Major histocompatibility complex (MHC) class II molecules are heterodimeric cell surface glycoproteins comprised of both a heavy (alpha) chain and a light (beta) chain. MHC class II molecules (HLA-DR, -DP, and -DQ in humans) bind and display peptide antigens for recognition by CD4+
T lymphocytes. Recognition of the MHC class II heterodimer-antigen complex by the T-cell receptor and the accessory protein CD4 of T lymphocytes leads to the generation of an immune response. MHC class II molecules play an important role in antitumor immunity (1
). Specifically, transfection of tumor cells with syngeneic murine MHC class II genes immunizes mice against MHC class II-negative parental tumor cells (2
). Vaccination of mice using this protocol also leads to eradication of an MHC class II-negative, basement membrane-invasive tumor (4
). Also, tumor-specific antigens capable of eliciting HLA class II-restricted activation of tumor-infiltrating T lymphocytes have been identified (46
MHC class II expression is constitutively activated during development in professional antigen-presenting cells, such as B cells, dendritic cells, and macrophages; it is inducible by cytokines, most importantly, gamma interferon (IFN-γ), in nearly all other types of cells. MHC class II expression is regulated primarily at the level of transcription through promoter elements that are conserved among the MHC class II genes and the genes encoding accessory molecules such as the invariant chain, the MHC class II chaperone. The elements are, from 5′ to 3′, S box, X1 box, X2 box, Y box, and TATA box. The transactivators RFX, X2BP (CREB), and NF-Y are required factors for MHC class II gene activation and bind the X1, X2, and Y boxes, respectively. Cooperative interactions between transactivators bound to the X and Y elements have been demonstrated to be essential for the establishment of promoter occupancy and the transcription of class II genes (60
). In particular, binding of the Y box factor, NF-Y, has been demonstrated to be required for occupancy of the other promoter elements and for IFN-γ-inducible MHC class II gene expression (60
). In addition to the promoter binding factors, the class II transactivator (CIITA) is a required coactivator that functions by interaction with and stabilization of the transcription factors previously assembled on MHC class II promoters (20
It has been shown that the retinoblastoma tumor suppressor protein (Rb) is also required for IFN-γ-inducible MHC class II gene expression (34
). Several Rb-defective human tumor cell lines exhibit a loss of IFN-γ-inducible MHC class II gene expression that is rescued by the reexpression of functional Rb (34
). Rb-defective tumor cell lines exhibit significantly reduced or complete loss of promoter occupancy at all of the known transactivator binding sites within the HLA-DRA promoter, as detected by in vivo footprinting (41
). The expression of exogenous Rb results in increased occupancy at these promoter elements, and this effect of Rb is independent of IFN-γ-mediated transcriptional activation (41
). Thus, Rb apparently relieves a block to efficient, transcriptionally productive transcription factor assembly at the HLA-DRA promoter. There is also significantly reduced or absent promoter occupancy in cells from patients with bare lymphocyte syndrome (BLS), where RFX is defective or missing (26
). In BLS cells, the HLA-DRA promoter DNase I-hypersensitive site is absent (17
), indicating a close association of nucleosomes with promoter DNA.
In this report, we demonstrate that the HLA-DRA promoter retains the DNase I-hypersensitive site in non-IFN-γ-inducible, Rb-defective tumor cells. This observation separates the formation of the hypersensitive site and presumably a nucleosome-free promoter region from the transcriptional competency of the promoter. The distinction between the lack of the DNase I-hypersensitive site and the lack of transcriptionally productive transactivator binding establishes two stable levels of repression of the HLA-DRA promoter in situ. While histone deacetylase (HDAC) activity is generally accepted as mediating repression by nucleosomes, its role in other levels of promoter repression is unknown. We were interested in determining whether HDAC activity could be involved in maintaining repression following the partial derepression of chromatin that is represented by the establishment of the promoter DNase I-hypersensitive site. Here we show that the lack of HLA-DR expression in Rb-defective tumor cells was due to the repression of HLA-DRA and -DRB promoter activation by HDAC activity. We also show that the repression of HLA-DRA promoter activation by HDAC activity is facilitated by YY1 and octamer elements. The approach of mapping HDAC function to distinct states of chromatin has the potential of identifying specific target proteins that mediate stable, intermediate states of promoter repression.