HLA-DRA Promoter Research Articles

Blockade of endothelin-1 receptor B regulates molecules of the major histocompatibility complex in sickle cell disease.

Major Histocompatibility Complex (MHC) molecules have been proposed to play a role in Sickle Cell Disease (SCD) pathophysiology. Endothelial cells express MHC molecules following exposure to cytokines. SCD is characterized, in part, by vascular endothelial cell activation, increased oxidative stress, sickle cell adhesion, and excess levels of endothelin-1 (ET-1) contributing to vaso-occlusive crises. ET-1 activates endothelial cells, induces oxidative stress and inflammation, and alters erythrocyte volume homeostasis. However, the role of ET-1 on MHC regulation in SCD is unclear. We first studied two sickle transgenic knockout mouse models of moderate to severe disease phenotype, βS-Antilles and Berkeley (BERK) mice. We observed significant increases in H2-Aa mRNA levels in spleens, lungs, and kidneys from transgenic sickle mice when compared to transgenic knockout mice expressing human hemoglobin A (HbA). Mice treated for 14 days with ET-1 receptor antagonists significantly reduced H2-Aa mRNA levels. We characterized the effect of ET-1 on MHC class II expression in the human endothelial cell line EA.hy926. We observed dose-dependent increases in the expression of MHC class II (HLA-DRA) and MHC transcription factor (CIITA) that were significantly blocked by treatment with BQ788, a selective blocker of ET-1 type B receptors. Chromatin immunoprecipitation studies in EA.hy926 cells showed that ET-1 increased Histone H3 acetylation of the HLA-DRA promoter, an event blocked by BQ788 treatment. These results implicate ET-1 as a novel regulator of MHC class II molecules and suggest that ET-1 receptor blockade represents a promising therapeutic approach to regulate both immune and vascular responses in SCD.

Degradation, Promoter Recruitment and Transactivation Mediated by the Extreme N-Terminus of MHC Class II Transactivator CIITA Isoform III.

Multiple relationships between ubiquitin-proteasome mediated protein turnover and transcriptional activation have been well documented, but the underlying mechanisms are still poorly understood. One way to induce degradation is via ubiquitination of the N-terminal α-amino group of proteins. The major histocompatibility complex (MHC) class II transactivator CIITA is the master regulator of MHC class II gene expression and we found earlier that CIITA is a short-lived protein. Using stable and transient transfections of different CIITA constructs into HEK-293 and HeLa cell lines, we show here that the extreme N-terminal end of CIITA isoform III induces both rapid degradation and transactivation. It is essential that this sequence resides at the N-terminal end of the protein since blocking of the N-terminal end with an epitope-tag stabilizes the protein and reduces transactivation potential. The first ten amino acids of CIITA isoform III act as a portable degron and transactivation sequence when transferred as N-terminal extension to truncated CIITA constructs and are also able to destabilize a heterologous protein. The same is observed with the N-terminal ends of several known N-terminal ubiquitination substrates, such as Id2, Cdt1 and MyoD. Arginine and proline residues within the N-terminal ends contribute to rapid turnover. The N-terminal end of CIITA isoform III is responsible for efficient in vivo recruitment to the HLA-DRA promoter and increased interaction with components of the transcription machinery, such as TBP, p300, p400/Domino, the 19S ATPase S8, and the MHC-II promoter binding complex RFX. These experiments reveal a novel function of free N-terminal ends of proteins in degradation-dependent transcriptional activation.

Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Inhibits Major Histocompatibility Complex Class II Expression by Disrupting Enhanceosome Assembly through Binding with the Regulatory Factor X Complex.

Major histocompatibility complex class II (MHC-II) molecules play a central role in adaptive antiviral immunity by presenting viral peptides to CD4(+) T cells. Due to their key role in adaptive immunity, many viruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), have evolved multiple strategies to inhibit the MHC-II antigen presentation pathway. The expression of MHC-II, which is controlled mainly at the level of transcription, is strictly dependent upon the binding of the class II transactivator (CIITA) to the highly conserved promoters of all MHC-II genes. The recruitment of CIITA to MHC-II promoters requires its direct interactions with a preassembled MHC-II enhanceosome consisting of cyclic AMP response element-binding protein (CREB) and nuclear factor Y (NF-Y) complex and regulatory factor X (RFX) complex proteins. Here, we show that KSHV-encoded latency-associated nuclear antigen (LANA) disrupts the association of CIITA with the MHC-II enhanceosome by binding to the components of the RFX complex. Our data show that LANA is capable of binding to all three components of the RFX complex, RFX-associated protein (RFXAP), RFX5, and RFX-associated ankyrin-containing protein (RFXANK), in vivo but binds more strongly with the RFXAP component in in vitro binding assays. Levels of MHC-II proteins were significantly reduced in KSHV-infected as well as LANA-expressing B cells. Additionally, the expression of LANA in a luciferase promoter reporter assay showed reduced HLA-DRA promoter activity in a dose-dependent manner. Chromatin immunoprecipitation assays showed that LANA binds to the MHC-II promoter along with RFX proteins and that the overexpression of LANA disrupts the association of CIITA with the MHC-II promoter. These assays led to the conclusion that the interaction of LANA with RFX proteins interferes with the recruitment of CIITA to MHC-II promoters, resulting in an inhibition of MHC-II gene expression. Thus, the data presented here identify a novel mechanism used by KSHV to downregulate the expressions of MHC-II genes. Kaposi's sarcoma-associated herpesvirus is the causative agent of multiple human malignancies. It establishes a lifelong latent infection and persists in infected cells without being detected by the host's immune surveillance system. Only a limited number of viral proteins are expressed during latency, and these proteins play a significant role in suppressing both the innate and adaptive immunities of the host. Latency-associated nuclear antigen (LANA) is one of the major proteins expressed during latent infection. Here, we show that LANA blocks MHC-II gene expression to subvert the host immune system by disrupting the MHC-II enhanceosome through binding with RFX transcription factors. Therefore, this study identifies a novel mechanism utilized by KSHV LANA to deregulate MHC-II gene expression, which is critical for CD4(+) T cell responses in order to escape host immune surveillance.

Cohesin Regulates MHC Class II Genes through Interactions with MHC Class II Insulators

Cohesin is a multiprotein, ringed complex that is most well-known for its role in stabilizing the association of sister chromatids between S phase and M. More recently, cohesin was found to be associated with transcriptional insulators, elements that are associated with the organization of chromatin into regulatory domains. The human MHC class II (MHC-II) locus contains 10 intergenic elements, termed MHC-II insulators, which bind the transcriptional insulator protein CCCTC-binding factor. MHC-II insulators interact with each other, forming a base architecture of discrete loops and potential regulatory domains. When MHC-II genes are expressed, their proximal promoter regulatory regions reorganize to the foci established by the interacting MHC-II insulators. MHC-II insulators also bind cohesin, but the functional role of cohesin in regulating this system is not known. In this article, we show that the binding of cohesin to MHC-II insulators occurred irrespective of MHC-II expression but was required for optimal expression of the HLA-DR and HLA-DQ genes. In a DNA-dependent manner, cohesin subunits interacted with CCCTC-binding factor and the MHC-II-specific transcription factors regulatory factor X and CIITA. Intriguingly, cohesin subunits were important for DNA looping interactions between the HLA-DRA promoter region and a 5' MHC-II insulator but were not required for interactions between the MHC-II insulators themselves. This latter observation introduces cohesin as a regulator of MHC-II expression by initiating or stabilizing MHC-II promoter regulatory element interactions with the MHC-II insulator elements, events that are required for maximal MHC-II transcription.

Enhancement of immunomodulatory activity by liposome-encapsulated natural phosphodiester bond CpG-DNA in a human B cell line

Natural phosphodiester bond CpG-DNA that contains immunomodulatory CpG motifs (PO-DNA) upregulates the expression of proinflammatory cytokines and induces an Ag-driven Th1 response in a CG sequence-dependent manner in mice. In humans, only phosphorothioate backbone-modified CpG-DNA (PS-DNA) and not PO-DNA has immunomodulatory activity. In this study, we found that liposome-encapsulated PO-DNA upregulated the expression of human Beta-defensin-2 (hBD-2) and major histocompatibility class II molecules (HLA-DRA) in a CG sequence-dependent and liposome- dependent manner in human B cells. Of the three different liposomes, DOTAP has the unique ability to enhance the immunomodulatory activity of PO-DNA. In contrast, HLA-DRA and hBD-2 promoter activation can be induced by liposomeencapsulated PS-DNA in a CG sequence-independent manner, depending on the CpG-DNA species. Our observations demonstrate that, when encapsulated with a proper liposome in the immune system, natural PO-DNA has the potential to be a useful therapy for the regulation of the innate immune response.

A haplotype of the HLA-DRA gene promoter region was associated with susceptibility to gastric cancer in a Korean population (88.1)

Abstract Gastric cancer, the second leading cause of mortality by cancer in the world, shows particularly high incidence and mortality in Korea. To screen the causative genes or loci that control the transition from gastritis to gastric cancer, whole genome approach was applied using Illumina's 510S SNP chip technology. One of the interesting associated genes, HLA-DRA1 was investigated. HLA-DRA gene is very limited in polymorphism compared to the highly polymorphic HLA-DRB1, 3, 4, and 5 loci. HLA-DR alpha protein has roles as a non-polymorphic partner for HLA DR-beta for transport and surface expression as a functional HLA-class II protein. The expression level of the HLA-DR alpha/beta on the cell surface may be controlled by the transcriptional level of the HLA-DR alpha. To test this hypothesis, we tested susceptibility to gastric cancer compared to gastritis in terms of SNPs, haplotypes, and diplotypes covering the HLA-DRA promoter region. To find causative SNP(s), the whole linkage disequilibrium (LD) region of the significant haplotype using more than 20 Koreans were sequenced. We found that a diplotype composed of significantly associated SNPs and a haplotype was associated with susceptibility to gastric cancer significantly. These data suggested that the expression level of HLA-DRA might control the HLA-DR protein levels on the cell surface.

Technological advances in the study of HLA-DRA promoter regulation: Extending the functions of CIITA, Oct-1, Rb, and RFX

Several advances were established in examining the interaction of transcriptional factors with the HLA-DRA promoter. First, hydrodynamic injection was used to demonstrate the activation of the promoter by class II transactivator in a live mouse. Second, the Oct-1 DNA-binding site in the HLA-DRA promoter is a negative element in many cells, but here we show that Oct-1 activates the promoter independently of the Oct-1-binding site. Third, the retinoblastoma (Rb) protein is required for the induction of the endogenous HLA-DRA gene, due to a poorly understood, pleiotropic effect on the Oct-1 and YY1 repressive functions at the HLA-DRA promoter. There has never been an indication that direct promoter activation, by Rb, is possible. Here, we report that the first HLA-DRA intron has an Rb-responsive element, as indicated by a transient transfection/promoter reporter assay. Finally, RFX activates a methylated version of an HLA-DRA promoter reporter construct, consistent with the role of RFX in rescuing the expression of the methylated, endogenous HLA-DRA gene. Here, we report that this RFX function is not limited to a specific RFX-binding sequence or to the HLA-DRA promoter. These advances provide bases for novel investigations into the function of the major histocompatibility class II promoter.

Rescue of Major Histocompatibility-DR Surface Expression in Retinoblastoma-Defective, Non-small Cell Lung Carcinoma Cells by the MS-275 Histone Deacetylase Inhibitor

Major histocompatibility (MHC) class II expression is ordinarily inducible by interferon-gamma (IFN-gamma), but the induction is repressed in retinoblastoma protein (Rb)-defective cells. The repression can be rescued by histone deacetylase (HDAC) inhibitor treatment, but this has never been shown for an HDAC inhibitor that is suitable for clinical trials and eventual patient therapy. Here we demonstrate that the HDAC inhibitor, MS-275, can rescue the IFN-gamma inducibility of human leukocyte antigen (HLA)-DR in non-small cell lung cancer cells. This HDAC inhibitor is currently being tested in phase I/II clinical trials for non-small cell lung cancer. We further verified that the MS-275 effect is related to an HDAC tethered to the HLA-DRA promoter by the transcription factor, YY1. HDAC inhibitors that can be used to treat patients may augment the expression of tumor cell MHC class II, and the results suggest an opportunity to determine the immunological consequences of HDAC inhibitor treatment in tumor therapy.

CREB and phospho-CREB interact with RFX5 and CIITA to regulate MHC class II genes

Major histocompatibility class II (MHC-II) genes are coordinately regulated by conserved, upstream promoter elements that are bound cooperatively by cyclic AMP response element binding protein (CREB), regulatory factor X (RFX), and nuclear factor Y (NF-Y). These DNA-binding proteins serve as a scaffold for the transcriptional coactivator class II transactivator (CIITA). To determine how CREB interacts with RFX and CIITA, co-immunoprecipitations and reporter assays were performed using a variety of CREB mutants. These assays demonstrated that CREB interacted with CIITA and the RFX5 subunit of RFX through the C-terminal portion of CREB. This C-terminal portion of CREB was fully functional in MHC-II promoter reporter assays. Phosphorylation of CREB enhanced transcription from the reporter, but was not required for transcription. Phospho-CREB was found at the HLA-DRA promoter by chromatin immunoprecipitation, providing evidence for its role. Together, these data provide genetic and biochemical evidence of the specific associations between CREB and two elements of the MHC-II regulatory complex and of the role played by phosphorylated CREB at MHC-II promoters.

Activation of a Methylated Promoter Mediated by a Sequence-specific DNA-binding Protein, RFX

The roles of eukaryotic DNA methylation in the repression of mRNA transcription and in the formation of heterochromatin have been extensively elucidated over the past several years. However, the role of DNA methylation in transcriptional activation remains a mystery. In particular, it is not known whether the transcriptional activation of methylated DNA is promoter-specific, depends directly on sequence-specific DNA-binding proteins, or is facilitated by the methylation. Here we report that the sequence-specific DNA-binding protein, RFX, previously shown to mediate the transition from an inactive to an active chromatin structure, activates a methylated promoter. RFX is capable of mediating enhanceosome formation on a methylated promoter, thereby mediating a transition from a methylation-dependent repression of the promoter to a methylation-dependent activation of the promoter. These results indicate novel roles for DNA methylation and sequence-specific DNA-binding proteins in transcriptional activation.

X Box-Like Sequences in the MHC Class II Region Maintain Regulatory Function

Sequences homologous to the canonical MHC class II (MHC-II) gene X box regulatory elements were identified within the HLA-DR subregion of the human MHC and termed X box-like (XL) sequences. Several XL box sequences were found to bind the MHC class II-specific transcription factors regulatory factor X and CIITA and were transcriptionally active. The histone code associated with the XL boxes and that of the HLA-DRA X box was determined. Using CIITA-positive and -negative B cell lines, CIITA-specific histone modifications were identified and found to be consistent among the active XL boxes. Although a remarkable similarity was observed for most modifications, differences in magnitude between the HLA-DRA promoter for modifications associated with the assembly of the general transcription factors, such as histone H3 lysine 9 acetylation and H3 lysine 4 trimethylation, distinguished the very active HLA-DRA promoter from the XL box regions. In response to IFN-gamma, XL box-containing histones displayed increased acetylation, coincident with CIITA expression and that observed in B cells, suggesting that the end point mechanisms of chromatin remodeling for cell type-specific MHC-II expression were similar. Lastly, an interaction between one XL box and the HLA-DRA promoter was observed in a chromatin-looping assay. Therefore, these data provide evidence that certain XL box sequences contribute to a global increase in chromatin accessibility of the HLA-DR region in B lymphocytes and in response to IFN-gamma and supports the involvement of these XL sequences in the regulation of MHC-II genes.

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

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.

Inhibition of Major Histocompatibility Complex Class II Gene Transcription by Nitric Oxide and Antioxidants

Interferon (IFN)-gamma facilitates cellular immune response, in part, by inducing the expression of major histocompatibility complex class II (MHC-II) molecules. We demonstrate that IFN-gamma induces the expression of HLA-DRA in vascular endothelial cells via mechanisms involving reactive oxygen species. IFN-gamma-induced HLA-DRA expression was inhibited by nitric oxide (NO) and antioxidants such as superoxide dismutase, catalase, pyrrolidine dithiocarbamate, and N-acetylcysteine. Nuclear run-on assays demonstrated that NO and antioxidants inhibited IFN-gamma-induced HLA-DRA gene transcription. Transient transfection studies using a fully functional HLA-DRA promoter construct ([-300]DR alpha.CAT) showed that inhibition of endogenous NO synthase activity by N(omega)-monomethyl-l-arginine or addition of exogenous hydrogen peroxide (H(2)O(2)) augmented basal and IFN-gamma-stimulated [-300]DR alpha.CAT activity. However, H(2)O(2) and N(omega)-monomethyl-l-arginine could induce HLA-DRA expression suggesting that H(2)O(2) is a necessary but not a sufficient mediator of IFN-gamma-induced HLA-DRA expression. Electrophoretic mobility shift assay and Western blotting demonstrated that NO and antioxidants had little or no effect on IFN-gamma-induced IRF-1 activation or MHC-II transactivator (CIITA) expression but did inhibit IFN-gamma-induced activation of STAT1 alpha (p91) and Y box transcription factors, NF-Y(A) and NF-Y(B). These results indicate that NO and antioxidants may attenuate vascular inflammation by antagonizing the effects of intracellular reactive oxygen species generation by IFN-gamma, which is necessary for MHC-II gene transcription.

Histone deacetylase activity represses gamma interferon-inducible HLA-DR gene expression following the establishment of a DNase I-hypersensitive chromatin conformation.

Expression of the retinoblastoma tumor suppressor protein (Rb) is required for gamma interferon (IFN-gamma)-inducible major histocompatibility complex class II gene expression and transcriptionally productive HLA-DRA promoter occupancy in several human tumor cell lines. Treatment of these Rb-defective tumor cell lines with histone deacetylase (HDAC) inhibitors rescued IFN-gamma-inducible HLA-DRA and -DRB mRNA and cell surface protein expression, demonstrating repression of these genes by endogenous cellular HDAC activity. Additionally, Rb-defective, transcriptionally incompetent tumor cells retained the HLA-DRA promoter DNase I-hypersensitive site. Thus, HDAC-mediated repression of the HLA-DRA promoter occurs following the establishment of an apparent nucleosome-free promoter region and before transcriptionally productive occupancy of the promoter by the required transactivators. Repression of HLA-DRA promoter activation by HDAC activity likely involves a YY1 binding element located in the first exon of the HLA-DRA gene. Chromatin immunoprecipitation experiments localized YY1 to the HLA-DRA gene in Rb-defective tumor cells. Additionally, mutation of the YY1 binding site prevented repression of the promoter by HDAC1 and partially prevented activation of the promoter by trichostatin A. Mutation of the octamer element also significantly reduced the ability of HDAC1 to confer repression of inducible HLA-DRA promoter activation. Treatment of Rb-defective tumor cells with HDAC inhibitors greatly reduced the DNA binding activity of Oct-1, a repressor of inducible HLA-DRA promoter activation. These findings represent the first evidence that HDAC activity can repress IFN-gamma-inducible HLA class II gene expression and also demonstrate that HDAC activity can contribute to promoter repression following the establishment of a DNase I-hypersensitive chromatin conformation.

The Histone Acetyltransferase Domains of CREB-binding Protein (CBP) and p300/CBP-associated Factor Are Not Necessary for Cooperativity with the Class II Transactivator

The class II transactivator (CIITA) is a transcriptional co-activator regulating the constitutive and interferon-gamma-inducible expression of class II major histocompatibility complex (MHC) and related genes. Promoter remodeling occurs following CIITA induction, suggesting the involvement of chromatin remodeling factors. Transcription of numerous genes requires the histone acetyltransferase (HAT) activities of CREB-binding protein (CBP), p300, and/or p300/CBP-associated factor (pCAF). These co-activators cooperate with CIITA and are hypothesized to promote class II major histocompatibility complex transcription through their HAT activity. To directly test this, we used HAT-defective CBP and pCAF. We demonstrate that cooperation between CIITA and CBP is independent of CBP HAT activity. Further, although pCAF enhances CIITA-mediated transcription, pCAF HAT domain dependence appears contingent upon the concentration of available CIITA. When HAT-defective CBP and pCAF are both present, cooperativity with CIITA is maintained. Consistent with a recent report, we show that nuclear localization of CIITA is enhanced by lysine 144, an in vitro target of pCAF-mediated HAT. Yet we find that neither mutation of lysine 144 nor deletion of residues 132-209 affects transcriptional cooperation with CBP or pCAF. Thus, acetylation of this residue may not be the primary mechanism for pCAF/CBP cooperation with CIITA. In conclusion, the HAT activities of the co-activators are not necessary for cooperation with CIITA.

CIITA coordinates multiple histone acetylation modifications at the HLA-DRA promoter.

We present here an in vivo view of major histocompatibility complex (MHC) class II promoter assembly, nucleosome modifications and gene expression mediated by the class II transactivator (CIITA). Acetylation and deacetylation of histones H3 and H4 at the HLA-DRA promoter were found to occur during a time-course that depended on CIITA expression and binding. Expression of a CIITA mutant, which lacked the activation domain, induced H4 but not H3 histone acetylation. This suggested that multiple histone acetyltransferase activities are associated with MHC class II expression. H4 acetylation was mapped to Lys8, which implicated several histone acetyltransferases as possible modulators of this activity.

Targeted transgene expression using the human HLA-DRA promoter in a retroviral vector

Targeted transgene expression using the human HLA-DRA promoter in a retroviral vector

HLA-DMB gene and HLA-DRA promoter region polymorphisms in Australian multiple sclerosis patients

The MHC region has been shown to contain a susceptibility locus for multiple sclerosis (MS). While the strongest association to date has been between HLA-DRB1∗1501 and MS, the exact nature of the MHC association in MS remains unclear. Two candidate polymorphic loci within the MHC class II region, the HLA-DMB gene and the HLA-DRA promoter, which lie close to HLA-DRB1, were therefore examined in an Australian MS population. The HLA-DMB∗0103 phenotype was increased in the MS patients (46% vs. 30%) and the frequency of the HLA-DRA promoter A allele was also increased (81% vs. 68%). When the subjects were stratified into HLA-DRB1∗1501 positive and negative individuals these associations were not significantly different. This is a result of the strong linkage disequilibrium between HLA-DRB1∗1501 and both HLA-DMB∗0103 and the HLA-DRA promoter A allele. The complete linkage between DRB1∗1501 and the HLA-DRA promoter A allele indicates that the MS susceptibility haplotype (DRB1∗1501-HLA-DQB1∗0602-HLA-DQA1∗0102) can be extended out to promoter of the HLA-DRA locus. Interactions between both HLA-DMB and the HLA-DRA promoter and other reported MS susceptibility loci were examined (TCRBV polymorphisms, HLA-DQA1 and HLA-DQB1). Some interactions between specific TCRBV polymorphisms and the HLA-DRA promoter were observed, which is consistent with other published reports suggesting an epistatic interaction between TCRBV and HLA-DRB1.

Retinoblastoma protein expression facilitates chromatin remodeling at the HLA-DRA promoter.

The major histocompatibility complex (MHC) class II genes encode a series of heterodimeric cell surface glycoproteins that bind peptide antigen. The MHC class II/peptide complex is bound by the T-cell receptor of CD4(+) T cells, thereby stimulating an immune response. The MHC class II genes are coordinately regulated by conserved promoter elements and are inducible by IFN-gamma. Furthermore, IFN-gamma induction of the MHC class II genes in solid human tumor lines requires retinoblastoma protein (Rb). In vivo footprinting analyses of the HLA-DRA gene, which encodes the heavy chain subunit of the human MHC class II molecule, HLA-DR, revealed that Rb facilitates occupancy of multiple HLA-DRA promoter elements. Detecting the effect of Rb on HLA-DRA promoter occupancy in vivo required IFN-gamma treatment. However, use of a variation on the in vivo footprinting technique, nuclei footprinting, which assays for promoter occupancy in isolated nuclei, revealed that expression of Rb facilitates promoter occupancy even in the absence of IFN-gamma. These results indicate that expression of Rb leads to modification of the chromatin environment of the HLA-DRA promoter independently of transcription.

HLA-DRA promoter polymorphism and diversity generation within the immune system.

Multiple major histocompatibility complex (MHC) alleles exist at most class I and II loci. Polymorphism of MHC polypeptides may reflect either different levels of selective pressure operating on each molecule or different mutation rates at different loci. To gain further insight into this issue, we sequenced the non-coding promoter region of the HLA-DRA gene from several Epstein-Barr virus-transformed B cell lines and compared the extent of polymorphism found in this region with the known polymorphism of the HLA-DQB promoter. Our results indicate that the HLA-DRA promoter displays a low level of polymorphism while the promoter of HLA-DQB exhibits a nucleotide substitution rate fivefold greater than that of DRA. Moreover, through phylogenetic analysis, the HLA-DRA promoter was found to have diverged much less than the associated alleles of HLA-DRB1 and -DQA1. Taken together, these results suggest that the HLA-DRA promoter is highly conserved and may be under a stronger functional constraint than the promoter regions of other MHC class II genes.