Knockdown of the insulator factor CCCTC binding factor (CTCF), which binds XL9, an intergenic element located between HLA-DRB1 and HLA-DQA1, was found to diminish expression of these genes. The mechanism involved interactions between CTCF and class II transactivator (CIITA), the master regulator of major histocompatibility complex class II (MHC-II) gene expression, and the formation of long-distance chromatin loops between XL9 and the proximal promoter regions of these MHC-II genes. The interactions were inducible and dependent on the activity of CIITA, regulatory factor X, and CTCF. RNA fluorescence in situ hybridizations show that both genes can be expressed simultaneously from the same chromosome. Collectively, the results suggest a model whereby both HLA-DRB1 and HLA-DQA1 loci can interact simultaneously with XL9, and describe a new regulatory mechanism for these MHC-II genes involving the alteration of the general chromatin conformation of the region and their regulation by CTCF.

Programmed cell death-1 (PD-1) is a crucial negative regulator of CD8 T cell development and function, yet the mechanisms that control its expression are not fully understood. Through a non-biased DNase I hypersensitivity assay, four novel regulatory regions within the Pdcd1 locus were identified. Two of these elements flank the locus, bind the transcriptional insulator protein CTCF, and interacted with each other, creating a potential regulatory compartmentalization of the locus. In response to T cell activation signaling, NFATc1 bound to two of the novel regions that function as independent regulatory elements. STAT binding sites were identified in these elements as well. In splenic CD8 T cells, TCR-induced PD-1 expression was augmented by interleukin 6 and 12, inducers of STAT3 and STAT4 activity, respectively. IL-6 or IL-12 on its own did not induce PD-1. Importantly, STAT3/4 and distinct chromatin modifications were associated with the novel regulatory regions following cytokine stimulation. The NFATc1/STAT regulatory regions were found to interact with the promoter region of the Pdcd1 gene providing a mechanism for their action. Together these data add multiple novel distal regulatory regions and pathways to the control of PD-1 expression and provide a molecular mechanism by which proinflammatory cytokines, such as IL-6 or IL-12 can augment PD-1 expression.

The major histocompatibility complex class II (MHC-II) locus includes a dense cluster of genes that function to initiate immune responses. Expression of insulator CCCTC binding factor (CTCF) was found to be required for expression of all MHC class II genes associated with antigen presentation. Ten CTCF sites that divide the MHC-II locus into apparent evolutionary domains were identified. To define the role of CTCF in mediating regulation of the MHC II genes, chromatin conformation capture assays, which provide an architectural assessment of a locus, were conducted across the MHC-II region. Depending on whether MHC-II genes and the class II transactivator (CIITA) were being expressed, two CTCF-dependent chromatin architectural states, each with multiple configurations and interactions, were observed. These states included the ability to express MHC-II gene promoter regions to interact with nearby CTCF sites and CTCF sites to interact with each other. Thus, CTCF organizes the MHC-II locus into a novel basal architecture of interacting foci and loop structures that rearranges in the presence of CIITA. Disruption of the rearranged states eradicated expression, suggesting that the formation of these structures is key to coregulation of MHC-II genes and the locus.Spanning nearly 700 kb of the short arm of chromosome 6 at 6p21.31, the major histocompatibility complex class II (MHC-II) locus contains a dense cluster of highly polymorphic genes that encode the alpha and beta chains of the classical MHC-II heterodimeric molecules (reviewed in references 8, 21, and 59). Three MHC-II isotypes, HLA-DR, -DQ, and -DP, can be formed, and they present antigenic peptides to CD4 T lymphocytes in order to initiate, control, and/or maintain adaptive immune responses (20). This antigen presentation process is aided by two MHC-II region-encoded molecules, HLA-DM and HLA-DO, which are also alpha/beta heterodimers with sequence and structural homology with MHC-II proteins (55, 64). The locus includes several MHC-II homologous pseudogenes that represent defunct duplications of ancient MHC-II genes (29). Also within the locus are five genes that do not bear structural homology to MHC-II proteins, the TAP1 and TAP2 genes involved in peptide transport for MHC-I antigen presentation, the proteasome 20s core beta subunit genes PSMB8 and PSMB9, and the bromodomain and extra terminal domain transcriptional regulator gene BRD2 (25,38,67).MHC-II genes are expressed in B lymphocytes, macrophages, and dendritic cells and certain cells within the thymus. Gamma interferon (IFN-␥) is a potent inducer of MHC-II gene expression in most cell types (12). The transcription factors RFX (regulatory factor X), CREB (cyclic AMP [cAMP] response element binding protein), and NF-Y (nuclear factor Y) are constitutively and ubiquitously expressed and bind to highly conserved MHC-II gene-and related gene promoterproximal sequences termed the X1, X2, and Y boxes (9, 51, 59). CIITA (class II transactivator) interacts with the X-Y box-bound factors and mediates the i...

The intrinsic enhancer–promoter specificity and chromatin boundary/insulator function are two general mechanisms that govern enhancer trafficking in complex genetic loci. They have been shown to contribute to gene regulation in the homeotic gene complexes from fly to mouse. The regulatory region of the Scr gene in the Drosophila Antennapedia complex is interrupted by the neighboring ftz transcription unit, yet both genes are specifically activated by their respective enhancers from such juxtaposed positions. We identified a novel insulator, SF1, in the Scr–ftz intergenic region that restricts promoter selection by the ftz‐distal enhancer in transgenic embryos. The enhancer‐blocking activity of the full‐length SF1, observed in both embryo and adult, is orientation‐ and enhancer‐independent. The core region of the insulator, which contains a cluster of GAGA sites essential for its activity, is highly conserved among other Drosophila species. SF1 may be a member of a conserved family of chromatin boundaries/insulators in the HOM/Hox complexes and may facilitate the independent regulation of the neighboring Scr and ftz genes, by insulating the evolutionarily mobile ftz transcription unit.

The human major histocompatibility complex class II (MHC-II) region encodes a cluster of polymorphic heterodimeric glycoproteins HLA-DR, -DQ, and -DP that functions in antigen presentation. Separated by ϳ44 kb of DNA, the HLA-DRB1 and HLA-DQA1 encode MHC-II proteins that function in separate MHC-II heterodimers and are diametrically transcribed. A region of high acetylation located in the intergenic sequences between HLA-DRB1 and HLA-DQA1 was discovered and termed XL9. The peak of acetylation coincided with sequences that bound the insulator protein CCCTC-binding factor as determined by chromatin immunoprecipitations and in vitro DNA binding studies. XL9 was also found to be associated with the nuclear matrix. The activity of the XL9 region was examined and found to be a potent enhancer-blocking element. These results suggest that the XL9 region may have evolved to separate the transcriptional units of the HLA-DR and HLA-DQ genes.The human major histocompatibility complex (MHC) 3 encodes a dense cluster of genes that spans almost 4 megabases of human chromosome 6 (1). The MHC is divided into the following three regions: class I, II, and III. Many of the genes encoded in these subregions function in adaptive and innate immunity. The MHC-II locus consists of a group of 7-10 highly polymorphic genes that code for the ␣ and ␤ chains of the classical MHC-II heterodimeric molecules (reviewed in Ref. 2). In total, three MHC class II isotypes HLA-DR, HLA-DQ, and HLA-DP can be formed. MHC class II molecules function by presenting antigenic peptides to CD4ϩ T lymphocytes and are critical to the development of the T cell repertoire and the proliferation and differentiation of antigen-specific CD4 T cells during adaptive immune responses (3, 4). This process is aided by two MHC class II-associated molecules, HLA-DM and -DO, which are also ␣/␤ heterodimers with sequence and structural homology to MHC-II proteins (5, 6).MHC-II genes are regulated in a cell type-specific manner and are constitutively expressed in B lymphocytes, macrophages, dendritic cells, and thymic epithelia (reviewed in Refs. 2 and 7). However, in response to interferon-␥, most other cell types can be induced to express MHC class II genes. Regulation of MHC class II genes is coordinated by a group of conserved sequence elements termed the W/Z, X1, X2, and Y boxes, located at a promoter proximal region upstream of all MHC-II genes. The factors RFX, CREB, and NF-Y bind cooperatively to the X1-X2-Y box sequences, respectively, but are not sufficient for gene expression (8 -10). Expression requires the class II transactivator, CIITA, a non-DNA binding co-activator (11). CIITA mediates interactions between the DNA-bound X-Y box factors, chromatin remodeling machinery, additional co-activators, and various components of the general transcription machinery to allow for MHC class II transcription (2, 7).Despite the requirement of the W-X-Y box conserved sequences for MHC class II gene expression, a number of observations suggest that other regulatory components ...