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Originally published In Press as doi:10.1074/jbc.M403118200 on April 27, 2004 Originally published In Press as doi:10.1074/jbc.M403118200 on April 22, 2004

J. Biol. Chem., Vol. 279, Issue 28, 28911-28919, July 9, 2004
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Oct-1 Maintains an Intermediate, Stable State of HLA-DRA Promoter Repression in Rb-defective Cells

AN Oct-1-CONTAINING REPRESSOSOME THAT PREVENTS NF-Y BINDING TO THE HLA-DRA PROMOTER*

Aaron R. Osborne{ddagger}, Hongquan Zhang§, Gyorgy Fejer{ddagger}||, Kimberly M. Palubin{ddagger}, Melissa I. Niesen{ddagger}, and George Blanck{ddagger}§**{ddagger}{ddagger}

From the Departments of {ddagger}Biochemistry and Molecular Biology and §Pathology and Laboratory Medicine, College of Medicine, University of South Florida and the **Immunology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612

Received for publication, March 19, 2004 , and in revised form, April 19, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cell surface HLA-DR molecule binds foreign peptide antigen and forms an intercellular complex with the T cell receptor in the course of the development of an immune response against or immune tolerance to the antigen represented by the bound peptide. The HLA-DR molecule also functions as a receptor that mediates cell signaling pathways, including as yet poorly characterized pathway(s) leading to apoptosis. Expression of HLA-DR mRNA and protein is ordinarily inducible by interferon-{gamma} but is not inducible in tumor cells defective for the retinoblastoma tumor suppressor protein (Rb). In the case of the HLA-DRA gene, which encodes the HLA-DR heavy chain, previous work has indicated that this loss of inducibility is attributable to Oct-1 binding to the HLA-DRA promoter. In this report, we used Oct-1 antisense transformants to determine that Oct-1 represses the interferon-{gamma} response of the endogenous HLA-DRA gene. This determination is consistent with results from a chromatin immunoprecipitation assay, indicating that Oct-1 occupies the endogenous HLA-DRA promoter when the HLA-DRA promoter is inactive in Rb-defective cells but not when the promoter is converted to a previously defined, transcriptionally competent state, induced by treatment of the Rb-defective cells with the HDAC inhibitor, trichostatin A. In vitro DNA-protein binding analyses indicated that Oct-1 prevents HLA-DRA promoter activation by mediating the formation of a complex of proteins, termed DRAN (DRA negative), that blocks NF-Y access to the promoter.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Major histocompatibility complex (MHC)1 class II proteins are involved in cellular immune responses directed against exogenously derived peptide antigens. MHC class II proteins (HLA-DR, -DP, and -DQ in humans) present these peptide antigens to CD4+ T cells for interaction with the T cell receptor. Interaction of the MHC class II-peptide/T cell receptor-CD4 complex results in T cell activation and secretion of various cytokines and can lead to the elicitation of a strong immune response. Aberrant expression or the loss of expression of MHC class II proteins results in a variety of autoimmune and immunodeficiency diseases. Additionally, MHC class II proteins play an important role in anti-tumor immunity, and tumor-specific antigens capable of eliciting HLA class II-restricted activation of tumor infiltrating lymphocytes have been identified (1–5).

Constitutive MHC class II gene expression occurs primarily in professional antigen presenting cells such as B cells, macrophages, monocytes, and dendritic cells. However, MHC class II gene expression is inducible by cytokines such as interferon-{gamma} (IFN-{gamma}) and tumor necrosis factor-{alpha} in nearly all cells types. Regulation of MHC class II expression is controlled primarily at the level of transcription and is strictly dependent both quantitatively and temporally by the expression of the MHC class II transactivator (CIITA) (6–8).

Transcription of MHC class II genes is also dependent on the binding of RFX, CREB, and NF-Y to the X1, X2, and Y promoter elements, respectively (9–11). However, binding of these proteins to their cognate elements in MHC class II promoters is insufficient for the transcriptional activation of MHC class II genes. Recruitment of the non-DNA-binding co-activator, CIITA, to MHC class II promoters through interaction with RFX and NF-Y is essential for promoter activation (12–16). Prior to recruitment of CIITA, both RFX and NF-Y occupy the MHC class II promoter DNA (17–24); however, upon interaction with CIITA, their binding to the promoter is greatly enhanced (16, 23, 27). Because of its essential role in the activation of MHC class II transcription, the complex of MHC class II sequence-specific transacting factors and CIITA has been termed the MHC enhanceosome (16).

As noted, normal cells possess a chromatin environment at MHC class II promoters that is apparently nucleosome-free and accessible to binding by sequence-specific transcription factors. However, the MHC class II promoter is inaccessible to the promoter-binding transactivators in cells derived from patients with a form of bare lymphocyte syndrome, where RFX is defective, and in in vitro derived B cell mutants, where RFX is defective (17, 28).

We and others have demonstrated that the retinoblastoma tumor suppressor protein (Rb) is required for IFN-{gamma}-inducible MHC class II gene expression in human tumor cells and in mouse embryo fibroblasts lacking Rb (21, 29–31). In human tumor cells that lack functional Rb protein, occupancy of promoter elements within the HLA-DRA promoter is greatly reduced or eliminated entirely (21). However, unlike cells that are defective for RFX, the HLA-DRA promoter is not associated with nucleosomes, i.e. is accessible to DNase I (32). This distinction defines a stable, repressed state of chromatin intermediate to the formation of promoter chromatin accessible to NF-Y and to the other sequence-specific transactivators. This state of repression involves the interaction of the repressors Oct-1 and YY1 with the HLA-DRA promoter (32).

YY1 associates with histone deacetylase (HDAC) activities such as HDAC1/2 (33, 34), and treatment of Rb-defective tumor cells with HDAC inhibitors rescues expression of HLA-DRA and -DRB mRNA and cell surface protein expression (32). Overexpression of HDAC1 leads to a pronounced reduction in IFN-{gamma}-inducible HLA-DRA promoter activation that can be relieved by mutation of the YY1-binding site (32).

The way in which Oct-1 functions to repress HLA-DRA expression and contribute to the maintenance of the intermediate state of HLA-DRA promoter repression remains to be determined. For example, the HLA-DRA Oct-1-binding site does not overlap any of the binding sites for required transactivators such as RFX and NF-Y, and Oct-1 is not known to physically interact with HDACs. It is known that Rb-defective cells have a high level of Oct-1 DNA binding activity compared with Rb-transformed cells (32, 35). Furthermore, Oct-1 is known to be hypophosphorylated in Rb-defective cells, and phosphorylation of Oct-1 has been demonstrated to reduce its ability to bind DNA (35). Treatment of these Rb-defective tumor cells with HDAC inhibitors leads to a pronounced and specific reduction in Oct-1 DNA binding activity, although it is not known whether Oct-1 is acetylated or whether the reduction of Oct-1 DNA binding activity is a direct or indirect effect of the HDAC inhibitors (32).

In this report we demonstrate that Oct-1 interacted with the endogenous HLA-DRA promoter in Rb-defective tumor cells and that treatment of these cells with HDAC inhibitors prevented this interaction. Additionally, using Rb-defective tumor cells that have been stably transformed with an expression vector encoding Oct-1 antisense mRNA, we demonstrate that Oct-1 repressed HLA-DRA expression in vivo. Furthermore, using EMSAs, we identified a multi-protein "repressosome" complex that contains Oct-1, interacts with the HLA-DRA promoter Oct-1-binding site, is disrupted by Rb transformation and HDAC inhibitor treatment, and prevents binding of NF-Y with the HLA-DRA promoter in vitro. Accordingly, Oct-1 may maintain stable repression of the HLA-DRA promoter by facilitating the assembly of this repressosome.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Tissue Culture—1A4 and 12-27 are subclones of the human bladder carcinoma cell line, 5637 (ATCC HTB9) (30), and MDA-468-S4 (S4) and MDA-468-MTRB1 (Rb1) are subclones of the human breast carcinoma cell line MDA-468 (36). 12-27 and Rb1 cells are transformed with an Rb expression vector and the G418 resistance gene. The 1A4 cells are transformed only with the G418 resistance gene, and the S4 cells are the parent of Rb1. 5637 (HTB-9)-derived cell lines were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 units of penicillin-streptomycin/milliliter, 3 mM L-glutamine, and 1 mM sodium pyruvate. MDA-468-derived cell lines were grown in Dulbecco's modified Eagle's medium/Ham's F-12 medium supplemented with 10% calf serum, 100 units of penicillin-streptomycin/milliliter, and 3 mM L-glutamine. The cells were maintained at 37 °C and 5% CO2 in a humidified tissue culture incubator.

Chromatin Immunoprecipitation Assay—5637 cells on 100-mm plates were treated with IFN-{gamma} and TSA separately and in combination as described (32) and as indicated in the figures. The cells were treated with 1% formaldehyde for 10 min at room temperature, and the reaction was stopped by adding glycine to 0.125 M. The cells were harvested by scraping and then washed twice in PBS at 4 °C, followed by resuspension in 1 ml of sample buffer (100 mM KCl, 50 mM NaCl, 5 mM MgCl2, 10 mM Tris-HCl, pH 7.4) at 4 °C. The cells were lysed by the addition of 0.1 volume of 10% Igepal for 5 min on ice. The nuclei were collected by centrifugation and then lysed in 1 volume of nuclear suspension buffer (100 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 0.1% Igepal) by the addition of 0.1% volume of 20% Sarkosyl. The nuclear lysates were sonicated, and the chromatin-cross-linked protein fraction was separated from free proteins by CsCl ultracentrifugation. The DNA-containing fractions were pooled and dialyzed against three changes of PBS and kept frozen at –80 °C in aliquots. Immunoprecipitated chromatin was precleared with salmon sperm DNA, and protein bovine serum albumin-blocked, A/G beads before overnight chromatin immunoprecipitations were done using equal amounts of purified chromatin incubated with different antibodies (anti-Oct-1 from Santa Cruz and control normal rabbit IgG) in a buffer containing 0.1% SDS, 1% Triton X-100, 0.2 mg/ml sonicated salmon sperm DNA, and 0.2 mg/ml yeast tRNA and collected with protein A/G beads (Santa Cruz) blocked with 0.4 mg/ml salmon sperm DNA and 1 mg/ml bovine serum albumin. The beads were washed with low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl), LiCl detergent buffer (0.25 M LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1), and Tris-EDTA buffer. DNA was eluted with 0.1 M NaHCO3 containing 1% SDS, and the cross-links were reversed by heating the samples at 65 °C overnight. The immunoprecipitated DNA was purified by proteinase K treatment, phenol/chloroform:isoamyl-alcohol extraction, and ethanol precipitation. DNA was also purified from a small portion of the chromatin mixture before the addition of the antibody (unselected DNA), which served as a positive control for the subsequent PCR analysis. Primers used for amplification of the HLA-DRA promoter region were: 5'-CTAGCACAGGGACTCCACTTATG-3' (exon 1) and 5'-AACCCTTCCCCTAGCAACAGAT-3' (promoter)

Nuclear Extract Preparation—Crude nuclear extracts were prepared as previously described (35). Briefly, cultured cells were washed three times with 10 ml of cold 1x PBS and once with 3 ml of hypotonic buffer (20 mM HEPES, pH 7.9, 1 mM EDTA, 1 mM EGTA, 20 mM NaF) and 300 µl of lysis buffer (20 mM HEPES, pH 7.9, 1 mM EDTA, 1 mM EGTA, 20 mM NaF, 1 mM Na3VO4, 1 mM Na4P2O7, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 0.2% Igepal, 0.5 µg/ml leupeptin, 2 µg/ml aprotinin, and 50 µg/ml antipain) to each 100-mm tissue culture plate. The lysed cells were collected by scraping, incubated on ice for 10 min, and centrifuged at 8,400 x g for 30 s at 4 °C to pellet the nuclei. Following centrifugation, the supernatant was removed, and the pelleted nuclei were resuspended in 100 µl of high-salt buffer (420 mM NaCl, 20 mM HEPES, pH 7.9, 1 mM EDTA, 1 mM EGTA, 20 mM NaF, 1 mM Na3VO4, 1 mM Na4P2O7, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, 2 µg/ml aprotinin, and 50 µg/ml antipain). The resuspended nuclei were placed on a rotating stand and incubated at 4 °C for 1 h to extract the nuclear proteins. The samples were centrifuged at 8,400 x g for 1 min to pellet the nuclei. The supernatants were removed by pipetting, transferred to fresh tubes, and stored at –70 °C until required. The concentration of total protein in the supernatant was performed using a BCA assay kit according to the vendor's (Pierce) instructions.

In Vitro Transcription and Translation—Oct-1 was generated by using pBS-Oct-1 (gift of Winship Herr) as a template for in vitro transcription and subsequent translation using the TNT-coupled in vitro transcription and translation kit (Promega). NF-Y proteins (NF-YA, NF-YB, and NF-YC) were generated by using pCITE-2a-CBF-A, pCITE-2b-CBF-B, and pCITE-2a-CBF-C, respectively (27, 28) as templates for in vitro transcription and translation using the TNT-coupled in vitro transcription and translation kit. Briefly, to generate each protein, 1 µg of its template plasmid was added to a reaction mixture containing 25 µl of TNT rabbit reticulocyte lysate, 2 µl of TNT reaction buffer, 1 µl of TNT T7 RNA polymerase, 0.5 µl of 1 mM amino acid mixture minus leucine, 0.5 µl of 1 mM amino acid mixture minus methionine, and 40 units of RNasin ribonuclease inhibitor in a total reaction volume of 50 µl. The reaction mixture was incubated at 30 °C for 90 min and stored at –70 °C until required. Prior to use in EMSA (see below), equal volumes of the in vitro transcription and translation reactions for the three NF-Y subunits (NF-YA, NF-YB, and NF-YC) were mixed.

Electrophoretic Mobility Shift Assay—EMSAs were performed by the procedure of Yu et al. (37), except that all of the binding reactions were performed at room temperature for 30 min. The –62/–37 HLA-DRA Oct-1-binding site probe and competitor oligonucleotides have been described previously (35). The HLA-DRA Y-box competitor oligonucleotides have been described previously (35). The –176/+45 HLA-DRA promoter fragment probe was generated by digestion of pUC18DRA with EcoRI and XbaI. Several other HLA-DRA promoter fragments used as competitors were also generated by restriction endonuclease digestion of pUC18DRA (see Fig. 4A).



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  		FIG. 4.
DRAN interacts with multiple regions of the HLA-DRA promoter and contains Oct-1. A, schematic diagram indicating the various HLA-DRA promoter fragments used as competitors in EMSAs and the restriction endonucleases used to generated them. B, EMSA was performed using nuclear protein extracts from 5637 cells and labeled –176/+45 HLA-DRA promoter fragment probe. EMSAs were separated by electrophoresis on a 5% polyacrylamide gel. Lane 1, labeled probe only. Lanes 2–10 represent nuclear extract from 5637 cells. Unlabeled competitor HLA-DRA promoter fragments at 5x and 20x molar excess relative to the probe were added to each reaction as follows: –176/+45 (lanes 3 and 4), –176/SspI (lanes 5 and 6), –176/Tsp509I (lanes 7 and 8), and Tsp509I/+45 (lanes 9 and 10). The slowly migrating DRAN complex is indicated. C, EMSA was performed using nuclear protein extracts from 5637 cells and labeled –176/+45 HLA-DRA promoter fragment probe in the presence of isotype matched antibodies. Lanes 1 and 2, control antibody (Ab) specific for the interferon consensus sequence-binding protein (ICSBP), a protein not expected to interact with the HLA-DRA promoter. Lanes 3 and 4, control antibody specific for c-Fos, another protein not expected to interact with the HLA-DRA promoter. Lanes 5 and 6, antibody specific for Oct-1. D, EMSA was performed using either nuclear protein extract from 5637 cells or in vitro translated Oct-1 protein in the presence of the labeled –176/+45 HLA-DRA promoter fragment probe. Lane 1, labeled probe only. Lanes 2 and 3, 1 and 2 µl, respectively, of in vitro translated Oct-1 and labeled probe. Lane 4, nuclear protein extract and labeled probe.

 
The –176/+45 HLA-DRA promoter fragment and the –62/–37 HLA-DRA octamer element probe were labeled with [{alpha}-32P]dATP or [{alpha}-32P]dCTP by the Klenow fill-in procedure. Antibody supershift reactions were performed by including the indicated amounts of anti-NF-Y(A) (Rockland Immunochemicals, Gilbertsville, PA), anti-Oct-1, anti-c-Fos, and anti-interferon consensus sequence-binding protein antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) to the appropriate EMSA binding reactions. All of the EMSAs were separated by 4–5% (DRAN) and 7% (Oct-1) polyacrylamide gels at 12 V/cm for ~3 h in 0.25x Tris-borate-EDTA running buffer.

Western Blots—Fifteen µg of nuclear protein extracts electrophoresed on 4–15% Tris-HCl, SDS-PAGE gels (Bio-Rad) at 100 volts for 1.5 h. The proteins were transferred to polyvinylidene difluoride membranes by wet transfer at 100 V for 1 h and then blocked in PBSM (1x PBS containing 3% (w/v) nonfat dry milk) for 25 min at room temperature with mild agitation. After blocking, the membranes were rinsed two times in deionized water. The membranes were incubated overnight at 4 °C with mild agitation in PBSM containing a 1:1000 dilution of either anti-Oct-1 (Santa Cruz) or anti-NF-YA antibodies (Rockland). The membranes were washed twice with deionized water and incubated at room temperature for 1 h with mild agitation in PBSM containing a 1:3000 dilution of goat-anti-rabbit IgG conjugated to horseradish peroxidase. The membranes were then washed once in PBST (1x PBS containing 0.02% (v/v) Tween 20) followed by four washes in deionized water. The proteins were visualized using an ECL kit according to the vendor's (Amersham Biosciences) instructions.

Plasmid Construction—cDNA encoding full-length Oct-1 was obtained by HindIII and BamHI digestion of the pCG-Oct-1 vector followed by gel purification of the Oct-1 cDNA insert. The pcDNA3 expression vector was also digested with HindIII and BamHI, treated with calf-intestinal alkaline phosphatase, and gel-purified. To generate the pcDNA3-AS-Oct-1 vector, the digested Oct-1 cDNA insert and pcDNA3 vector were ligated using T4 DNA ligase, and the ligation products were used to transform JM109 Escherichia coli cells.

Generation of Stably Transformed Cells Expressing Oct-1 Antisense mRNA—5637 cells were seeded onto 100-mm tissue culture dishes at a density of 1.0 x 106 cells/dish and incubated overnight at 37 °C. Twenty-four hours post-seeding, the cells were transfected with either the empty pcDNA3 vector or the recombinant pcDNA3-AS-Oct-1 expression vector using the cationic lipid reagent, TransIT-LT1 (Panvera), according to the vendor's instructions. Twenty-four hours post-transfection, the 5637 cells were detached from the tissue culture dishes and diluted 1:10,000 onto fresh 100-mm dishes in complete medium supplemented with 500 µg/ml G418. This concentration of G418 efficiently killed all cells on a mock transfected control plate. The transfected cells were maintained in G418 for approximately 2 weeks, at which time individual colonies were isolated and transferred to separate tissue culture vessels for further expansion of the clonal cultures. The details of the phenotype of the Oct-1 antisense transformants will constitute a separate report.

Isolation of Total mRNA and Reverse Transcriptase-PCR—Total cytoplasmic RNA was prepared by the Nonidet P-40 lysis method as previously described (38). Where indicated, each sample was treated with 400 units/ml IFN-{gamma} for 48 h prior to harvesting total cytoplasmic RNA. Five micrograms of total cytoplasmic RNA from each sample was primed using random hexameric primers (Invitrogen) and reverse transcribed with Superscript II reverse transcriptase according to the vendor's (Invitrogen) instructions. PCR was performed for each sample in a 50-µl reaction mixture using 5 µl of reverse transcription reaction product, 5 µl of 10x PCR assay buffer B (Fisher), 1.5 mM MgCl2, 5% Me2SO, 10 pmol of DRA, DRB, or {gamma}-actin-specific primers (39), 0.2 mM dNTP, and 1 unit of Taq polymerase (Fisher). Each sample was incubated successively at 95 °C for 30 s, 53.5 °C for 30 s, and 72 °C for 45 s for a total of 30 (DRA and DRB) or 25 ({gamma}-actin) cycles, followed by a final extension at 72 °C for 5 min. The PCR products were visualized on a 2.2% agarose gel containing ethidium bromide.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
HDAC Inhibitor Treatment Leads to a Reduction of Oct-1 Associated with the Endogenous HLA-DRA Promoter—As noted in the Introduction, evidence indicates that Oct-1 binding to the HLA-DRA promoter prevents HLA-DRA mRNA inducibility in Rb-defective cells (32), but this model has never been tested for the endogenous HLA-DRA gene. HLA-DRA inducibility is rescued by treatment of Rb-defective human tumor cells with the HDAC inhibitors sodium butyrate and TSA (32) more efficiently than by exogenous Rb expression. Treatment of Rb-defective cells with the HDAC inhibitors also eliminates the Oct-1 DNA binding activity from nuclear extracts prepared from the treated cells far more efficiently than does exogenous expression of Rb (32). Therefore, we performed an Oct-1 chromatin immunoprecipitation assay to determine whether Oct-1 was present at the promoter of the endogenous HLA-DRA gene and to determine whether TSA treatment abolished the Oct-1/HLA-DRA promoter interaction. HLA-DRA promoter sequences were immunoprecipitated by the Oct-1 antibody but not by control antibody (Fig. 1). However, no detectable HLA-DRA promoter sequences were immunoprecipitated in 5637 cells treated with TSA (Fig. 1), a treatment that establishes the transcriptionally competent state (see Fig. 7) of the HLA-DRA promoter in these cells (32). Thus, although the mechanism of the TSA effect leading to reduced DNA binding activity of Oct-1 is unknown, this well established TSA effect as detected by in vitro experiments is also relevant to the endogenous HLA-DRA promoter. The presence of Oct-1 at the endogenous promoter correlates with the noninducible state of the endogenous promoter. Removal of Oct-1 from the endogenous promoter, in this case by TSA treatment, correlates with reacquisition of the inducibility of the endogenous HLA-DRA gene.



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  		FIG. 1.
Oct-1 is present at the HLA-DRA promoter in vivo. Prior to harvesting chromatin, the Rb-defective bladder carcinoma cell line, 5637, was treated with 400 units/ml IFN-{gamma}, 200 nM TSA, or both as indicated in the figure. Chromatin immunoprecipitation was performed using either an Oct-1-specific antibody (Ab) or an isotype-matched control antibody. PCR specific for the HLA-DRA promoter was then performed on each sample using either 2 or 6 µl of the immunoprecipitate as a template. No DNA represents a negative control PCR in which water was substituted for immunoprecipitated chromatin as a template. Input DNA represents a positive control PCR using 1% of the total chromatin introduced in each immunoprecipitation reaction as a template.

 



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  		FIG. 7.
Proposed model for HLA-DRA promoter chromatin transitioning through two distinct states of chromatin and repression by the DRAN repressosome. The basis of this model was originally proposed in Ref. 32. The model is refined here to take into account DRAN, the dependence of DRAN on Oct-1, and the specific effect of DRAN in preventing NF-Y access to the HLA-DRA promoter.

 
Expression of Oct-1 Antisense mRNA Results in Increased HLA-DR mRNA Expression Following Treatment of Cells with IFN-{gamma}—To determine whether Oct-1 can repress endogenous HLA-DRA promoter activation, we transformed the Rb-defective 5637 cells with an expression vector that encodes full-length Oct-1 antisense mRNA. Several Oct-1 antisense 5637 clones expressing reduced levels of Oct-1 protein relative to control, G418-resistant transformants, were identified by Western blotting (Fig. 2A) and confirmed by immunohistochemistry (data not shown). To determine whether the Oct-1 antisense transformants had increased HLA-DRA mRNA expression in response to IFN-{gamma}, we performed reverse transcriptase-PCR for HLA-DRA using total RNA isolated from an Oct-1 antisense transformant, termed A1, and a control, G418-resistant transformant, C1. The A1 cells exhibited a 2.2–3.1-fold increase in the HLA-DRA response to IFN-{gamma} treatment relative to the C1 cells in two experiments, respectively (Fig. 2, B–D). Also, we assayed for IFN-{gamma}-induced HLA-DRA mRNA by real time PCR (Fig. 2E), which indicated that the A1 cells had about a 2.7-fold greater response than did the C1 cells, consistent with Fig. 2 (B–D). Finally, we assayed for HLA-DRA promoter luciferase activity in the A1 and C1 cells, respectively, as described in Ref. 32. The results of this experiment (Fig. 2F) are consistent with an increased level of HLA-DRA promoter activity in the A1 cells. In sum, these observations are consistent with Oct-1 maintaining repression of the HLA-DRA promoter in Rb-defective human tumor cells.



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  		FIG. 2.
HLA-DRA mRNA expression is increased in permanently transformed 5637 cells expressing Oct-1 antisense mRNA. A, Western analysis of Oct-1 expression in control vector transformed cells (C1 and C2) and the Oct-1 antisense transformed cells, A1 (AS1 in this panel). B, Rb-defective bladder carcinoma 5637 cells were permanently transformed with a mammalian expression vector encoding full-length Oct-1 antisense cDNA or a control expression vector. RNA was harvested from the control vector transformed cells (C1) and the Oct-1 antisense transformed cells (A1) following treatment of the cells where indicated with 400 units/ml of IFN-{gamma} for 48 h. Template cDNA was generated by reverse transcription, and the samples were analyzed by PCR using primers specific for the HLA-DRA, HLA-DRB, and actin coding sequences. The potential role of Oct-1 in the repression of the HLA-DRB gene is unknown. C, each bar represents the relative ratio of the intensity of the HLA-DRA or -DRB amplification product from the indicated cells when treated with IFN-{gamma} over the intensity of the amplification product from the same cells when left untreated. This ratio was arbitrarily set to 1.0 for the C1 control cells, and the value for the Oct-1 antisense A1 cells is shown relative to this value. D, quantification of a separate, independent experiment with identical treatment conditions. E, real time PCR results of an assay for HLA-DRA mRNA in IFN-{gamma} treated C1 and A1 cells normalized to 18S rRNA. F, transient transfection of an HLA-DRA luciferase reporter construct, described in Ref. 32, into C1 and A1 cells.

 
Identification of a Novel HLA-DRA Promoter-specific Complex in Rb-defective, IFN-{gamma} Noninducible Tumor Cells—Rescue of IFN-{gamma}-inducible HLA-DRA gene expression in Rb-defective tumor cells is at least partly attributable to a reduction in Oct-1 binding following reconstitution of these cells with functional Rb (32, 35), HDAC inhibitor treatment (32), or expression of Oct-1 antisense mRNA (Fig. 2). Furthermore, we have shown that the endogenous HLA-DRA promoter in Rb-defective cells is not occupied by the positive acting promoter-binding proteins, either with or without IFN-{gamma} treatment (21). Because NF-Y facilitates the binding of RFX and CREB to the HLA-DRA promoter (20), at least in the absence of CIITA expression (40), the unoccupied promoter of the Rb-defective cells likely reflects the lack of NF-Y access. However, the HLA-DRA promoter Oct-1-binding element does not overlap any of the binding sites required for the transcriptional activators. The closest positive element is the NF-Y-binding site (Y-element), which is separated from the Oct-1-binding site by 14 nucleotides. To resolve this paradox, we considered that the loss of Rb expression in these human tumor cells leads to the formation of a higher order complex involving a region of the HLA-DRA promoter that is larger than the basic Oct-1-binding site.

We performed an EMSA using a probe spanning the HLA-DRA promoter from positions –175 to +45 relative to the start site of transcription (Fig. 3A) and encompassing all known binding sites for transacting factors, including the W-, X1-, X2-, and Y-elements and the Oct-1-binding site (Fig. 3A; see also Fig. 7). Using extracts from Rb-defective, non-IFN-{gamma}-inducible, bladder and breast carcinoma cells (Fig. 3B, lanes 2 and 4), we observed a prominent protein complex, termed DRAN (HLA-DRA negative). The DRAN-probe complex migrated significantly slower than the probe-protein complex observed when using extracts from Rb-transformed, IFN-{gamma}-inducible, cells (Fig. 3B, lanes 3 and 5).



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  		FIG. 3.
Identification of the DRAN complex. A, schematic diagram of the –176/+45 HLA-DRA promoter fragment used as a probe in EMSAs. B, nuclear protein extracts were isolated from Rb-defective and Rb-transformed breast carcinoma cells (MDA-468-S4 and MDA-468-MTRB1) as well as Rb-defective and Rb-transformed bladder carcinoma (5637 and 1227) cells. EMSA was performed using 3 µg of nuclear protein and 25 fmol of labeled –176/+45 HLA-DRA promoter fragment per reaction. Lane 1, labeled probe only. Lanes 2 and 3, labeled probe and nuclear protein extract, respectively, from breast carcinoma cells. Lanes 4 and 5, labeled probe and nuclear protein extract, respectively, from bladder carcinoma cells. The slowly migrating complex in lanes 2 and 4 will be referred to as DRAN henceforth because these cells are noninducible for HLA-DR mRNA expression by IFN-{gamma}. C, EMSA was performed using nuclear protein extracts from 5637 cells and the labeled –176/+45 HLA-DRA promoter fragment probe. EMSAs were separated by electrophoresis on a 5% polyacrylamide gel. Lane 1, labeled probe only. Lane 2, nuclear protein extract and labeled probe. Lanes 3 and 4, nuclear protein extract and labeled probe in the presence of 20x and 80x, respectively, molar excess of unlabeled –176/+45 HLA-DRA promoter fragment. Lanes 5 and 6, nuclear protein extract and labeled probe in the presence of 20x and 80x, respectively, molar excess of unlabeled DNA representing a nucleosome positioning sequence.

 
Detection of the DRAN-probe complex was prevented by including 20- and 80-fold molar excess of nonradioactive –176/+45 HLA-DRA promoter sequences in the DRAN-probe binding reaction (Fig. 3C, compare lanes 2-4). However, a 20- or 80-fold molar excess of an unrelated DNA fragment of about the same length as the –176/+45 HLA-DRA probe was unable to compete with the HLA-DRA probe in formation of the DRAN complex (Fig. 3C, lanes 5 and 6).

DRAN Interacts with the Oct-1-binding Site and Other Promoter Sequences—To identify specific regions within the HLA-DRA promoter that facilitate formation of the DRAN-probe complex, we used restriction endonuclease digestion fragments, representing portions of the HLA-DRA promoter as competitor DNA in ESMA experiments (Fig. 4A). The –176/SspI fragment spanning the W- and X-elements was unable to prevent formation of the DRAN complex (Fig. 4B, lanes 5 and 6). This suggests that the W- and X-element sequences, as well as the factors known to interact with them, are not required for formation of the DRAN-probe complex. However, the –176/Tsp509I fragment, including the Y-element, competed with the full-length radioactive –176/+45 probe (Fig. 4B, lanes 7 and 8), although the –176/Tsp509I fragment was not as efficient as the full-length –176/+45 probe at preventing formation of the DRAN complex. Additionally, the Tsp509I/+45 competitor fragment, encompassing only the Oct-1 binding site and down-stream sequences, which do not include any known sites required for HLA-DRA expression or HLA-DRA repression, was able to prevent formation of the DRAN complex (Fig. 4B, lanes 9 and 10). However, like the –176/Tsp509I fragment, the Tsp509I/+45 competitor fragment was not as efficient as the full-length –176/+45 competitor fragment at disrupting the DRAN complex (Fig. 4B, lanes 3 and 4). Finally, a 30-base pair synthetic oligonucleotide spanning the HLA-DRA Oct-1-binding site was also able to prevent formation of the DRAN complex. However, a 200-fold molar excess of this oligonucleotide is required for efficient disruption of the DRAN-probe complex (data not shown). In sum, these observations suggest that DRAN contacts the HLA-DRA promoter at or near the Oct-1-binding site and Y-element and that these interactions may be cooperative, which would explain the reduced binding efficiency of competitor DNAs that have fewer DRAN-binding sites. The W- and X-elements are apparently not required for interaction of the DRAN complex with the HLA-DRA promoter.

DRAN Is Comprised of Oct-1 and Additional Factors— Within the regions of the HLA-DRA promoter that interact with DRAN, only the Y-element and the Oct-1-binding site have been shown to interact with proteins. To attempt to identify components of the DRAN complex, we performed EMSAs with antibody specific for Oct-1, which was capable of decreasing the mobility of the DRAN complex (Fig. 4C, lanes 5 and 6).

To verify that DRAN is not comprised solely of one Oct-1 molecule, we compared the mobility of in vitro translated Oct-1 protein complexed with the –176/+45 probe to the DRAN complex in an EMSA. As expected, the in vitro translated Oct-1-probe complex migrated much more rapidly than did the DRAN complex (Fig. 4D, compare lanes 2 and 4). Furthermore, we were unable to reconstitute the DRAN complex by adding increased amounts of in vitro translated Oct-1 to the binding reactions with the –176/+45 probe (Fig. 4D, compare lanes 3 and 4).

HDAC Inhibitor Treatment of Rb-defective Tumor Cells Prevents Formation of the DRAN Complex and Allows for Interaction of NF-Y with the HLA-DRA Promoter—The DRAN complex can be formed using nuclear extracts from the Rb-defective, noninducible, 5637 cells but not when using extracts from their Rb-transformed, transcriptionally competent subclones. Also, extracts from cells treated with the TSA or sodium butyrate HDAC inhibitors have no Oct-1 DNA binding activity. To verify the apparent requirement of Oct-1 DNA binding activity for the formation of the DRAN-probe complex, we took advantage of the HDAC inhibitor function that leads to elimination of Oct-1 DNA binding activity (32). We performed an EMSA with the full-length –176/+45 HLA-DRA promoter probe and with extracts from cells exposed to concentrations of sodium butyrate (1 mM) and TSA (50, 100, and 200 nM) that eliminate Oct-1 binding activity and that rescue endogenous HLA-DRA mRNA and protein expression (32). The HDAC treatments efficiently prevented DRAN-probe complex formation (Fig. 5, lanes 3 and 6), consistent with the role of Oct-1 in the formation of DRAN. Furthermore, the lack of the DRAN complex was associated with the appearance of a more rapidly migrating, HLA-DRA promoter probe complex (Fig. 5, lanes 3 and 6, labeled A).



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  		FIG. 5.
Treatment of 5637 cells with HDAC inhibitors results in disruption of DRAN/HLA-DRA promoter probe complex. Rb-defective human bladder carcinoma cells, 5637, were either left untreated or were treated with the HDAC inhibitors sodium butyrate (1 mM) or increasing amounts of TSA (50, 100, and 200 nm) as indicated. EMSAs were performed using 3 µg of nuclear protein extracts from 5637 cells and 25 fmol of labeled –176/+46 HLA-DRA promoter probe. Lane 1, labeled probe with no nuclear protein extract. Lanes 2 and 3, probe and extract from untreated and sodium butyrate-treated (1 mM) cells, respectively. Lanes 4–6, probe and extract from TSA-treated (50, 100, and 200 nM, respectively) cells. The slowly migrating probe-protein complex (lanes 2, 4, and 5) represents DRAN, whereas the identity of the most rapidly migrating probe-protein complex (indicated as A, lanes 3 and 6) is established in Fig. 6.

 
Because treatment of Rb-defective tumor cells with HDAC inhibitors leads to restoration of IFN-{gamma} inducible HLA-DRA expression, we hypothesized that this rapidly migrating complex (Fig. 5, labeled A) represented NF-Y, which is the initial requirement for a transcriptionally competent promoter (20), particularly in the absence of CIITA (40). Accordingly, we performed an EMSA using extracts from sodium butyrate-treated cells as were used in Fig. 5. A competitor oligonucleotide encompassing the HLA-DRA Y-element prevented formation of complex A with the probe (Fig. 6, lanes 4–6), and the NF-Y(A)-specific antibody resulted in a decrease in mobility of complex A (Fig. 6, lane 7). Finally, in vitro translated NF-Y(A, B, and C) resulted in a complex with mobility similar to complex A (Fig. 6, lane 1). Thus, complex A is identified as NF-Y.



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  		FIG. 6.
HDAC inhibitors disrupt DRAN and allow for interaction of NF-Y with the HLA-DRA promoter. An EMSA was performed using the same labeled –176/+45 HLA-DRA promoter probe and the same nuclear protein extracts as were used in figure 5 under identical assay conditions. The extracts used were from 5637 cells left untreated or treated with sodium butyrate (1 mM) as indicated in the figure. Lane 1, labeled probe only. Lanes 3 and 4, labeled probe and nuclear protein extracts from untreated and sodium butyrate-treated cells, respectively. Lanes 4–6, labeled probe and nuclear protein extracts in the presence of increasing concentrations (10x, 50x, and 100x molar excesses, respectively) of an unlabeled competitor HLA-DRA promoter fragment spanning the Y-box. Lane 7, labeled probe and nuclear protein extract in the presence of an antibody specific for NF-Y. Lanes 8 and 9, labeled probe and nuclear protein extract in the presence of a 100x molar excess of unlabeled competitor HLA-DRA promoter fragments spanning the octamer element (as indicated, these promoter fragments were either wild-type or mutated at the octamer element). Lane 2, labeled probe and equal amounts of the in vitro translated NF-Y(A), NF-Y(B), and NF-Y(C) subunits.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previous experiments have indicated that transiently transfected HLA-DRA promoter constructs are repressed by Oct-1 DNA binding activity. The current data indicate that this is also true for the endogenous HLA-DRA promoter. Interestingly, Ting and co-workers (22) have recently reported that TSA treatment of E1A transformed, 293 cells (in the absence of IFN-{gamma} and CIITA) resulted in a pronounced increase in promoter-associated NF-Y and RFX. Because E1A effectively renders cells Rb-defective by binding to Rb, the TSA treatment of the 293 cells likely prevents Oct-1 binding to the HLA-DRA promoter and thereby facilitates the loading of NF-Y and RFX onto the promoter.

The mechanism of the repression of the promoter by Oct-1 is unknown and is particularly perplexing because the Oct-1-binding site does not overlap with any known HLA-DRA promoter element, likely ruling out a simple blockage of the activator sites by Oct-1. This type of activator site blockage does appear to function in the interleukin-8 promoter (41), which, like the MHC class II promoters, also functions at a low level in Rb-defective cells but at a high level in Rb transformants, where the Oct-1 DNA binding activity is highly reduced (35).

Although the mechanism of the Oct-1-mediated repression of the HLA-DRA promoter is unknown, the data above indicate the existence of a higher order DRAN complex that is only detectable when Oct-1 DNA binding activity is high and that prevents NF-Y binding in vitro. Note that TSA treatment was used to eliminate DRAN and thus to permit NF-Y interaction with the HLA-DRA promoter probe. Fig. 3 indicates that DRAN cannot be formed when using extracts from Rb-transformed cells, and as with TSA treatment, a complex of reduced sized is present. However, because this complex is less well defined than the NF-Y complex (complex A) of Figs. 5 and 6, we were not able to make a determination that this complex (i.e. complex A of the Rb-transformed cells) represents NF-Y bound to the HLA-DRA promoter probe. These results are consistent with the differing efficiencies of Rb expression and HDAC inhibitor treatment, respectively, with regard to rescue of HLA-DRA and HLA-DRB mRNA induction. The HDAC inhibitors appear to be much more efficient (32), and thus the clear replacement of DRAN with NF-Y would be expected. In the case of the extracts prepared from the Rb transformants (Fig. 3), DRAN binding to the HLA-DRA promoter may not be completely abolished, and NF-Y binding activity in the extracts from the Rb transformants may not be as extensive as it is in the case of the extracts prepared from the cells treated with the HDAC inhibitors.

If future work supports the existence of this complex in vivo, the Oct-1-dependent formation of DRAN may represent a mechanism for the repression of the endogenous HLA-DRA gene in the Rb-defective cells. In this scenario, NF-Y binding would be prevented by DRAN, and because NF-Y binding is required for formation of the remainder of the MHC class II enhanceosome, there would be no HLA-DRA promoter activation when the promoter is bound to DRAN (Fig. 7).

Oct-1 repression in Rb-defective cells occurs despite the presence of the canonical HLA-DRA promoter DNase I-hypersensitive site, previously described for IFN-{gamma}-inducible cells (28, 32). Work by others has indicated that this hypersensitive site forms as a result of RFX expression (28), consistent with the relatively high affinity of RFX for nucleosomal versus naked DNA (42). Furthermore, NF-Y, which is composed of histone-like motifs, also facilitates RFX binding to the HLA-DRA promoter (42). Thus, it is likely that RFX destabilizes an HLA-DRA promoter nucleosome that then permits Oct-1-mediated repression of the promoter. If there is no Oct-1 DNA binding activity, then NF-Y facilitates a stable association of RFX with the HLA-DRA promoter, setting the stage for formation of an enhanceosome stabilized by IFN-{gamma}-induced CIITA (Fig. 7).

We also note that Oct-1 mediates (apparently) intermediate stages of repression for other promoters where NF-Y functions as an activator and where the NF-Y site does not overlap the Oct-1-binding site (25, 26, 43), raising the question of whether DRAN may mediate the repression of multiple promoters?


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant R01 CA81497 (to G. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Present address: Dept. of Pathology, Harper University Hospital, Wayne State University, Detroit, MI 48201. Back

|| Present address: Veterinary Medical Research Institute, Hungarian Academy of Sciences, Budapest, Hungary. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of South Florida College of Medicine, MDC Box 7, 12901 Bruce B. Downs Blvd., Tampa, FL 33612. Tel.: 813-974-9585; Fax: 813-974-7280; E-mail: gblanck{at}hsc.usf.edu.

1 The abbreviations used are: MHC, major histocompatibility complex; IFN, interferon; TSA, trichostatin A; CIITA, MHC class II transactivator; HDAC, histone deacetylase; PBS, phosphate-buffered saline; EMSA, electrophoretic mobility shift assay. Back


   ACKNOWLEDGMENTS
 
The real time PCR data were obtained with the assistance of Mary Beth Colter and the Molecular Biology Core facility at the H. Lee Moffitt Cancer Center and Research Institute.



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