. By 4 h, in pCR3 vector-transfected cells there was 100% weak nuclear localization of STAT1 with 200 and 66 U/ml IFN-{gamma} treatment, and at 20 U/ml some strong nuclear localization remained. By 40 h of continuous IFN-{gamma} treatment, all STAT1 was localized to the cytoplasm. Transfectants expressing TSU showed no strong nuclear localization after 0.4 h of 200, 66, or 20 U/ml IFN-{gamma} treatment, 75% and 87% weak nuclear localization at 200 and 66 U/ml, respectively, and 78% cytoplasmic localization at 20 U/ml. By 4 h, 20%, 85%, and 84% of TSU transfectants showed cytoplasmic localization of STAT1 at 200, 66, and 20 U/ml, respectively, compared to 0%, 2%, and 15% in the controls. As in vector-transfected control HeLa cells, the weak nuclear localization of STAT1 observed with 200 and 66 U/ml IFN-{gamma} returned to 100% cytoplasmic localization at 40 h. The differences at 0.4 h between the mean percentages of strong nuclear localization for TSU-pCR3-transfected cells and pCR3 vector-transfected control cells were compared by t-test and found to be significant, with p < 0.001, under the conditions of treatment with 20, 66, and 200 U/ml IFN-{gamma} (Fig. 4, solid bars at 0.4 h). The trophoblast cell line Jar exhibited cytoplasmic and weak nuclear localization of STAT1 when treated with 200 U/ml IFN-{gamma} for 0.4 h.



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  		FIG. 4. Inhibition of nuclear translocation of STAT1 by transfected TSU. Morphometric analysis of localization of STAT1 antigen was performed on cell pools stably transfected with TSU-pCR3 expression construct or pCR3 vector alone and either untreated or treated at 20, 66, or 200 U/ml IFN-{gamma} for the times shown. Transfectants were grown and treated in chamber slides, fixed with acetone, and stained for cytoplasmic and nuclear STAT1 antigen with the mAb G16920 (Transduction Laboratories) and Ni2+-enhanced avidin-biotin-peroxidase immunocytochemistry [4]. The photomicrographs demonstrate the three distinct patterns of antigen localization. Gray cells indicate cytoplasmic localization. Gray cells with stained nuclei indicate weak nuclear localization, and stained nuclei alone indicate strong nuclear localization.

To define a mechanistic role for TSU RNA that was independent of the transfected DNA, samples of purified, recombinant TSU RNA were microinjected [38] directly into HeLa cells that were challenged with a 0.5-h IFN-{gamma} treatment and then stained for STAT1 localization (Fig. 5). Injection of 1 µg/µl TSU RNA caused inhibition of IFN-{gamma}-stimulated STAT1 nuclear localization (Fig. 5, open arrows). Negative control injections of KCl-Tris buffer alone (Fig. 5, solid arrows) or of 1 µg/µl tRNA (results not shown) allowed the cells to respond to IFN-{gamma} with strong nuclear localization of STAT1. The recombinant TSU RNA was not capped, and effects were seen at short times of incubation postinjection (8, 17, and 20 min). This suggests that the TSU RNA is the active moiety in the inhibition of STAT1 nuclear translocation. No DNA was microinjected in these experiments.



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  		FIG. 5. Function of microinjected TSU RNA in HeLa cells. HeLa cells grown on coverslips were individually identified by phase-contrast optics, bathed in HEPES-buffered medium, and microinjected at 25°C before short-term incubations as shown. IFN-{gamma} (200 U/ml) was added to the medium, and the cells were incubated at 37°C for 30 min before acetone fixation and immunocytochemistry for STAT1 antigen as in Figure 4. Several dozen cells were successfully microinjected over 4 independent experiments. Scale bar indicates 10 µm. The TSU-injected cells exhibited cytoplasmic (open arrows) or weak nuclear localization of STAT1, while the buffer-injected cells responded to IFN-{gamma} treatment with strong nuclear localization (solid arrows).

TSU and Synthetic Loop-Loop Utrons Bound STAT1 In Vitro

One possible mechanism of action involves direct interaction of TSU RNA with STAT1 protein. Consistent with this, crude extracts from STAT1-expressing cells (Fig. 6, lane 2), but not from STAT1-deficient cells (lane 3), were observed to retard the mobility of labeled TSU RNA in agarose gels. The binding was saturable (lanes 2, 4, 6). Sequence-specific binding was demonstrated by synthetic constructs designed as 80-nucleotide TSU model structures that consisted of recombinant RNA with two predicted stem-loops and a central hairpin (depicted schematically in Fig. 7). The electrophoretic mobility of the loop-loop structure with the partially complementary motif 1 (5'-GUAAAGUAA-3') and motif 8 (5'-UUACGUCAU-3') (Fig. 7) was retarded with the formation of two complexes A and B (Fig. 6, lanes 12–14), similar to those formed by TSU and STAT1 (lanes 2, and 8–10). Replacement by mutant GAS motifs (5'-CGAAAAUCC-3' and 5'-UAACGGACA-3') reduced the binding to trace levels (lanes 16–18). A {gamma}-actin mRNA fragment served as negative control (lanes 20–22). These loop-loop model structures represent deletion of > 80% of the sequence of TSU, and they have no open reading frames in common with TSU.



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  		FIG. 6. Specificity and saturability of TSU-STAT1 binding determined by electrophoretic mobility shift assay. Radiolabeled TSU probe (20 ng) was mixed with 0 ng (lane 1) or 300 ng of protein in extracts made from IFN-{gamma}-treated 3B5 cells (lanes 2, 4, and 6) or from IFN-{gamma}-treated U3A cells (lanes 3, 5, and 7), or with 30x (lanes 4 and 5) or 100x (lanes 6 and 7) nonradioactive TSU competitor. TSU probe (lanes 8–11), loop-loop model RNA probes with GAS motifs (lanes 12–15) or mutant GAS motifs (lanes 16–19), and a negative control {gamma}-actin mRNA fragment (lanes 20–23) were used at 20 ng in each reaction with 0 ng, 300 ng, 900 ng, or 3000 ng of 3B5 cell extract protein. Two independent experiments gave identical results. Autoradiographic exposures: 3 h (lanes 1–7) and 5 h (lanes 8–23).



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  		FIG. 7. Possible base-pairing of promoter-like motifs 1 and 8 in TSU and synthetic TSU model RNA. The predicted folding pattern is shown of a synthetic construct that contains GAS motifs in loop-loop bent-helical structures.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The strong repression of MHC class II genes and the weak repression of MHC class I and ICAM-1 by TSU, together with the observed inhibition of STAT1 strong nuclear localization, but not weak nuclear localization, suggests that there is a balance of positive signal transduction that drives STAT into the nucleus and of TSU inhibition that retains the bulk of the STAT1 in the cytoplasm. Weak nuclear localization of STAT1 may be sufficient to induce transcription of MHC class I and ICAM-1 genes, but not MHC class II genes. Indeed, ICAM-1 is expressed constitutively on many cell types, and high-level expression is rapidly induced by cytokines such as IFN-{gamma}, but there is reduced cytokine responsiveness in the expression of ICAM-1 in cultured cytotrophoblasts from term placenta [40]. The phenotype of TSU-pCR3-transfected HeLa cells reproduces partially the characteristics of trophoblast cell lines like Jar—repressed MHC class II genes, reduced MHC class I and ICAM-1 expression, and reduced STAT1 signaling from the IFN-{gamma} receptor to the cell nucleus.

The identification of an untranslated RNA, or utron, that functions as a trophoblast-derived suppressor factor was facilitated by the application of mammalian expression cloning methods under the sole constraint that single gene products effected a reduction in MHC class II antigen expression. There are precedents for functions of RNA as cis-acting, but not trans-acting, gene repressors. The expression of the H19 RNA blocks insulin-like growth factor-2 expression in cis [41], and the encoded Xist RNA silences other X-chromosome loci in cis [42]. These studies have not suggested RNA binding to transcription factors. Protein binding by other RNA loop-loop structures has been observed, however, in bacterial and viral systems [43, 44]. RNA loop-loop structures have been described that take on a bent helix rather than the standard A-form RNA helix [45]. Therefore, the possibility of binding of RNA in this conformation to a DNA-binding protein would not be ruled out a priori.

A model is proposed in Figure 8 that takes into account the observed reduction in STAT1 translocation in TSU stable transfectants as well as the saturable and sequence-specific binding of TSU and small loop-loop model RNA to extracts containing STAT1. The apparent titration of activated STAT1 by TSU leads to reduced, but not absent, signaling from the IFN-{gamma} receptor. Downstream from this blunted signaling, there is strong reduction of MHC class II antigen levels versus weak reduction of MHC class I and ICAM-1 antigen levels. The model (Fig. 8) shows binding of the loop-loop RNA structures to the DNA-binding site of an activated STAT1 dimer to produce RNA-protein complex B observed by gel shift assay. The formation of a dimer of dimers representing RNA-protein complex A may also occur. In the proposed mechanism, the TSU-STAT1 complexes would not be substrates for nuclear import. The mechanism of inhibition of nuclear translocation of TSU-STAT1 complexes is not known, and the possible role of additional cytoplasmic or nuclear components remains to be elucidated.



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  		FIG. 8. Model for binding of folded TSU RNA to cytokine-activated STAT1 dimer leading to retention of dimers and tetramers in the cytoplasm. STAT1 dimer forms a GAS binding site and is normally targeted to the nucleus. TSU binds the STAT1 dimer, and these complexes may form a dimer of dimers. Nuclear translocation is blocked for TSU-STAT1 complexes.

Analysis of the TSU cDNA sequence provided no long open reading frame but pointed to the possibility of sequences similar to IFN-{gamma}-response elements in the folded RNA gene product that functions to inhibit MHC expression. A search of the promoter database with the Findpatterns program revealed the TSU GAS motifs 8 and 10, the IL-4RE motif 9, and the ISRE motif 6; but the GAS motifs 1, 5, and 7, the IL-4RE motif 2, and the ISRE motifs 3 and 4 were missed. The last bases in GAS motifs 1, 5, 7, 8, and 10 and the IL-4RE motif 9—AC, TA, AC, AT, AC, and CA, respectively—are different from the AA of the consensus. Some of these variants were absent from the promoter database. Variant GAS motifs have been found to play a functional role in the determination of the specificity of binding of STATs to gene promoters [46]. The conservation of the promoter motifs, but not of a polypeptide coding region, in the human TSU and the TSU-related goat EST provides a strong evolutionary argument that supports the idea that the RNA itself inhibits STAT1 function and suppresses MHC expression without the participation of a TSU-encoded polypeptide.

Another example of utrons with significant physiological impact is found in the studies of Blau and coworkers [47, 48]. The 3'-untranslated regions of {alpha}-cardiac actin, {alpha}-tropomyosin, and troponin I mRNAs are found to cause mitotic arrest in fibroblasts and to stimulate differentiation in muscle cells, and the 3'-untranslated region of the {alpha}-tropomyosin mRNA suppresses anchorage-independent growth of a neoplastic myogenic cell line. It is tempting to speculate that the interaction of one or more transcription factors with these well-described muscle cell-related utrons may mediate these transcriptional events. Indeed, a preliminary computer-assisted survey of the sequence of the 3'-UTR of {alpha}-tropomyosin yielded a list of candidate promoter sequences that included a number of growth factor response elements and histone promoter elements.

Several lines of evidence presented in this paper suggest strongly that the trophoblast STAT utron is a small RNA that functions to inhibit STAT1 translocation to the nucleus and repress MHC gene expression: 1) The human 481-base TSU cDNA and the TSU-related goat EST had no common open reading frames but showed conservation of sequences similar to IFN-signal transduction target motifs. 2) The placenta normally produces a moderately abundant, small, poly(A)+ TSU RNA, and a trophoblast cell line expresses TSU RNA and constitutively inhibits STAT1 function and MHC expression. 3) TSU cDNA expression constructs suppressed MHC class II, MHC class I, and ICAM-1 antigen expression to varying degrees with particular IFN-{gamma} treatments and inhibited STAT1 function in transfected cells. 4) Microinjection of recombinant TSU RNA into HeLa cells blocked STAT1 function in several minutes. 5) TSU bound STAT1 in a saturable manner, and synthetic RNAs consisting solely of predicted loop-loop structures formed RNA-protein complexes in a sequence-specific manner.

Aided by this understanding of the repression of MHC genes by TSU, we may soon be able to answer the question, "How does the pregnant mother contrive to nourish within itself, for many weeks or months, a foetus that is an antigenically foreign body?" [49]. It will be interesting to determine whether utrons repress other genes. Little is known about the mechanisms that comprise selective gene repression during embryogenesis, organogenesis, and differentiation, yet most genes are repressed in most cell types [50]. Gene repression by utrons is a novel mechanism by which a cell can simultaneously produce specialized protein-encoding mRNAs and a selection of gene repressors, namely, utrons derived from the mRNAs, both of which can contribute to the cell's developmental program.


   ACKNOWLEDGMENTS
 
The technical assistance of these individuals is gratefully acknowledged: Pam J. Nelson for transfection, mammalian cell cloning, and other cell culture procedures; Elaine Coupal for the preparation of the cDNA library; Sonia Mumford for additional mammalian cell cloning; Monica Talmor for synthesis of oligonucleotides; and the staff of the W. M. Keck Foundation Biotechnology Resource Laboratory in the Boyer Center for Molecular Medicine at Yale for the DNA sequencing service. I am thankful to Graeme L. Hammond, in whose laboratory some of this work was performed; and to David G. Schatz, in whose laboratory the work was completed, for critical reading of the manuscript. I thank Ari Helenius for the use of the microinjection facilities. I am grateful to Peter Cresswell, David R. Johnson, Nancy H. Ruddle, Joan A. Steitz, Haren A. Vasavada, David C. Ward, and Sherman M. Weissman for helpful discussions.


   FOOTNOTES
 
1 Correspondence and current address: CuraGen Corporation, 555 Long Wharf Drive, New Haven, CT 06511. FAX: 203 401 3351; jpeyman{at}curagen.com Back

Accepted: August 17, 1998.

Received: April 21, 1998.


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