MHC class II enhanceosome: how is the class II transactivator recruited to DNA-bound activators?

Nabila Jabrane-Ferrat1, Nada Nekrep2,3, Giovanni Tosi2,4, Laura Esserman1 and B. Matija Peterlin2

1 Department of Surgery and Comprehensive Cancer Center, and 2 Rosalind Russell Medical Research Center, and Departments of Medicine, Microbiology and Immunology, University of California San Francisco, CA 94115-0703, USA 3 Institute of Biochemistry, Medical Faculty, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Republic of Slovenia 4 Department of Clinical and Biological Sciences, University of Insubria, Varese, Italy

Correspondence to: B. M. Peterlin, N215, UCSF Mt Zion Cancer Center, 2340 Sutter Street, San Francisco, CA 94115-0703, USA. E-mail: matija{at}itsa.ucsf.edu
Transmitting editor: L. Steinman


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgments
 References
 
MHC class II (MHCII) determinants play a crucial role in the immune response by presenting antigenic peptides to T cells. Their expression is controlled from compact promoters at the transcriptional level. Pre-assembled regulatory factor X (RFX) and nuclear factor Y (NFY) complexes form a platform on DNA. The class II transactivator (CIITA) can then be recruited through multiple protein–protein interactions. In this report, we defined domains of CIITA that are responsible for its interactions with these DNA-bound factors. Furthermore, using DNA-affinity precipitation, we demonstrated that although CIITA binds at least five activators, RFX5, RFXAP, RFXANK/B, NFYB and NFYC, its assembly on the promoter requires the addition of nuclear extracts. We conclude that not only does the platform bind DNA via multiple, spatially constrained nteractions, but that it can recruit only modified and/or complexed CIITA to MHCII promoters.

Keywords: activator, assembly, nuclear factor Y, regulatory factor X, transcription


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgments
 References
 
The expression of MHC class II (MHCII) genes is critical for the function of the immune system (1,2). Thus, the congenital lack of these determinants at the cell surface results in an autosomal and recessive severe combined immunodeficiency called the bare lymphocyte syndrome (3,4). Moreover, their inappropriate expression on target tissues facilitates organ-specific autoimmunity (5).

The expression of MHCII genes is regulated primarily from compact promoters at the transcriptional level. These include conserved upstream sequences (CUS) that occupy a stretch of 75 nucleotides. Regulatory factor X (RFX) binds S and X boxes (6), and nuclear factor Y (NFY) binds the Y box (7,8). In addition, AP1, X2BP and CREB interact with the X2 box. RFX is a complex of three subunits: RFX5, RFXANK/B (hereafter RFXANK) and RFXAP (911). NFY contains NFYA, NFYB and NFYC. RFX and NFY are ubiquitous, and their presence alone is not sufficient for the B cell-specific or IFN-{gamma}-inducible expression of MHCII determinants.

The MHCII platform starts to assemble off CUS and then binds DNA via multiple, spatially constrained interactions (6,12,13). CUS and the MHCII platform play an important role in the recruitment of the class II transactivator (CIITA) (1416). CIITA, which is not a DNA-binding protein, is the master integrator for the immune response that coordinates several steps of transcription (17,18). Unlike RFX and NFY, the expression of CIITA is highly regulated (4). Through its tight interactions with TFIID components (TAFII32 and TAFII70) and TFIIB, CIITA directs the initiation of transcription (19). Similarly, via its interaction with CREB-binding protein (CBP) (17,20), p300/CBP-associated factor (pCAF) (21), CIITA plays a role in chromatin remodeling (22). Finally, CIITA promotes steps of promoter clearance and transcriptional elongation via its interaction with the cylin T1 of the positive transcription elongation factor b (P-TEFb) (18). Despite many studies on CIITA and different factors of the MHCII platform, the assembly of a transcriptionally active enhanceosome has not been observed in vitro.

Previous reports addressed the formation of a macromolecular complex, where CIITA interacted with RFX, NFY and CREB (16), and chromatin immunoprecipitation assays co-precipitated CIITA and CUS (15). Nevertheless, these studies were all carried out in cells. Thus, the binding of CIITA to a complex rather than single proteins could not be excluded. Moreover, its interactions could be direct or via multiple protein–protein interactions. To this end, we wanted to study the binding of CIITA to individual subunits of RFX and NFY, and their roles in the recruitment of CIITA and the assembly of an active enhanceosome in a cell-free system. Indeed, CIITA also bound subunits of RFX and NFY in vitro. By deletional analyses, we mapped domains in CIITA that bind individual subunits of RFX and NFY. Even though CIITA bound five of these subunits, these interactions were not sufficient to recruit unmodified CIITA to the MHCII platform in vitro. Additionally, the complementation with nuclear extracts was required. These findings suggest that post-translational modification of CIITA or additional proteins are required for the recruitment of CIITA to MHCII promoters.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgments
 References
 
Plasmids
Plasmid target pG5bCAT and plasmid effectors RFX1VP16 and RFX5VP16 were described previously (19,23). Mammalian and bacterial expression vectors that contained cDNA for RFX5, RFXANK, RFXAP, NFYA, NFYB and NFYC as well as their corresponding glutathione-S-transferase (GST)-fusion proteins were described elsewhere (13). Wild-type Flag epitope-tagged CIITA was under the control of the cytomegalovirus promoter (fCIITA) and kindly provided by J. Ting. CIITA (1–980) was generated by deletion of CIITA C-terminal fragments after the BamHI site. CIITA (1–408), CIITA (1–630), CIITA (409–1130) and CIITA (630–1130) were generated by PCR and confirmed by DNA sequencing. CIITA (1–138), CIITA (1–321), CIITA (1–748) and CIITA (322–1130) were described elsewhere (18).

Cell culture, transient transfection and CAT assays
COS cells were maintained in DMEM supplemented with 10% (v/v) heat-inactivated FCS, 100 mM L-glutamine, and 50 µg/ml each of penicillin and streptomycin. Cells were grown in the presence of 5% (v/v) CO2 at 37°C.

A total of 5 µg of DNA was used in all transfections. Empty plasmid vector was used to keep the amount of total transfected DNA constant. Transfections were carried out with Lipofectamine reagent (Life Technologies, Grand Island, NY) as described previously (13). At 48 h post-transfection, cells were lysed and CAT activities were determined. Mean ± SEM values of two independent transfections were calculated and represented.

Coupled transcription and translation reactions using the rabbit reticulocyte lysate (RRL) in vitro
Recombinant proteins were synthesized in RRL in vitro using TnT T7-coupled transcription and translation system (Promega, Madison, WI) according to the manufacturer’s protocol. Labeled proteins were synthesized in the presence of excess [35S]cysteine or [35S]methionine (NEN Life Science, Boston, MA). Recombinant proteins were analyzed by SDS–PAGE.

Protein extracts and Western blotting
Total protein extracts were prepared from transfected COS cells as described before (12). SDS–PAGE (10 or 12%) gels were loaded with equivalent amounts of protein as determined with the Bradford assay (Bio-Rad, Hercules, CA). After electrophoresis, proteins were blotted onto nitrocellulose (Amersham Pharmacia, Arlington Heights, IL) using wet transfer. Membranes were washed, saturated with 5% dry milk and incubated with specific primary antibody for 4 h at 4°C. After extensive washes, membranes were incubated with the secondary antibody coupled to horseradish peroxidase for 1 h and proteins were visualized by the chemiluminescence assay using the ECL Plus substrate solution (NEN Life Science).

GST-fusion proteins and affinity pull-down assays
GST and GST-fusion proteins were produced as described (13). Equivalent amounts of GST or GST-fusion proteins (1 µg) were combined with specific translated proteins in vitro. Proteins were allowed to bind overnight at 4°C in 500 µl of binding buffer (50 mM Tris–HCl, pH 8.0, 5% glycerol, 0.5 mM EDTA, 5 mM MgCl2, 1% BSA, 137 mM NaCl, 1% Triton X-100, 0.5% NP-40, 1 mM dithiothreitol and 0.1 mM PMSF). After extensive washing in the presence of 250–500 mM salt, bound proteins were denatured in Laemmli buffer, resolved on SDS–PAGE and revealed by autoradiography.

DNA-affinity capture assay
Biotinylated double-stranded oligonucleotide containing sequences for S, X and Y boxes of DRA promoter was incubated with recombinant proteins synthesized in RRL. Either 5 or 10 µg of HeLa nuclear extracts, prepared as described previously (6), was added. The mixture was incubated overnight at 4°C in 500 µl of binding buffer (20 pmol of biotinylated oligonucleotide, 10 pmol of irrelevant oligonucleotide, 15 µg of sonicated denatured salmon sperm DNA and 100 µg of fraction V BSA). DNA–protein complexes were recovered by binding to streptavidin-coupled agarose beads (Life Technologies) and proteins retained on the DNA were resolved under denaturing conditions as described above.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgments
 References
 
Subunits of RFX and NFY bind CIITA in vivo
Previous studies demonstrated that CIITA associates with RFX and NFY in vivo. To confirm these interactions, CIITA was co-expressed transiently with individual subunits of RFX or NFY in COS cells. Cells were transfected with constant amounts of plasmid DNA encoding the wild-type flag epitope-tagged CIITA protein (fCIITA) and combinations of myc epitope-tagged subunits of RFX or NFY. Next, we performed co-immunoprecipitations with lysates prepared from these transfected cells. To detect CIITA, immunoprecipitations with the anti-myc antibody were followed by anti-flag Western blotting (Fig. 1A). When CIITA was expressed alone in COS cells, no interaction could be detected by Western blotting (Fig. 1A, lane 1). However, when CIITA and RFX or NFY proteins were co-expressed, CIITA was found to associate with RFX5 (Fig. 1A, lane 2), RFXANK (Fig. 1A, lane 3), RFXAP (Fig. 1A, lane 4), NFYA (Fig. 1A, lane 5), NFYB (Fig. 1A, lane 6) and NFYC (Fig. 1A, lane 7). We conclude that CIITA interacts with all subunits of RFX and NFY in vivo. Our previous work on the assembly of the MHCII platform demonstrated that RFX and/or NFY start to assemble independently of CUS in cells (13). Thus, interactions between CIITA and all subunits of RFX and NFY could reflect the binding of CIITA to pre-assembled complexes rather than individual subunits in vivo.



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Fig. 1. CIITA interacts with subunits of RFX and NFY in vivo and in vitro. A schematic representation of CIITA is diagrammed on top. Boxes depict different CIITA domains from positions 1–1130. They are the activation domain (AD) rich in aspartic and glutamic acid residues (amino acids 26–140), the proline/serine/threonine (P/S/T)-rich region (amino acids 163–319), the GTP-binding motif (GBD) (amino acids 420–561) and the LRR (amino acids 988–1086). (A) CIITA interacts with all RFX and NFY subunits in COS cells. Cells were co-transfected with plasmid DNA expressing flag epitope-tagged CIITA, and the indicated myc epitope-tagged subunits of RFX and NFY. Mammalian protein expression was directed from the cytomegalovirus promoter. Cell lysates were subjected to immunoprecipitation with anti-myc polyclonal antibody followed by Western blotting with the anti-flag mAb. Levels of expression of different proteins were determined by Western blotting on 5% of pre-cleared total cell lysates. (B) CIITA interacts with subunits of RFX and NFY in a cell-free system. GST and GST-fusion proteins were expressed in E. coli and purified by glutathione–Sepharose beads. Recombinant proteins were combined with 35S-labeled CIITA produced in RRL and allowed to interact overnight under stringent conditions. Bound proteins were separated under denaturing conditions. The arrow indicates the position of CIITA revealed by autoradiography. Input lane represents 10% of CIITA separated on 7.5% SDS–PAGE.

 
Subunits of RFX and NFY bind CIITA in vitro
To pinpoint the specific subunits of NFY and RFX that bind CIITA, we performed GST pull-down assays in vitro. GST or GST-fusion proteins were expressed in Escherichia coli. Proteins were purified with glutathione–Sepharose beads and 35S-labeled CIITA was produced in the TnT T7-coupled transcription and translation system using the RRL in the presence of [35S]cysteine and [35S]methionine in vitro. These GST or GST-fusion proteins (RFX5, RFXANK, RFXAP, NFYA, NFYB or NFYC) were incubated with the labeled CIITA. When GST alone (Fig. 1B, lane 1) or GSTNFYA chimera (Fig. 1B, lane 6) were used, no binding could be detected. However, strong interactions were detected between GSTRFX5 (Fig. 1B, lane 2), GSTRFXAP (Fig. 1B, lane 3), GSTRFXANK (Fig. 1B, lane 4), GSTNFYB (Fig. 1B, lane 5) and GSTNFYC (Fig. 1B, lane 7) fusion proteins and CIITA. We conclude that CIITA binds directly five of the six subunits of RFX and NFY.

Interactions with subunits of RFX or NFY map to the N-terminal half of CIITA
CIITA is attracted to the promoter via numerous protein–protein interactions. Since they must occur at the same time, we decided to map regions of CIITA that bind to single subunits of RFX and NFY. To this end, we expressed in RRL three different N-terminal deletion mutant CIITA proteins (Fig. 2A) and assayed them for the binding to subunits of RFX and NFY (Fig. 2). Levels of expression of different CIITA proteins are given in Fig. 2(B).



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Fig. 2. Mapping of the domains that target CIITA to the promoter. (A) Schematic representation of the N-terminal deletions. To define domains of CIITA that interact with subunits of RFX or NFY, three N-terminal mutant CIITA proteins were expressed. GST or GST-fusion proteins were combined with mutant CIITA proteins in a GST pull-down assay (C, D and E). Bound proteins were separated on SDS–PAGE and revealed by autoradiography as described in Fig. 1. The presence of proteins is indicated by the plus signs. Arrow indicates the position of CIITA fragments. All cDNAs were cloned in pCDNA3.1 and in vitro expression was directed from the T7 bacterial promoter. (B) Input represents 10% of 35S-labeled mutant CIITA proteins: CIITA (322–1130) (lane 1), CIITA (409–1130) (lane 2) and CIITA (630–1130) (lane 3). (C) Sequences from positions 1 to 321 are required for CIITA to bind NFYB. (D and E) Further deletions of CIITA revealed the role of the N-terminal half of the protein in the promoter targeting.

 
35S-Labeled N-terminal deletion mutant CIITA proteins were combined with GST or GST-fusion proteins. The mutant CIITA (322–1130) protein bound GSTNFYC (Fig. 2C, lane 2), GSTRFXAP (Fig. 2C, lane 4), GSTRFXANK (Fig. 2C, lane 5) and GSTRFX5 (Fig. 2C, lane 6) chimeras. However, no interaction was detected between the mutant CIITA (322–1130) protein and GST (Fig. 2C, lane 1) or the GSTNFYB (Fig. 2C, lane 3) chimera. We conclude that NFYB binds the first 321 N-terminal residues of CIITA that include the acidic activation (AD) and the proline, serine, threonine-rich (P/S/T) domains. Further N-terminal deletion mutant CIITA (409–1130) protein no longer supported the binding of RFXAP (Fig. 2D, lane 2) or NFYC (Fig. 2D, lane 5). However, the mutant CIITA (409–1130) protein still bound RFXANK (Fig. 2D, lane 4) and RFX5 (Fig. 2D, lane 6). When an additional 221 residues were deleted from the N-terminus of CIITA [mutant CIITA (630–1130) protein], binding to RFXANK was lost completely (Fig. 2E, lane 4) and the binding to RFX5 was diminished (Fig. 2E, lane 6). We conclude that the first half of CIITA is responsible for its binding to activators and mediates its targeting to CUS.

C-terminal deletions mapped sequences within CIITA that bind activators on CUS
To delineate further sequences of CIITA that are involved in interactions with subunits of RFX and NFY, six C-terminal deletion mutant CIITA proteins were expressed and assayed in vitro (Fig. 3A). Levels of expression are presented in Fig. 3(B).



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Fig. 3. C-terminal deletions of CIITA delineate further binding domains of CIITA. C-terminal deletion mutant CIITA proteins were expressed and used to delineate domains of CIITA that interact with subunits of RFX and NFY. CIITA fragments synthesized in vitro were combined with GST or GST-fusion proteins (C–H) as described in Fig. 2. (A) Schematic representation of mutant CIITA proteins. (C and D) Large deletions including the LRR do not affect the binding of CIITA to subunits of RFX or NFY. Mutant CIITA (1–980) (C) and CIITA (1–748) (D) proteins were used in the GST pull-down assay as described in Fig. 2. Mutant CIITA (1–630) protein was used in (E), mutant CIITA (1–408) protein in (F), mutant CIITA (1–321) protein in (G) and mutant CIITA (1–138) protein in (H). Inputs are given in (B) and correspond to 10% of various CIITA proteins separated on SDS–PAGE. Mutant CIITA (1–980), (1–748), (1–630), (1–408), (1–321) and (1–138) proteins are represented in lanes 1–6 respectively. Arrows indicate the position of CIITA.

 
Mutant CIITA proteins were combined with GST or GSTNFY and RFX fusion proteins (Fig. 3). The mutant CIITA (1–980) or CIITA (1–748) proteins retained all the binding with subunits of RFX and NFY (Fig. 3C and D). These findings confirm that the first half of CIITA is sufficient for promoter targeting. When additional 118 residues were deleted from the C-terminus of CIITA [mutant CIITA (1–630) protein] the binding between CIITA and the GSTRFX5 chimera was greatly diminished (Fig. 3E, lane 6). No binding could be detected between the mutant CIITA (1–408) protein and GSTRFXANK or GSTRFX5 chimeras (Fig. 3F, lanes 5 and 6). When the mutant CIITA (1–321) protein was used, only the binding to the GSTNFYB chimera could be detected (Fig. 3G, lane 3). The mutant CIITA (1–138) protein did not bind any of our chimeras (Fig. 3H). We conclude that the region between positions 139 and 748 of CIITA binds RFX and NFY. Thus, the N-terminal AD and the C-terminal leucine-rich repeats (LRR) are available to bind other proteins.

CIITA does not increase interactions between NFY and RFX or RFX dimers in the mammalian two-hybrid system
Since CIITA is the only limiting factor, its expression results in optimal transcription from MHCII promoters in COS cells. To determine if CIITA can increase the formation of RFX dimers or interactions between RFX and NFY we used a mammalian two-hybrid system in these cells. In this assay, the CAT reporter gene was under the control of a minimal promoter that contained five palindromic Gal4 DNA-binding sites (UAS), upstream of TATA (T) and Initiator (I) sequences (Fig. 4B). Chimeras that contained the Gal4 DNA-binding domain served as bait and bound UAS sites. Hybrid proteins that expressed the VP16 activation domains were used as prey in this system. Interactions between bait and prey were scored by the expression of the CAT reporter gene.



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Fig. 4. (A) CIITA does not facilitate interactions between NFY and RFX or RFX dimers in the mammalian two-hybrid system. COS cells were co-transfected with the plasmid reporter pG5bCAT (0.5 µg) (lanes 1–8) with or without 1 µg of the plasmid effectors encoding the hybrid GalRFX5 (lanes 1–4) or GalNFYC (lanes 5–8) proteins used as the bait. The hybrid prey protein RFX5VP16 was also expressed in lanes 3, 4, 7 and 8. CIITA was expressed from the cytomegalovirus promoter (lanes 2, 4, 6, 8 and 9). Co-expression of CIITA and pDRASCAT plasmid reporter gene (4:1 ratio) was used as the positive control (lane 9). The total amount of DNA was held constant at 5 µg. Plus signs on the top indicate the combination of plasmids transfected. Cell lysates from transfected cells were used to measure CAT activities. Fold transactivation was calculated (see Methods). Error bars represent the SEM from three different transfections. (B) Schematic representation of the mammalian two-hybrid system. pG5bCAT plasmid reporter where five Gal4 DNA-binding sites (UAS) were cloned upstream of a minimal promoter, which included the TATA box (T) and an Initiator (I), directed the expression of the CAT reporter gene (CAT) and terminated with a poly(A) signal (pA). The right side of (B) represents the association between NFY and RFX. The left side of (B) represents the RFX dimer. CIITA was co-expressed in both conditions and is represented as a homodimer.

 
We performed transient expression assays with pG5bCAT and plasmid effectors, which encoded GalRFX5, GalNFYC and RFX5VP16 chimeras in COS cells. The co-expression of CIITA and the hybrid GalRFX5 or GalNFYC proteins did not activate transcription of the CAT reporter gene (Fig.4A, cf. lanes 1 and 5 to 2 and 6). In contrast, the interaction between GalRFX5 and RFX5VP16 chimeras resulted in 6-fold increased CAT activity (Fig. 4A, cf. lanes 1 and 3). The additional co-expression of CIITA did not change this CAT activity (Fig. 4A, cf. lanes 3 and 4). Similarly, interactions between RFX and NFY translated into 15-fold increased CAT activity (Fig. 4A, cf. lanes 5 and 7). However, the additional co-expression of CIITA did not change this level of expression from pG5bCAT (Fig. 4A, cf. lanes 7 and 8). pDRASCAT, where the CAT gene was under the control of the DRA promoter, was used to control for the activity of CIITA and resulted in 19-fold increased CAT activity (Fig. 4A, lane 9). These data suggest that although CIITA binds at least five subunits of RFX and NFY, the presence of CIITA does not increase interactions between RFX dimers or RFX and NFY.

Additional steps are required for the assembly of an active MHCII enhanceosome in vitro
CIITA does not interact directly with DNA, but rather binds RFX and NFY. It is well established that CIITA is the master switch for MHCII gene transcription. While RFX and NFY occupy the promoter and play a role in its recruitment, CIITA is the only protein in the enhanceosome that activates transcription. Nevertheless, its direct physical association with DNA-bound proteins has not been demonstrated in electrophoretic mobility shift assays in vitro. However, CIITA has been shown to associate with DNA-bound factors in living cells by chromatin immunoprecipitation assays (15).

To examine whether the assembly between DNA-bound factors and CIITA can occur in a cell-free system, we developed a DNA-affinity capture assay using CUS from the DRA promoter in vitro. 35S-labeled CIITA, RFX and NFY subunits were synthesized, combined with the biotinylated SXY oligonucleotide and allowed to interact overnight. Tightly associated complexes were recovered by incubation with streptavidin–Sepharose beads. After extensive washing, proteins were resolved on SDS–PAGE. When CIITA was combined with RFX and NFY bound to DNA, CIITA was not detected (Fig. 5A, lane 2). However, when different amounts of HeLa nuclear extracts were added alone (Fig. 5A, lanes 3 and 4) or in combination with RFX and NFY (Fig. 5A, lane 5), CIITA was present in the DNA–protein complex. Since CIITA is a phospho-protein that forms dimers, we also examined the possibility that phosphorylation might be required for the recruitment of CIITA by our DNA-bound activators. However, even when phosphorylated, CIITA could not be assembled on the platform (data not shown) in the absence of nuclear extracts. We conclude that additional steps are necessary for the assembly of the enhanceosome in vitro. Nuclear extracts might cause other modifications of CIITA such as acetylation or ubiquitination that cannot occur in vitro or provide additional proteins that might be necessary for this binding.



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Fig. 5. DNA-affinity capture assay. (A) 35S-labeled CIITA, RFX and NFY proteins produced in the RRL were combined with the biotinylated SXY double-stranded oligonucleotide and allowed to interact overnight. The assembled enhanceosome was captured on streptavidin–agarose beads and after washing under stringent conditions, proteins were resolved on SDS–PAGE and revealed by autoradiography. Nuclear extracts were added to the reactions (lanes 1 and 3–6. Inputs are given in (B) and correspond to 10% of total proteins separated on SDS–PAGE.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgments
 References
 
In this study we demonstrated that NFY and RFX interact with CIITA in vivo, and their individual subunits bind the N-terminal half of CIITA in vitro. Since all individual subunits could immunoprecipitate CIITA, it is likely that complex assembly occurred in cells. In vitro, NFYA did not bind CIITA and RFXAP bound CIITA weakly. On CIITA the binding to NFYB mapped to sequences down-stream of the AD from positions 138 to 321, NFYC and RFXAP to positions 322–408, RFXANK to positions 408–630 and RFX5 to positions 630–748 (Fig. 6). Thus, subunits of NFY bound sequences in CIITA that were more proximal than those that bound RFX allowing the orientation of free N-terminal AD of CIITA in the direction of the start site of transcription (Fig. 6). The LRR in CIITA also remained free to form other inter- or intra-molecular interactions. Despite these numerous contact points, CIITA did not increase interactions between NFY and RFX or RFX dimers off DNA. Nuclear extracts had to be added for the stable association between CIITA and the MHCII platform in vitro. Thus, further post-translational modifications or other proteins are required for the assembly of the MHCII enhanceosome.



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Fig. 6. A model for the assembly of the MHCII enhanceosome. (A) Schematic representation of CIITA is diagrammed on top. Boxes depict different structural and functional CIITA domains from positions 1 to 1130. Bellow are given CIITA sequences that bind subunits of NFY and RFX: NFYB binds sequences from positions 138 to 321, NFYC and RFXAP bind between positions 322 and 408, RFXANK binds sequences from positions 408 to 630, and RFX5 binds sequences from 630 to 748. (B) The N-terminal half of CIITA binds RFX and NFY. (C) Recruitment of CIITA to MHCII promoters. CUS contain S, X and Y boxes. RFX binds S and X boxes. NFY binds to the Y box. Black circles indicate interactions between NFY and RFX or RFX dimers. Exposed regions attract CIITA to the MHCII platform.

 
We did not map sequences on subunits of NFY and RFX that bind CIITA. In part, we performed some of this mapping previously with RFXAP and RFXANK (12,24), and the mapping on RFX5 was reported elsewhere (16). Of more interest was the correlation between binding studies in vivo and in vitro, and the mapping of sequences on CIITA that participate in its recruitment to the MHCII platform. This mapping was performed with N- and C-terminal deletions of CIITA in classical GST pull-down assays. Non-overlapping sequences of CIITA bound subunits of NFY and RFX. Sequences defined in our study differed from those defined in a previously published report in vivo (16). Differences between these studies most likely reflect the formation of higher-order complexes of NFY, RFX and other host cell proteins that bind CIITA with any over-expressed subunit in cells. Thus, in vivo studies do not differentiate between direct binding, inter- or intra-molecular interactions and/or the participation of several intermediary proteins or complexes. Importantly, our mapping left the N-terminal AD and LRR domains free to bind other co-activators and/or bridging proteins. Indeed, this AD had been demonstrated to bind general transcription factors (22), co-activators such as Bob1/OBF/OCA-B (25), histone acetyltransferases CBP/p300 (17,20) and P-CAF (21) as well as P-TEFb (18). On the other end, the LRR binds a cellular protein of 33 kDa (p33) (14).

Although CIITA binds multiple subunits of NFY and RFX, it neither increases the binding between NFY and RFX nor does it affect interactions between RFX dimers off DNA. This finding argues for the sequential recruitment of CIITA by these DNA-bound activators and against the requirement of CIITA for the formation of the MHCII platform. Moreover, these individual interactions are not sufficient to retain CIITA in electrophoretic mobility shift assays or DNA-affinity approaches. Additionally, nuclear extracts are required to stabilize CIITA on NFY and RFX bound to DNA. This requirement does not reflect the phosphorylation of CIITA, but might be its acetylation and/or ubiquitylation. Indeed, although it does not increase its stability, the former modification increases the nuclear retention of CIITA (21). On the other hand, nuclear extracts could provide additional proteins, such as p33 that might stabilize the enhanceosome (14).

Taken together, our findings paint a complex picture of how platforms and enhanceosomes are formed on DNA. In the case of MHCII promoters, the MHCII platform (NFY and RFX) begins to assemble off DNA and then binds via multiple contact points on CUS, where the spacing between S, X and Y boxes is constrained. RFX dimers dictate that the spacing between S and X boxes is invariant (13). Pre-assembled complexes between NFY and RFX target MHCII promoters with high affinity and specificity. With the help of proteins that bind the X2 box and possibly the pyrimidine tract, CIITA is then attracted to the MHCII platform. Again, multiple protein–protein contacts, post-translational modifications and/or additional cellular proteins participate in this recruitment and the stabilization of the enhanceosome. Finally, CIITA binds co-activators that initiate and elongate MHCII transcription. Given the complexity of individual steps, it remains a formidable challenge to reproduce MHCII transcription in a cell-free system in vitro.


    Acknowledgments
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgments
 References
 
We thank members of the laboratory for help and critical comments on the manuscript. N. J. F. was supported by the Breast Cancer California Program (BCRP 6KB-0116). This work was supported by the Nora Eccles Treadwell Foundation.


    Abbreviations
 
CIITA—class II transactivator

CBP—CREB-binding protein

GST—glutathione-S-transferase

CUS—conserved upstream sequences

LRR—leucine-rich repeats

MHCII—MHC class II

NFY—nuclear factor Y

pCAF—p300/CBP-associated factor

P-TEFb—positive transcription elongation factor b

RRL—rabbit reticulocyte lysate

RFX—regulatory factor X


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgments
 References
 

  1. Cresswell, P. 1994. Assembly, transport, and function of MHC class II molecules. Annu. Rev. Immunol. 12:259.[CrossRef][ISI][Medline]
  2. Nelson, C. A. and Fremont, D. H. 1999. Structural principles of MHC class II antigen presentation. Rev. Immunogenet. 1:47.[Medline]
  3. DeSandro, A., Nagarajan, U. M. and Boss, J. M. 1999. The bare lymphocyte syndrome: molecular clues to the transcriptional regulation of major histocompatibility complex class II genes. Am. J. Hum. Genet. 65:279.[CrossRef][ISI][Medline]
  4. Reith, W. and Mach, B. 2001. The bare lymphocyte syndrome and the regulation of MHC expression. Annu. Rev. Immunol. 19:331.[CrossRef][ISI][Medline]
  5. Bottazzo, G. F., Pujol-Borrell, R., Hanafusa, T. and Feldmann, M. 1983. Role of aberrant HLA-DR expression and antigen presentation in induction of endocrine autoimmunity. Lancet 2:1115.[ISI][Medline]
  6. Jabrane-Ferrat, N., Fontes, J. D., Boss, J. M. and Peterlin, B. M. 1996. Complex architecture of major histocompatibility complex class II promoters: reiterated motifs and conserved protein-protein interactions. Mol. Cell. Biol. 16:4683.[Abstract]
  7. Maity, S. N. and de Crombrugghe, B. 1998. Role of the CCAAT-binding protein CBF/NF-Y in transcription. Trends Biochem. Sci. 23:174.[CrossRef][ISI][Medline]
  8. Mantovani, R. 1999. The molecular biology of the CCAAT-binding factor NF-Y. Gene 239:15.[CrossRef][ISI][Medline]
  9. Durand, B., Kobr, M., Reith, W. and Mach, B. 1994. Functional complementation of major histocompatibility complex class II regulatory mutants by the purified X-box-binding protein RFX. Mol. Cell. Biol. 14:6839.[Abstract]
  10. Masternak, K., Barras, E., Zufferey, M., Conrad, B., Corthals, G., Aebersold, R., Sanchez, J. C., Hochstrasser, D. F., Mach, B. and Reith, W. 1998. A gene encoding a novel RFX-associated transactivator is mutated in the majority of MHC class II deficiency patients. Nat. Genet. 20:273.[CrossRef][ISI][Medline]
  11. Steimle, V., Durand, B., Barras, E., Zufferey, M., Hadam, M. R., Mach, B. and Reith, W. 1995. A novel DNA-binding regulatory factor is mutated in primary MHC class II deficiency (bare lymphocyte syndrome). Genes Dev. 9:1021.[Abstract]
  12. Nekrep, N., Jabrane-Ferrat, N. and Peterlin, B. M. 2000. Mutations in the bare lymphocyte syndrome define critical steps in the assembly of the regulatory factor X complex. Mol. Cell. Biol. 20:4455.[Abstract/Free Full Text]
  13. Jabrane-Ferrat, N., Nekrep, N., Tosi, G., Esserman, L. J. and Peterlin, B. M. 2002. Major histocompatibility complex class II transcriptional platform: assembly of nuclear factor Y and regulatory factor X (RFX) on DNA requires RFX5 dimers. Mol. Cell. Biol. 22:5616.[Abstract/Free Full Text]
  14. Hake, S. B., Masternak, K., Kammerbauer, C., Janzen, C., Reith, W. and Steimle, V. 2000. CIITA leucine-rich repeats control nuclear localization, in vivo recruitment to the major histocompatibility complex (MHC) class II enhanceosome, and MHC class II gene transactivation. Mol. Cell. Biol. 20:7716.[Abstract/Free Full Text]
  15. Masternak, K., Muhlethaler-Mottet, A., Villard, J., Zufferey, M., Steimle, V. and Reith, W. 2000. CIITA is a transcriptional coactivator that is recruited to MHC class II promoters by multiple synergistic interactions with an enhanceosome complex. Genes Dev. 14:1156.[Abstract/Free Full Text]
  16. Zhu, X. S., Linhoff, M. W., Li, G., Chin, K. C., Maity, S. N. and Ting, J. P. 2000. Transcriptional scaffold: CIITA interacts with NF-Y, RFX, and CREB to cause stereospecific regulation of the class II major histocompatibility complex promoter. Mol. Cell. Biol. 20:6051.[Abstract/Free Full Text]
  17. Fontes, J. D., Kanazawa, S., Jean, D. and Peterlin, B. M. 1999. Interactions between the class II transactivator and CREB binding protein increase transcription of major histocompatibility complex class II genes. Mol. Cell. Biol. 19:941.[Abstract/Free Full Text]
  18. Kanazawa, S., Okamoto, T. and Peterlin, B. M. 2000. Tat competes with CIITA for the binding to P-TEFb and blocks the expression of MHC class II genes in HIV infection. Immunity 12:61.[ISI][Medline]
  19. Fontes, J. D., Jiang, B. and Peterlin, B. M. 1997. The class II trans-activator CIITA interacts with the TBP-associated factor TAFII32. Nucleic Acids Res. 25:2522.[Abstract/Free Full Text]
  20. Kretsovali, A., Agalioti, T., Spilianakis, C., Tzortzakaki, E., Merika, M. and Papamatheakis, J. 1998. Involvement of CREB binding protein in expression of major histocompatibility complex class II genes via interaction with the class II transactivator. Mol. Cell. Biol. 18:6777.[Abstract/Free Full Text]
  21. Spilianakis, C., Papamatheakis, J. and Kretsovali, A. 2000. Acetylation by PCAF enhances CIITA nuclear accumulation and transactivation of major histocompatibility complex class II genes. Mol. Cell. Biol. 20:8489.[Abstract/Free Full Text]
  22. Fontes, J. D., Kanazawa, S., Nekrep, N. and Peterlin, B. M. 1999. The class II transactivator CIITA is a transcriptional integrator. Microbes Infect. 1:863.[CrossRef][ISI][Medline]
  23. Ghosh, S., Selby, M. J. and Peterlin, B. M. 1993. Synergism between Tat and VP16 in trans-activation of HIV-1 LTR. J. Mol. Biol. 234:610.[CrossRef][ISI][Medline]
  24. Nekrep, N., Geyer, M., Jabrane-Ferrat, N. and Peterlin, B. M. 2001. Analysis of ankyrin repeats reveals how a single point mutation in RFXANK results in bare lymphocyte syndrome. Mol. Cell. Biol. 21:5566.[Abstract/Free Full Text]
  25. Fontes, J. D., Jabrane-Ferrat, N., Toth, C. R. and Peterlin, B. M. 1996. Binding and cooperative interactions between two B cell-specific transcriptional coactivators. J. Exp. Med. 183:2517.[Abstract]