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The Journal of Immunology, 2001, 166: 3966-3974.
Copyright © 2001 by The American Association of Immunologists

A 3'-Transcribed Region of the HLA-A2 Gene Mediates Posttranscriptional Stimulation by IFN-{gamma}1

Steven R. Snyder2,*,{ddagger}, Jeffrey F. Waring2,3,{dagger}, Sheng Zu Zhu*, Sarah Kaplan*, Julie Schultz*,{dagger} and Gordon D. Ginder4,*,{dagger},{ddagger}

* Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298; and {dagger} Institute of Human Genetics and {ddagger} Department of Medicine, University of Minnesota, Minneapolis, MN 55455


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The expression of several MHC class I genes is up-regulated at the transcriptional level by IFN-{gamma}. Posttranscriptional mechanisms also have been implicated, but not well characterized. To investigate the mechanism of IFN-{gamma} stimulation of the human MHC class I gene HLA-A2, several human tumor cell lines were transfected with reporter gene constructs driven by the HLA-A2 promoter. We have previously shown that the extended 525-bp HLA-A2 promoter alone, which includes a 5' IFN-stimulated response element consensus sequence, is not sufficient for IFN-{gamma} response in either K562 or Jurkat cells. In the current study, stable transfection of a genomic HLA-A2 gene construct, containing both 5'- and 3'-flanking sequences, resulted in stimulation of the gene by IFN-{gamma}. Nuclear run-on assays revealed that, unlike other class I genes, IFN-{gamma} stimulation of HLA-A mRNA accumulation occurs almost entirely through posttranscriptional mechanisms. RNA stability assays showed that the effect is not mediated by alteration of the half-life of the HLA-A2 mRNA. Formation of the 3' end was unaffected by IFN-{gamma} treatment. Sequences that mediate the majority of IFN-{gamma} induction of HLA-A2 mRNA reside in a 127-bp 3'-transcribed region of the gene. This region contains the terminal splice site, the usage of which is not affected by IFN-{gamma} treatment. These results demonstrate a novel posttranscriptional mechanism of regulation of MHC class I genes by IFN-{gamma}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interferons influence a number of cellular processes, such as cell growth, differentiation, and immune reactivity. The two types of IFNs ({alpha}{beta} and {gamma}) bind to unique cell surface receptors, which induce the transcription of a number of distinct, but overlapping sets of genes (1, 2, 3). Among the genes that are inducible by both {alpha}{beta} and {gamma} IFNs are the MHC class I genes (1 , 4, 5, 6, 7). The MHC class I genes encode for a membrane spanning polypeptide heavy chain, which associates noncovalently on the cell surface with an invariant light chain called {beta}2 microglobulin (8). Classical MHC class I Ags function in the presentation of foreign antigenic peptides to CD8+ CTL and are the principle targets for cell-mediated lysis by CTLs during the rejection of allogenic tissue transplants (9, 10, 11).

In most cases the up-regulation of MHC class I Ag expression by IFNs is mediated by transcriptional mechanisms (4, 6, 12, 13, 14). Transcriptional induction by IFN is thought to be mediated, at least in part, through the IFN-stimulated response element (ISRE)5 located in the 5' promoter of most MHC class I genes. The mechanism of IFN-{alpha} induction through the ISRE is well-characterized (reviewed in Ref. 15). The mechanism of IFN-{gamma} stimulation of class I genes through the ISRE is less well understood, but appears to be mediated by IFN regulatory factor-1 (IRF-1; Ref. 16).

Although the ISRE, and the proteins that bind to it, may be necessary for IFN induction of MHC class I gene expression, they do not appear to be sufficient to explain the entire effect of IFN stimulation. Studies of the mouse H-2L gene have shown that while the ISRE was necessary for full IFN induction, the H-2L mRNA was still up-regulated 2- to 4-fold by IFN-{gamma} when the entire 5' promoter of the gene was replaced with a feline leukemia virus promoter (17, 18, 19). A similar result was obtained in the human HLA-B7 gene, in that a truncated HLA-B7 gene lacking the 5' promoter region was IFN-inducible when transfected into mouse L cells (20). Further, HLA-B7 expression from constructs that lacked the 5' ISRE were up-regulated severalfold by IFN-{gamma} in transgenic mice (21). In none of these cases was it demonstrated whether IFN stimulation of class I gene expression was due to a transcriptional effect mediated by sequences downstream of the promoter, or by posttranscriptional mechanisms.

Studies from our laboratory (22), and others (23), have shown that the HLA-B locus contains a much stronger ISRE than the HLA-A locus. Yet, IFN-{gamma} up-regulates HLA-A locus gene expression variably, depending on the cell type (23, 24 , and this study). It has been reported that IFN-{gamma} stimulates HLA-A2 gene expression 4- to 5-fold at the level of transcription (24, 25). However, in such reports, the probe used to assay the level of endogenous HLA-A2 transcription in nuclear run-on assays was a full length HLA-A2 gene that shares large hybridizable stretches of sequence with HLA-B and -C genes (26). Other investigators have suggested that genes in the HLA-A locus are only very weakly induced, if at all, by IFN-{gamma}, based on the fact that the HLA-A2 and HLA-A3 5' promoters are not induced by IFN-{gamma} (23).

In the course of studying the mechanisms of locus-specific IFN-induced regulation of HLA class I genes, we have examined IFN-{gamma} induction of the HLA-A2 gene. Our results show that the HLA-A2 promoter, including 525 bp of 5'-flanking sequences, is not sufficient for an IFN-{gamma} response in either Jurkat or K562 cells (22), in agreement with others (23). However, using probes specific for the HLA-A locus, we show in the current study that a stably transfected HLA-A2 gene, including 525 bp of 5'-flanking sequence and ~1300 bp of 3'-flanking sequence, is induced ~3-fold by IFN-{gamma} in K562 leukemia cells. In SK-N-MC neuroblastoma cells, IFN-{gamma} induces a 13-fold increase in endogenous HLA-A-specific mRNA. Nuclear run-on transcription assays demonstrate that in four different tumor cell lines, including K562 and SK-NM-C, IFN-{gamma}-induced increases in the level of HLA-A mRNA is due mostly to posttranscriptional effects, in contrast to previous studies of other HLA class I genes (4, 12, 13). Deletion constructs and heterologous chloramphenicol acetyl transferase (CAT) reporter gene constructs were used to demonstrate that the IFN-{gamma}-induced increase in HLA-A2 mRNA is dependent on 127 bp of 3'-transcribed sequences. However, IFN-{gamma} treatment does not significantly alter the half-life of HLA-A2 mRNA. Rather, these results document a previously undescribed posttranscriptional mechanism of IFN-{gamma} stimulation of MHC class I gene expression mediated by specific 3'-transcribed sequences.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
RNase protection assays

For RNase protection assays, single-stranded RNA for probes were generated from the plasmid constructs as described below. A 360-bp PvuII fragment from the gene that codes for neomycin resistance was isolated and subcloned into pGEM I. The HLA-A2 exon 4 probe was made by subcloning a 768-bp PstI fragment from pUC 9 HLA-A2 into pGEM I. The RNA probe protects 160 bp of exon 4 in HLA-A2. The plasmid for the RNA probe used for 3' HLA-A mRNA end formation determination was made by inserting a PCR-generated fragment of the extreme 3' end of the HLA-A2 gene (nt 3244–3497 from the 5' cap site) into the vector pTarget (Promega, Madison, WI). The plasmid for the RNA probe used for determining splice site usage was similar, except that it contained nt 2792–3112 of the HLA-2 gene. The plasmid for the CAT RNA probe was constructed by inserting 214 bp of the CAT cDNA into pGEM I. The human triosephosphate isomerase (TPI) control probe was generated from the plasmid pSPTPI/c and protects 110 bp of TPI mRNA (Ref. 27 , a gift from Dr. Lynne Maquat). The RNA isolation and RNase protection assays were performed as described previously (22).

Plasmid construction

The pCAT A2 construct was made as previously described (28). The A2 del 3' construct was made by digesting the plasmid pUC 9 HLA A2 (26) with SspI. This enzyme cuts the HLA-A2 gene 2918 bp from the 5' cap site, removing the entire 3'-flanking region and the terminal alternate exon 8, along with 15 bp of intron 7. The SV40 polyadenylation site from the plasmid pCATbasic (Promega) was then inserted at the SspI site. The HLA-A2 del 330, HLA-A2 del 530, HLA-A2 del 980, and HLA-A2 del 1245 clones were constructed from plasmid pUC 9 HLA-A2 using Bal 31 nuclease, according to the methods of Poncz et al. (28). The truncated HLA-A2 gene was removed from pUC 9 by digestion with HindIII and subcloned into pGem I. The number of nucleotides deleted from the HLA-A2 gene in each construct were determined by dideoxy sequencing (29). The pCMV-CAT-A2 3' deletion clones were constructed by inserting 500, 422, 300, 219, and 127 nt, respectively, of 3'-transcribed sequence from the HLA-A2 gene into the plasmid pTarget immediately upstream of the SV40 poly(A) addition site. The tetracycline (Tet)-inducible CAT-A2 3' plasmid was constructed by inserting the CAT cDNA, immediately followed by 422 bp of the 3'-transcribed sequence from the HLA-A2 gene, into pcDNA4/TO (Invitrogen, Carlsbad, CA; see Fig. 4GoA).



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  		FIGURE 4. A, Depiction of Tet-inducible T-REx construct used to measure mRNA half life. B, RNase protection assay of pulse-chase decay of RNA containing the HLA-A2 3' IRE transcribed from a CAT-HLA-A2 3' fusion gene. TPI mRNA was quantitated as an internal control. C, A representative plot of mRNA decay taken from phosphorimager analysis of the data from Fig. 4GoB. The following half-lives were determined (mean ± 95% confidence interval (p <= 0.05), calculated from three independent experiments): untreated, 1.13 ± 0.21 h; IFN-{gamma}-treated, 0.95 ± 0.15 h.

 
Cell culture and transfection

Cell culture for K562, Jurkat, HSB-2, Molt-4, SK-N-MC, HeLa, SK-BR-3, U937, and 721.144 cell lines and transfections for K562 cells were performed as described previously (22) with modifications described in the figure legends, when necessary.

Northern RNA blot hybridization

Northern blotting assays were performed as previously described (12, 30).

Nuclei isolation and nuclear run-on transcription assay

Nuclei were isolated from ~108 cells for transcription reactions as previously described (31). Transcription reactions were performed exactly as previously described (31, 32). Hybridization to slot-blotted cDNA probes on Zetaprobe membranes was performed at 65°C for 48 h in 3x SSC, 10x Denhardt’s, 0.4% SDS, 40 µg/ml yeast tRNA, 0.1 mg/ml salmon sperm DNA, and 0.5 µg/ml blank vector (pGEM3zf(-)) DNA. Filters were washed twice for 15 min at 65°C in 2x SSC, 0.1% SDS, then twice in 0.1x SSC, 0.1% SDS before autoradiography.

Probes for nuclear run-on and Northern analyses

The control probe for nuclear run-on transcription reactions is a human {alpha} actin 1.5-kb cDNA (pHM{alpha}-A1). The negative control is {lambda} DNA digested with HindIII. The positive control MHC class I probe is a 1.4-kb fragment from plasmid pHLA-B7 containing a human HLA-B7 cDNA sequence (33). The specific probe for HLA-A2 is a 490-bp PvuII-MspI subclone of the 3' untranslated region (UTR) of the HLA-A2 genomic clone (Ref. 34 ; a gift from Dr. Harry Orr). The control probe for the Northern blotting assay is a probe specific for 18S RNA, which has the following sequence: 5'-CGAACAGAGTTTCTAATTCGG-3' (a gift from Dr. Paul Siliciano, University of Minnesota, Minneapolis, MN).

mRNA half-life studies

Tet-inducible CAT-3' A2 mRNA was generated using the T-REx system (Invitrogen). SK-N-MC cells were stably cotransfected with the plasmid pcDNA6/TR, which expresses the Tet R repressor, and the Tet-inducible CAT-HLA-A2 3' fusion construct described above, according to the vendor’s instructions. After selection, the cells were treated with 1 µg/ml Tet, with or without 200 U/ml IFN-{gamma}, and incubated for 24 h. The cells were then washed free of Tet to stop transcription, and were maintained an additional 36 h with or without IFN. RNA was harvested at the indicated time points and analyzed by RNase protection using a CAT cRNA probe. The mRNA half-life was calculated from the slope of a logarithmic plot of mRNA, determined by phosphorimager analysis, vs time. Half-lives were determined for untreated and IFN-{gamma}-treated samples from three independent experiments. The data were analyzed using a two-tailed Student’s t test. The 95% confidence interval was determined (p <= 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A transfected full-length HLA-A2 gene is inducible with IFN-{gamma}

IFN-{gamma} induction of many class I genes is mediated, at least in part, by the ISRE located within the 5' promoter of the gene (17, 18, 35). CAT expression constructs consisting of 525 bp of the HLA-A2 promoter, either containing or lacking the ISRE, were transiently transfected into both K562 and Jurkat cells to determine whether IFN-{gamma} up-regulates expression of the HLA-A2 gene through the ISRE. Our results (28) confirmed previous observations that showed that the 5' promoter, and hence the HLA-A2 ISRE, is unable to confer IFN-{gamma} induction of transcription (23, 31). We have previously shown that total endogenous HLA class I gene transcription in both of these cell lines responds to IFN-{gamma} with a 3- to 10-fold increase under identical treatment conditions (30, 36).

To determine whether sequences downstream from the promoter confer responsiveness of the HLA-A2 gene to IFN-{gamma}, a 5.1-kb HLA-A2 genomic construct containing 525 bp of 5'-flanking sequences, all eight exons, and ~1300 bp of 3'-flanking sequences was stably cotransfected into K562 cells with an RSV-Neo drug selection plasmid. K562 cells were chosen because they completely lack mRNA expression from their endogenous HLA-A locus (37), thus ensuring that any HLA-A mRNA detected was transcribed from the transfected gene. After stimulation with IFN-{gamma}, cytoplasmic RNA was harvested from three independently transfected populations of K562 cells. The level of transfected HLA-A2 RNA produced was determined by RNase protection assay using a probe that hybridizes to the fourth exon of the HLA-A2 gene, resulting in a 160-bp protected fragment. This probe also cross-hybridizes with endogenous HLA class I RNA in K562 cells, giving a protected fragment of 120 bp. Fig. 1GoA shows that in stably transfected cells, an increase in HLA-A2 RNA levels can be seen following treatment with IFN-{gamma}. The level of induction was determined by densitometeric analysis of the resulting autoradiograms. Results were standardized relative to an internal constitutive control, RSV Neo. Analysis of three pools of cells showed an average 3.2-fold (range 2.3- to 4-fold) level of induction of HLA-A2 mRNA following treatment with IFN-{gamma}.



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  		FIGURE 1. A, RNase protection assay of three different pools of K562 cells stably transfected with the HLA-A2 genomic wild type gene using the exon 4 protection probe that protects 160 nt of exon 4 and cross-hybridizes with endogenous K562 class I mRNA, giving a 120 nt protected fragment. The cells were either not stimulated (0) or stimulated with 100 U/ml IFN-{gamma} (+). B, RNase protection assay of two pools of K562 cells stably transfected with the HLA-A2 del 1245 construct. The cells were either unstimulated (0) or stimulated with 100 U/ml IFN-{gamma} (+). The exon 4 probe was used. C, RNase protection assay of K562 cells stably transfected with the HLA-A2 del 3' clone. The cells were stimulated with 100 U/ml IFN-{gamma} for the indicated periods of time. The exon 4 probe was used.

 
IFN-{gamma} was also able to induce expression of endogenous HLA-A locus mRNA in six human cell lines including Jurkat, HeLa, SK-BR-3, SK-N-MC, U-937, and Molt 4, which are a T cell leukemia line, an epithelioid carcinoma cell line, a breast cancer cell line, a neuroblastoma cell line, a histiocytic lymphoma cell line, and an acute lymphoblastic leukemia cell line, respectively. As shown in Table IGo, the induction was greater than 5-fold in two of the cell lines tested. Untransfected K562 cells were included as a negative control for the probe because they do not express any endogenous HLA-A mRNA (37).


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  		Table I. HLA-A gene transcription rates and mRNA levels in response to IFN-{gamma}1

 
A specific 3'-transcribed sequence of the HLA-A2 gene is necessary and sufficient for IFN-{gamma} induction

Although previous investigations in the murine and human MHC class I systems have suggested that a component of IFN induction of class I genes is mediated by sequences located downstream from the transcription initiation site, neither the location of the relevant sequences nor the mechanism involved have been identified (17, 20, 21). To determine whether an IFN-{gamma} response element is located in the 3'-flanking region of the HLA-A2 gene, Bal 31 exonuclease deletion analysis was conducted on the 3'-flanking region of the gene. Four clones with 3' deletions of 330, 530, 980, and 1245 bp, respectively, were analyzed. The clone HLA-A2 del 1245 contains a deletion in the HLA-A2 3'-transcribed region to within 20 bp downstream of the polyadenylation signal that corresponds to 3352 bp relative to the 5' RNA cap site (30). These clones were stably transfected into K562 cells, which were then stimulated with IFN-{gamma}. Several pools of each truncated clone were analyzed for IFN-{gamma} induction by RNase protection assay. Two representative pools of the del 1245 clone are shown in Fig. 1GoB. The results show that IFN-{gamma} is able to stimulate expression from the HLA-A2 del 1245 deletion construct, despite the fact that only 20 bp 3' to the polyadenylation site remain. Clones with deletions of 330, 530, and 980 bp of 3'-flanking DNA were also fully responsive to IFN-{gamma} induction (data not shown).

To determine whether the polyadenylation site, or sequences directly surrounding it, mediate IFN-{gamma} induction of the HLA-A2 gene, the 3'-flanking region of the 5.1-kb HLA-A2 clone was removed by restriction enzyme cleavage with SspI. This enzyme cuts 2918 bp from the 5' cap site, which removes the last 15 bp of intron 7, all of the terminal alternate exon 8, and all of the 3'-flanking DNA. Because this deletion removes the HLA-A2 polyadenylation site, an SV40 polyadenylation site was inserted at the SspI site. The resulting clone, HLA-A2 del 3', was stably transfected into K562 cells under the same conditions described above. The cells were stimulated with IFN-{gamma} and expression levels were assayed by RNase protection. As illustrated in Fig. 1GoC, while the endogenous K562 class I mRNA showed a 4.8-fold level of induction after 48 h, mRNA from the transfected HLA-A2 del 3' construct showed no significant increase upon IFN-{gamma} induction. Similar results were obtained from other pools of K562 cells stably transfected with the HLA-A2 del 3' construct (data not shown).

To establish that the 3'-transcribed sequences of the HLA-A2 gene are sufficient to mediate induction by IFN-{gamma}, a construct containing a CAT reporter gene driven by the CMV promoter-enhancer and the 422-bp HLA-A2 3'-transcribed region (Fig. 2GoA) was stably transfected into SK-NM-C cells. This cell line was chosen because of the greater inducibility of endogenous HLA-A mRNA by IFN treatment (see Table IGo). These cells were not used for stable transfection of the genomic HLA-A2 construct because of the relatively high level of endogenous HLA-A expression, which would not be distinguishable from transcripts originating from the transfected genes. As illustrated in Fig. 2GoB, upon induction with 200 U/ml IFN-{gamma} for 40 h, the level of CAT mRNA increased 6-fold, whereas there was no increased expression from the construct lacking the 422-bp HLA-A2 3'-transcribed region. The magnitude of induction by IFN-{gamma} has been determined in multiple experiments and found to be 6-fold (Table IIGo). As shown in Fig. 2GoC and Table IIGo, the level of induction of the endogenous HLA-A genes in the same stably transfected SK-NM-C cell lines was ~12-fold. It should be noted that the decrease in TPI mRNA in IFN treated cells was found to reflect an overall decrease in mRNA and this effect was variable. The calculated fold induction mediated by the 3' IFN response element (IRE) did not vary irrespective of the presence or absence of decreased overall cellular mRNA, as reflected in the TPI signal. Thus, the majority of the fold induction is mediated by the 3'-transcribed region, and only about a 2-fold affect could be attributed to other sequences within and flanking the HLA-A gene. Deletion of the 3'-transcribed region resulted in complete loss of measurable IFN induction in K562 cells, which is consistent with the dominant contribution of that element in IFN induction of HLA-A mRNA in those cells as well. Therefore, the 3' HLA-A2-transcribed region appears to be both necessary and sufficient for the majority of IFN-{gamma} induction, excluding any requirement for the HLA-A2 promoter and 5' UTR. To further localize the IRE within the 3'-transcribed region, identical CMV promoter-CAT constructs containing 219 and 127 bp of the 3' HLA-A2-transcribed region (Fig. 2GoA) were stably transfected into SK-NM-C cells. The latter construct lacks the HLA-A2 poly(A) addition site. As shown in Fig. 2GoD, the IRE is entirely contained within the first 127 bp of the 3'-transcribed region. Consistent with this observation, IFN induction did not result in any alteration in 3' end formation of HLA-A2 mRNA (data not shown).



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  		FIGURE 2. A, Depiction of the construct containing a CMV promoter-enhancer driving a CAT reporter with 422, 219, or 127 bp of the HLA-A2 3'-transcribed region ligated downstream from the CAT coding sequence. B, RNase protection assay to demonstrate IFN-{gamma} response mediated by the HLA-A2 3'-transcribed sequence. SK-N-MC cells stably transfected with the pTarget-CAT-HLA-A2 3' 422-bp transcribed region plasmid (CAT-A2), or the pTarget vector alone (CAT), were either untreated or treated with 250 U/ml IFN-{gamma} for 40 h. CAT mRNA and control TPI mRNA was quantified by RNase protection. C, RNase protection assay showing response of the endogenous HLA-A mRNA in three separate pools of SK-NM-C cells stably transfected with CAT-A2. HLA-A mRNA was detected using the exon 4 probe. D, RNase protections, identical with Fig. 4GoB., using constructs containing 219 and 127 bp of the HLA-A2 3'-transcribed region

 

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  		Table II. Induction of endogenous HLA-A2 and transfected CAT 3'A2 by IFN-{gamma} in SK-N-MC cells1

 
Induction of HLA-A2 is primarily at the posttranscriptional level

To determine whether IFN-{gamma} induction of the HLA-A2 gene was mediated through transcriptional or posttranscriptional mechanisms, nuclear run-on assays were performed on K562 cells that had been stably transfected with the 5.1-kb HLA-A2 gene. The probe used to measure HLA-A2-specific transcription was the unique 3' probe that hybridizes specifically to genes from the HLA-A locus (34), while an HLA-B7 cDNA probe that cross-reacts with HLA-A, B, and C was used to measure endogenous class I gene transcription (12, 36). Cells, either not stimulated or stimulated for 2 or 16 h with 100 U/ml of IFN-{gamma}, were harvested for both nuclei for use in the nuclear run-on assay and cytoplasmic RNA for use in RNase protection assays. The results, shown in Fig. 3GoA, demonstrate that in the K562 cell line stably transfected with wild type HLA-A2, the endogenous K562 class I (non-HLA-A) genes showed a 3.9-fold transcriptional induction upon treatment with IFN-{gamma} at 2 h, which subsequently dropped to a 2.5-fold induction at 16 h. In contrast, HLA-A2-specific gene transcription was not induced at either 2 or 16 h of IFN-{gamma} treatment (Table IGo). This experiment was repeated three times with the same result. The cytoplasmic RNA harvested simultaneously from the same population of stably transfected K562 cells was assayed by an RNase protection to ensure that the HLA-A2 RNA levels did increase with IFN-{gamma} treatment. As shown in Fig. 3GoB and Table IGo, cytoplasmic HLA-A2 mRNA harvested from the stably transfected K562 cells showed a 2.2-fold and a 2.5-fold level of induction after 16 and 48 h, respectively, of IFN-{gamma} induction as compared with uninduced HLA-A2 mRNA. No increase in HLA-A2 mRNA was seen after 2 h, in contrast to the endogenous K562 class I RNA.



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  		FIGURE 3. A, Nuclear run-on transcription assay to determine the transcriptional response to IFN-{gamma} in K562 cells stably transfected with the wild type HLA-A2 clone shown in Fig. 3Go. The cells were either untreated (0) or treated with 100 U/ml for 2 or 16 h. The nuclei were isolated and transcription reactions were performed. The transcripts were hybridized to the HLA-A2 unique 3' probe (HLA-A2), {lambda} DNA, class I HLA-B7 probe, and {alpha}-actin probe which had been slot-blotted onto Zetaprobe membrane. B, RNase protection assay of cytoplasmic RNA harvested from stably transfected K562 cells used in Fig. 3GoA. The cells were either not stimulated (0) or stimulated with 100 U of IFN-{gamma} for the indicated time. The probe used was the HLA-A2 exon 4 probe. Shown in the assay are the HLA-A2-specific band at 160 bp and the K562 endogenous class I RNA at ~120 nt.

 
A nuclear run-on assay was also performed on HSB-2 cells, which express endogenous HLA-A2 mRNA, to determine whether IFN-{gamma} is able to transcriptionally induce an endogenous HLA-A2 gene. Table IGo shows that in agreement with the results in K562 cells stably transfected with full length HLA-A2, IFN-{gamma} was unable to transcriptionally activate the endogenous HLA-A2 gene, while the level of cytoplasmic HLA-A2 mRNA increased nearly 2-fold.

To determine whether other alleles of the HLA-A locus are transcriptionally induced with IFN-{gamma}, nuclear run-on assays were performed on additional cell lines. Table IGo shows the results of concomitant nuclear run-on and RNase protection assays performed on HeLa cells, which express HLA-A3 and HLA-A28 (38). These experiments show that IFN-{gamma} does not transcriptionally stimulate HeLa cell HLA-A genes, but does cause a ~5-fold increase in the level of HLA-A cytoplasmic mRNA. Identical expression assays performed on the neuroblastoma cell line SK-N-MC likewise revealed that while no transcriptional induction of endogenous HLA-A genes occurred, the levels of cytoplasmic HLA-A mRNA were increased by over 10-fold by treatment with IFN-{gamma} (Table IGo).

As a negative control, a nuclear run-on assay was performed on the cell line 721.144, a lymphoblastoid cell line that lacks both HLA-A alleles, but does express HLA-B5 (39). No HLA-A-specific signals were seen in the 721.144 cell line, but HLA transcripts hybridizing to the HLA-B7 probe were present, demonstrating that the HLA-A2 3' probe used in the nuclear run-on assay does not cross-hybridize with HLA-B transcripts (data not shown).

Posttranscriptional regulation of the HLA-A class I genes by IFN-{gamma} does not occur at the level of mRNA half-life

One possible mechanism whereby IFN-{gamma} could post-transcriptionally up-regulate the expression of the HLA-A2 gene is by stabilizing the HLA-A2 mRNA. To determine whether IFN-{gamma} stimulates the expression of HLA-A2 by increasing mRNA half-life, a Tet-inducible construct (T-REx), as depicted in Fig. 4GoA, containing a CMV promoter enhancer, a CAT reporter, and the 422-bp HLA-A2 3' IRE, was stably cotransfected with the Tet R repressor gene into SK-NM-C cells. Pulsing with Tet resulted in a 10- to 15-fold increase in CAT RNA transcription. After removing Tet, transcription is rapidly attenuated. The ensuing fall in RNA reflects the decay kinetics of the CAT-HLA-A2 3' IRE fusion RNA. As shown in Fig. 4Go, B and C, IFN-{gamma} treatment increased the level of CAT A2 3' IRE fusion RNA by 6-fold, but did not affect the half-life of decay (p <= 0.05).

IFN-{gamma} stimulation of HLA-A2 expression is not mediated by alternate 3'-terminal splice site selection

Upon examination of the GenBank sequence of the HLA-A2 gene, there appear to be two alternative 3'-terminal exon splice sites. The alternative downstream 3' exon 8 splice site is present in the 3' IRE, as shown in Fig. 5GoA. An RNase protection assay on RNA from untreated or IFN-{gamma}-treated SK-NM-C cells using a 320-bp probe spanning both alternative splice sites shows that only the downstream splice site is used in both uninduced and IFN-{gamma}-induced cells (Fig. 5GoB). These results show that the 3' IRE contains the predominant splice site, and the effect of IFN-{gamma} is not mediated by alternative splice site selection.



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  		FIGURE 5. A, Diagrammatic depiction of the alternate downstream splice acceptor and translation termination codon in the HLA-A2 3' region. The location of the minimal HL-A2 3' IRE is depicted by the thick hatched bar. B, RNase protection assay with a probe spanning from nt 2792 to nt 3112 showing only the splice site B RNase protection product of 179 bp in both untreated and IFN-{gamma}-treated SK-NM-C cells, indicating that only the downstream splice site is used.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we have examined the mechanism of IFN-{gamma} induction of the MHC class I gene, HLA-A2. In the case of other MHC class I genes, such as the murine H-2L and H-2D and human HLA-B7 genes, the mechanism of IFN-{gamma} stimulation has been shown to depend, in large part, on a conserved region called the ISRE, which is located in the 5' promoter and supports transcriptional induction. Evidence suggests this induction may be mediated by the factor IRF-1 in that previous studies have shown that IRF-1 is able to transactivate both endogenous and transfected MHC class I genes (16, 40). However, Pine et al. (41) showed that in HeLa cells, the endogenous IFN-{beta} was induced by dsRNA under circumstances in which there was no IRF-1 protein detectable, suggesting that IRF-1 is not necessary for induction of the IFN-{beta} gene. However, in the case of the HLA-A2 gene, we (22) and others (23) have shown that 525 bp of 5'-flanking DNA, including the ISRE consensus sequence, are insufficient to confer IFN-{gamma}-mediated stimulation of the gene in several cell types. These observations raised the question of whether sequences other than the 5'-flanking region of the HLA-A2 gene could be necessary for IFN-{gamma} induction. In this regard, studies in both the mouse H-2L gene and the human HLA-B7 gene have shown that sequences located 3' to the transcription initiation site are both necessary for full response to IFN-{gamma} and sufficient to mediate some IFN-{gamma} response (17, 18, 21).

Our results show that in K562 cells, which had been stably transfected with an HLA-A2 genomic clone containing 525 bp of the 5' promoter, all eight exons, and ~1300 bp of 3'-transcribed region, IFN-{gamma} stimulation increases the level of RNA from the transfected gene by 3-fold. Induction of the HLA-A2 gene was abrogated if the 3'-transcribed region, including the polyadenylation site, was removed and replaced with an SV40 polyadenylation site. Nuclear run-on assays performed using K562 cells stably transfected with the HLA-A2 gene, as well as in HSB-2 cells, which express endogenous HLA-A2, and other HLA-A-expressing lines, revealed that the transcription rate of the HLA-A genes in these cell lines was unaffected by treatment with IFN-{gamma} while transcription of other HLA class I genes was stimulated. However, the mRNA levels of HLA-A2 in these cell lines showed up to a 3-fold increase following IFN-{gamma} treatment.

In other cell lines, including SK-N-MC neuroblastoma cells and HeLa cells, which express HLA-A alleles other than HLA-A2, there was also no transcriptional induction of the HLA-A genes by IFN-{gamma}, although there was transcriptional induction of endogenous non-HLA-A MHC class I genes. In these two cell lines, there was a 5- to 13-fold increase in the levels of cytoplasmic HLA-A mRNA, indicating that IFN-{gamma} acts on various HLA-A locus genes in multiple cell types of different tissue origins through a posttranscriptional mechanism.

The results presented in this study suggest that weak or absent transcriptional stimulation by IFN-{gamma} may be a general characteristic of the alleles in the HLA-A locus. Of the HLA-A alleles that have been sequenced, including HLA-A1, A2, A3, A11, Aw24, and A26, there is a very high degree of sequence homology in the first 127 bp of the 3'-transcribed region (42), which has been shown in the current study to contain the 3' IRE that mediates the posttranscriptional stimulatory effects of IFN-{gamma}. Moreover, all of the sequenced HLA-A alleles share the same ISRE consensus sequence as the HLA-A2 gene (34, 43, 44, 45), which has been shown not to mediate a strong transcriptional response.

Although the transcriptional regulation of HLA class Ia genes by IFN has been studied extensively, very little is known about posttranscriptional mechanisms of stimulation of these, or any genes, by IFN-{gamma}. Indirect evidence has been reported that suggests a significant level of posttranscriptional up-regulation of HLA class Ia genes by IFN-{gamma} (4, 12). IFN-{gamma} also up-regulates a number of other genes at a posttranscriptional level, but the mechanisms involved have not been determined in most cases. Stabilization of cytoplasmic mRNA by IFN-{gamma} has been reported for a small number of these genes (46, 47, 48, 49). More recently, evidence for an effect of IFN-{gamma} on pre-mRNA splicing has been reported (50, 51). The precise sequences involved and the specific mechanisms have not been determined in any of these cases.

The mRNA stability studies reported in this study demonstrate that IFN-{gamma} does not stimulate HLA-A gene expression by increasing mRNA half-life. In addition, IFN-{gamma} treatment does not alter the location of mRNA 3' end formation, consistent with the lack of an observed effect on RNA half-life. Because the poly(A) addition site is not present in the 127-bp 3' HLA-A2 IRE, IFN-{gamma} does not up-regulate HLA-A2 expression by affecting poly(A) tail length. Taken together, these results demonstrate that the mechanism of posttranscriptional regulation of HLA-A gene expression by IFN-{gamma} does not involve a modulation of mRNA stability. These results also show that the mRNA half-life mediated by the HLA-A2 3' UTR is quite short.

Our results demonstrate that a 3' IRE is entirely contained within a 127-bp 3'-transcribed sequence and mediates the majority of the stimulatory effect of IFN-{gamma} on HLA-A2 expression. This region contains the predominant 3'-terminal exon splice site, which we show is the only detectable site used. Our results also show that terminal splice site selection is not affected by IFN-{gamma}. Therefore, it is possible that the IRE functions by enhancing splicing efficiency at the terminal 3' splice site, enhancing nuclear to cytoplasmic RNA transport, or by stabilizing nuclear pre-mRNA. The splice junction contained within the 3' IRE contains a 5' polypyrimidine tract flanking the splice acceptor site (52) and a potential purine-rich exonic splicing enhancer (53, 54), raising the possibility that the IRE functions to enhance splicing of the terminal 3' exon and, in turn, facilitates nuclear-cytoplasmic mRNA transport (56, 57).

The fact that HLA-A2 and other genes of the HLA-A locus are not stimulated transcriptionally by IFN-{gamma} in many cell types documents a potential mechanism of differential MHC class I regulation. Other class I genes, such as HLA-B7 and the murine H-2L and H-2K genes, have been shown to have a functional ISRE region which, by itself, can mediate a strong transcriptional response to IFN-{gamma} most likely involving the Janus kinase (Jak)/Stat family of protein kinases (18, 57). In addition, transcription of the class Ib gene HLA-E has been shown to be stimulated by IFN-{gamma} through a unique IFN response element, the interferon response region, through the Jak/Stat pathway (58). It is unclear whether the HLA-A locus genes are regulated by IFN-{gamma} posttranscriptionally through the Jak/Stat pathway. If so, this would be an as yet undescribed function of the Jak/Stat pathway. Alternatively, IFN-{gamma} could stimulate HLA-A gene expression through a different pathway. In this regard, we have previously shown that IFN-{gamma} stimulation of a transfected HLA-A2 gene is mediated by a non-A, non-C protein kinase (31). Whether the same posttranscriptional mechanisms that regulate IFN-{gamma} induction of HLA-A2 are also involved in posttranscriptional regulation of other MHC class I gene loci, or for that matter other non-class I genes, remains to be determined.


   Acknowledgments
 
We are grateful for the assistance of Catharine Tucker in preparing this manuscript and to Dr. Paul Siliciano for a critical review and suggestions.


   Footnotes
 
1 This work was supported by Grant R01CA45634 from National Institutes of Health and Masonic Cancer Center Fund, Inc. Back

2 S.R.S. and J.F.W. contributed equally to this work. Back

3 Current address: Abott Laboratories, Department 463, 100 Abott Park Road, Abott Park, IL 60064. Back

4 Address correspondence and reprint requests to Dr. Gordon D. Ginder, Massey Cancer Center, Virginia Commonwealth University, Box 980037, Richmond, VA 23298. Back

5 Abbreviations used in this paper: ISRE, IFN-stimulated response element; IRE, 3' IFN response element; TPI, triosephosphate isomerase; CAT, chloramphenicol acetyl transferase; Tet, tetracycline; UTR, untranslated region; IRF-1, IFN regulatory factor-1; Jak, Janus kinase. Back

Received for publication August 4, 2000. Accepted for publication January 4, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Basham, T., M. Bourgeade, A. Creasey, T. Merigan. 1982. Interferon increases HLA synthesis in melanoma cells: interferon-resistant and -sensitive cell lines. Proc. Natl. Acad. Sci. USA 79:3265.[Abstract/Free Full Text]
  2. Rosa, F., D. Hatat, A. Abadie, D. Wallach, M. Revel, M. Fellous. 1983. Differential regulation of HLA-DR mRNAs and cell surface antigens by interferon. EMBO J. 2:1585.[Medline]
  3. Wood, W. G., C. Bunch, S. Kelly, Y. Gunn, G. Breckon. 1985. Control of haemoglobin switching by a developmental clock?. Nature 313:320.[Medline]
  4. Friedman, R. L., S. P. Manly, M. McMahon, I. Karr, G. Stark. 1984. Transcriptional and posttranscriptional regulation of interferon-induced gene expression in human cells. Cell 38:745.[Medline]
  5. Sanderson, A., P. Beverly. 1983. Interferon, {beta}2 microglobulin and immunoselection in the pathway to malignancy. Immunol. Today 4:211.
  6. Cantley, L. C., K. R. Auger, C. Carpenter, B. Duckworth, A. Graziani, R. Kapeller, S. Soltoff. 1991. Oncogenes and signal transduction. Cell 64:281.[Medline]
  7. Decker, C. J., R. Parker. 1995. Diversity of cytoplasmic functions for the 3' untranslated region of eukaryotic transcripts. Curr. Opin. Cell Biol. 7:386.[Medline]
  8. Hood, L., M. Steinmetz, B. Malissen. 1983. Genes of the major histocompatibility complex of the mouse. Annu. Rev. Immunol. 1:529.[Medline]
  9. Bjorkman, P. J., M. A. Saper, B. Samraoui, W. S. Bennett, J. L. Strominger, D. C. Wiley. 1987. The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens. Nature 329:512.[Medline]
  10. Zinkernagel, R. M., P. C. Doherty. 1979. MHC-restricted cytotoxic T cells: studies on the biological role of polymorphic major transplantation antigens determining T-cell restriction-specificity, function, and responsiveness. Adv. Immunol. 27:51.[Medline]
  11. Bouche, G., M. Caizergues-Ferrer, B. Bugler, F. Amalric. 1984. Interrelations between the maturation of 100 kDa nucleolar protein and pre rRNA synthesis in cells. Nucleic Acids Res. 12:3025.[Abstract/Free Full Text]
  12. Chen, E., R. W. Karr, J. P. Frost, T. A. Gonwa, G. D. Ginder. 1986. {gamma} interferon and 5-azacytidine cause transcriptional elevation of class I major histocompatibility complex gene expression in K562 leukemia cells in the absence of differentiation. Mol. Cell. Biol. 6:1698.[Abstract/Free Full Text]
  13. Kimura, A., A. Israæl, O. Le Bail, P. Kourilsky. 1986. Detailed analysis of the mouse H-2kb promoter: enhancer-like sequences and their role in the regulation of class I gene expression. Cell 44:261.[Medline]
  14. Hooberman, A. L.. 1990. Molecular genetics of chronic myelogenous leukemia. Ann. Intern. Med. 113:898.
  15. Darnell, J. E., I. M. Kerr, G. R. Stark. 1994. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signalling proteins. Science 264:1415.[Abstract/Free Full Text]
  16. Reis, L. F., H. Harada, J. D. Wolchok, T. Taniguchi, J. Vilcek. 1992. Critical role of a common transcription factor, IRF-1, in the regulation of IFN-{beta} and IFN-inducible genes. EMBO J. 11:185.[Medline]
  17. Korber, B., L. Hood, L. Stroynowski. 1987. Regulation of murine class I genes by interferons is controlled by regions located both 5' and 3' to the transcriptional initiation site. Proc. Natl. Acad. Sci. USA 84:3380.[Abstract/Free Full Text]
  18. Korber, B., N. Mermod, L. Hood, I. Stroynowski. 1988. Regulation of gene expression by interferons: control of H-2 promoter responses. Science 239:1302.[Abstract/Free Full Text]
  19. Ngai, J., J. H. Stack, R. T. Moon, E. Lazarides. 1987. Regulated expression of multiple chicken erythroid membrane skeletal protein 4.1 variants is governed by differential RNA processing and translational control. Proc. Natl. Acad. Sci. USA 84:4432.[Abstract/Free Full Text]
  20. Yoshie, O., H. Schmidt, P. Lengyel, E. Reddy, W. Morgan, S. Weissman. 1984. Transcripts of human HLA gene fragments lacking the 5'-terminal region in transfected mouse cells. Proc. Natl. Acad. Sci. USA 81:649.[Abstract/Free Full Text]
  21. Chamberlain, J. W., H. A. Vasavada, S. Ganguly, S. M. Weissman. 1991. Identification of cis sequences controlling efficient position-independent tissue-specific expression of human major histocompatibility complex class I genes in transgenic mice. Mol. Cell. Biol. 11:3564.[Abstract/Free Full Text]
  22. Waring, J. F., J. E. Radford, L. J. Burns, G. D. Ginder. 1995. The human leukocyte antigen A2 interferon-stimulated response element consensus sequence binds a nuclear factor required for constitutive expression. J. Biol. Chem. 270:12276.[Abstract/Free Full Text]
  23. Hakem, R., P. Le Bouteiller, A. Jezo-Bremond, K. Harper, D. Campese, F. A. Lemonnier. 1991. Differential regulation of HLA-A3 and HLA-B7 MHC class I genes by IFN is due to two nucleotide differences in their IFN response sequences. J. Immunol. 147:2384.[Abstract]
  24. Blanar, M. A., E. C. Boettger, R. A. Flavell. 1988. Transcriptional activation of HLA-Dra by interferon {gamma} requires a trans-acting protein. Proc. Natl. Acad. Sci. USA 85:4672.[Abstract/Free Full Text]
  25. Dani, C., N. Mechti, M. Piechaczyk, B. Lebleu, P. Jeanteur, J. M. Blanchard. 1985. Increased rate of degradation of c-myc mRNA in interferon-treated Daudi cells. Proc. Natl. Acad. Sci. USA 82:4896.[Abstract/Free Full Text]
  26. Zemmour, J., P. Parham. 1991. HLA class 1 nucleotide sequences. Hum. Immunol. 31:195.[Medline]
  27. Chang, M. L., P. J. Artymiuk, X. Wu, S. Hollçn, A. Lammi, L. E. Maquat. 1993. Human triosephosphate isomerase deficiency resulting from mutation of Phe-240. Am. J. Hum. Genet. 52:1260.[Medline]
  28. Poncz, M., D. Solowiejczyk, M. Ballantine, E. Schwartz, S. Surrey. 1982. "Nonrandom" DNA sequence analysis in bacteriophage M13 by the dideoxy chain-termination method. Proc. Natl. Acad. Sci. USA 79:4298.[Abstract/Free Full Text]
  29. Sanger, F., S. Nicklen, A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463.[Abstract/Free Full Text]
  30. Burns, L. J., J. W. Waring, J. Reuter, M. F. Stinski, G. D. Ginder. 1993. Only the HLA class I gene minimal promoter elements are required for transactivation by human cytomegalovirus immediately early genes. Blood 81:1558.[Abstract/Free Full Text]
  31. Jr Radford, J. E., J. F. Waring, J. K. Pohlman, G. D. Ginder. 1993. Stimulation of MHC class I transcription by interferon-{gamma} involves a non-A, non-C kinase in addition to protein kinase C. J. Interferon Res. 13:133.[Medline]
  32. Chen, E., R. W. Karr, G. D. Ginder. 1987. Negative and positive regulation of HLA class I gene transcription in K562 leukemia cells. Mol. Cell. Biol. 7:4572.[Abstract/Free Full Text]
  33. Sood, A. K., D. Pereira, S. M. Weissman. 1981. Isolation and partial nucleotide sequence of a cDNA clone for human histocompatibility antigen HLA-B by use of an oligonucleotide primer. Proc. Natl. Acad. Sci. USA 78:616.[Abstract/Free Full Text]
  34. Koller, B. H., H. T. Orr. 1985. Cloning and complete sequence of an HLA-A2 gene: analysis of two HLA-A alleles at the nucleotide level. J. Immunol. 134:2727.[Abstract]
  35. Sugita, K., J. I. Miyazaki, E. Appella, K. Ozato. 1987. Interferons increase transcription of a major histocompatibility class I gene via a 5' interferon consensus sequence. Mol. Cell. Biol. 7:2625.[Abstract/Free Full Text]
  36. Jr Radford, J. E., E. Chen, R. Hromas, G. D. Ginder. 1991. Cell-type specificity of interferon-{gamma}-mediated HLA class I gene transcription in human hematopoietic tumor cells. Blood 77:2008.[Abstract/Free Full Text]
  37. Santamaria, P., A. L. Lindstrom, M. T. Boyce-Jacino, S. H. Myster, J. J. Barbosa, A. J. Faras, S. S. Rich. 1993. HLA class I sequence-based typing. Hum. Immunol. 37:39.[Medline]
  38. Blach-Olszewska, Z., J. Halasa, H. Matej, M. Cembrzynska-Nowak. 1977. Why HeLa cells do not produce interferon. Arch. Immunol. Ther. Exp. 25:683.
  39. Koller, B., L. J. Hagemann, T. Doetschman, J. R. Hagaman, S. Huang, P. J. Williams, N. L. First, N. Maeda, O. Smithies. 1989. Germ-line transmission of a planned alteration made in a hypoxanthine phosphoribosyltransferase gene by homologous recombination in embryonic stem cells. Proc. Natl. Acad. Sci. USA 86:8927.[Abstract/Free Full Text]
  40. Chang, C. H., J. Hammer, J. E. Loh, W. L. Fodor, R. A. Flavell. 1992. The activation of major histocompatibility complex class I genes by interferon regulatory factor-1 (IRF-1). Immunogenetics 35:378.[Medline]
  41. Pine, R., T. Decker, D. S. Kessler, D. E. Levy, Jr J. E. Darnell. 1990. Purification and cloning of interferon-stimulated gene factor 2 (ISGF2): ISGF2 (IRF-1) can bind to the promoters of both {beta} interferon- and interferfon-stimulated genes but is not a primary transcriptional activator of either. Mol. Cell. Biol. 10:2448.[Abstract/Free Full Text]
  42. Summers, C. W., V. J. Hampson, G. M. Taylor. 1993. HLA class I non-coding nucleotide sequences, 1992. Eur. J. Immunogenet. 20:201.[Medline]
  43. N'Guyen, C., R. Sodoyer, J. Trucy, T. Strachan, B. R. Jordan. 1985. The HLA-AW24 gene: sequence, surroundings and comparison with the HLA-A2 and HLA-A3 genes. Immunogenetics 21:479.[Medline]
  44. Strachan, T., R. Sodoyer, M. Damotte, B. R. Jordan. 1984. Complete nucleotide sequence of a functional class I HLA gene, HLA-A3: implications for the evolution of HLA genes. EMBO J. 3:887.[Medline]
  45. Zinszner, H., M. Masset, J. F. Bourge, J. Colombani, D. Cohen, L. Degos, P. Paul. 1990. Nucleotide sequence of the HLA-A26 class I gene: identification of specific residues and molecular mapping of public HLA class I epitopes. Hum. Immunol. 27:155.[Medline]
  46. Mitchell, T. J., M. Naughton, P. Norsworthy, K. A. Davies, M. J. Walport, B. J. Morley. 1996. IFN-{gamma} up-regulates expression of the complement components C3 and C4 by stabilization of mRNA. J. Immunol. 156:4429.[Abstract]
  47. Ohh, M., F. Takei. 1994. Interferon-{gamma}- and phorbol myristate acetate-responsive elements involved in intercellular adhesion molecule-1 mRNA stabilization. J. Biol. Chem. 269:30117.[Abstract/Free Full Text]
  48. Taylor, L. S., G. W. Cox, G. Melillo, M. C. Bosco, I. Espinoza-Delgado. 1997. Bryostatin-1 and IFN-{gamma} synergize for the expression of the inducible nitric oxide synthase gene and for nitric oxide production in murine macrophages. Cancer Res. 57:2468.[Abstract/Free Full Text]
  49. Curiel, R. E., C. S. Garcia, S. Rottschafer, M. C. Bosco, I. Espinoza-Delgado. 1999. Enhanced B7-2 gene expression by interferon-{gamma} in human monocytic cells is controlled through transcriptional and posttranscriptional mechanisms. Blood 94:1782.[Abstract/Free Full Text]
  50. Condino-Neto, A., P. E. Newburger. 2000. Interferon-{gamma} improves splicing efficiency of CYBB gene transcripts in an interferon-responsive variant of chronic granulomatous disease due to a splice site consensus region mutation. Blood 95:3548.[Abstract/Free Full Text]
  51. Tolstrup, A. B., A. Bejder, J. Fleckner, J. Justesen. 1995. Transcriptional regulation of the interferon-{gamma}-inducible triptophanyl-tRNA synthetase includes alternative splicing. J. Biol. Chem. 270:397.[Abstract/Free Full Text]
  52. Muhlemann, O., B.-G. Yue, S. Petersen-Mahrt, G. Akusjarvi. 2000. A novel type of splicing enhancer regulating adenovirus pre-mRNA splicing. Mol. Cell. Biol. 20:23317.
  53. Blencowe, B. J.. 2000. Exonic splicing enhancers: mechanism of action, diversity and role in human genetic diseases. Trends Biochem. Sci. 25:106.[Medline]
  54. Liu, H. X., M. Zhang, A. R. Krainer. 1998. Identification of functional exonic splicing enhancer motifs recognized by individual SR proteins. Genes Dev. 12:1998.[Abstract/Free Full Text]
  55. Cullen, B. R.. 2000. Nuclear RNA export pathways. Mol. Cell. Biol. 20:4181.[Free Full Text]
  56. Luo, M. J., R. Reed. 1999. Splicing is required for rapid and efficient mRNA export in metazoans. Proc. Natl. Acad. Sci. USA 96:14937.[Abstract/Free Full Text]
  57. Schmidt, H., V. Gekeler, H. Haas, G. Engler-Blum, I. Steiert, H. Probst, C. A. Muller. 1990. Differential regulation of HLA class I genes by interferon. Immunogenetics 31:245.[Medline]
  58. Gustafson, K. S., G. D. Ginder. 1996. Interferon-{gamma} induction of the human leukocyte antigen-E gene is mediated through binding of a complex containing STAT1a to a distinct interferon-{gamma}-responsive element. J. Biol. Chem. 271:20035.[Abstract/Free Full Text]



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