The Murine MHC Class II Super Enhancer IA/IE-SE Contains a Functionally Redundant CTCF-Binding Component and a Novel Element Critical for Maximal Expression.

In both humans and mice, CTCF-binding elements form a series of interacting loops across the MHC class II (MHC-II) locus, and CTCF is required for maximal MHC-II gene expression. In humans, a CTCF-bound chromatin insulator termed XL9 and a super enhancer (SE) DR/DQ-SE situated in the intergenic region between HLA-DRB1 and HLA-DQA1 play critical roles in regulating MHC-II expression. In this study, we identify a similar SE, termed IA/IE-SE, located between H2-Eb1 and H2-Aa of the mouse that contains a CTCF site (C15) and a novel region of high histone H3K27 acetylation. A genetic knockout of C15 was created and its role on MHC-II expression tested on immune cells. We found that C15 deletion did not alter MHC-II expression in B cells, macrophages, and macrophages treated with IFN-γ because of functional redundancy of the remaining MHC-II CTCF sites. Surprisingly, embryonic fibroblasts derived from C15-deleted mice failed to induce MHC-II gene expression in response to IFN-γ, suggesting that at least in this developmental lineage, C15 was required. Examination of the three-dimensional interactions with C15 and the H2-Eb1 and H2-Aa promoters identified interactions within the novel region of high histone acetylation within the IA/IE-SE (termed N1) that contains a PU.1 binding site. CRISPR/Cas9 deletion of N1 altered chromatin interactions across the locus and resulted in reduced MHC-II expression. Together, these data demonstrate the functional redundancy of the MHC-II CTCF elements and identify a functionally conserved SE that is critical for maximal expression of MHC-II genes.

PD-1 Expression during Acute Infection Is Repressed through an LSD1-Blimp-1 Axis.

During prolonged exposure to Ags, such as chronic viral infections, sustained TCR signaling can result in T cell exhaustion mediated in part by expression of programmed cell death-1 (PD-1) encoded by the Pdcd1 gene. In this study, dynamic changes in histone H3K4 modifications at the Pdcd1 locus during ex vivo and in vivo activation of CD8 T cells suggested a potential role for the histone H3 lysine 4 demethylase LSD1 in regulating PD-1 expression. CD8 T cells lacking LSD1 expressed higher levels of Pdcd1 mRNA following ex vivo stimulation as well as increased surface levels of PD-1 during acute, but not chronic, infection with lymphocytic choriomeningitis virus (LCMV). Blimp-1, a known repressor of PD-1, recruited LSD1 to the Pdcd1 gene during acute, but not chronic, LCMV infection. Loss of DNA methylation at Pdcd1's promoter-proximal regulatory regions is highly correlated with its expression. However, following acute LCMV infection, in which PD-1 expression levels return to near baseline, LSD1-deficient CD8 T cells failed to remethylate the Pdcd1 locus to the levels of wild-type cells. Finally, in a murine melanoma model, the frequency of PD-1-expressing tumor-infiltrating LSD1-deficient CD8 T cells was greater than in wild type. Thus, LSD1 is recruited to the Pdcd1 locus by Blimp-1, downregulates PD-1 expression by facilitating the removal of activating histone marks, and is important for remethylation of the locus. Together, these data provide insight into the complex regulatory mechanisms governing T cell immunity and regulation of a critical T cell checkpoint gene.

A super enhancer controls expression and chromatin architecture within the MHC class II locus.

Super enhancers (SEs) play critical roles in cell type-specific gene regulation. The mechanisms by which such elements work are largely unknown. Two SEs termed DR/DQ-SE and XL9-SE are situated within the human MHC class II locus between the HLA-DRB1 and HLA-DQA1 genes and are highly enriched for disease-causing SNPs. To test the function of these elements, we used CRISPR/Cas9 to generate a series of mutants that deleted the SE. Deletion of DR/DQ-SE resulted in reduced expression of HLA-DRB1 and HLA-DQA1 genes. The SEs were found to interact with each other and the promoters of HLA-DRB1 and HLA-DQA1. DR/DQ-SE also interacted with neighboring CTCF binding sites. Importantly, deletion of DR/DQ-SE reduced the local chromatin interactions, implying that it functions as the organizer for the local three-dimensional architecture. These data provide direct mechanisms by which an MHC-II SE contributes to expression of the locus and suggest how variation in these SEs may contribute to human disease and altered immunity.

The Histone Demethylase LSD1 Regulates B Cell Proliferation and Plasmablast Differentiation.

B cells undergo epigenetic remodeling as they differentiate into Ab-secreting cells (ASC). LSD1 is a histone demethylase known to decommission active enhancers and cooperate with the ASC master regulatory transcription factor Blimp-1. The contribution of LSD1 to ASC formation is poorly understood. In this study, we show that LSD1 is necessary for proliferation and differentiation of mouse naive B cells (nB) into plasmablasts (PB). Following LPS inoculation, LSD1-deficient hosts exhibited a 2-fold reduction of splenic PB and serum IgM. LSD1-deficient PB exhibited derepression and superinduction of genes involved in immune system processes; a subset of these being direct Blimp-1 target-repressed genes. Cell cycle genes were globally downregulated without LSD1, which corresponded to a decrease in the proliferative capacity of LSD1-deficient activated B cells. PB lacking LSD1 displayed increased histone H3 lysine 4 monomethylation and chromatin accessibility at nB active enhancers and the binding sites of transcription factors Blimp-1, PU.1, and IRF4 that mapped to LSD1-repressed genes. Together, these data show that LSD1 is required for normal in vivo PB formation, distinguish LSD1 as a transcriptional rheostat and epigenetic modifier of B cell differentiation, and identify LSD1 as a factor responsible for decommissioning nB active enhancers.

Genome-wide CIITA-binding profile identifies sequence preferences that dictate function versus recruitment.

The class II transactivator (CIITA) is essential for the expression of major histocompatibility complex class II (MHC-II) genes; however, the role of CIITA in gene regulation outside of MHC-II biology is not fully understood. To comprehensively map CIITA-bound loci, ChIP-seq was performed in the human B lymphoblastoma cell line Raji. CIITA bound 480 sites, and was significantly enriched at active promoters and enhancers. The complexity of CIITA transcriptional regulation of target genes was analyzed using a combination of CIITA-null cells, including a novel cell line created using CRISPR/Cas9 tools. MHC-II genes and a few novel genes were regulated by CIITA; however, most other genes demonstrated either diminished or no changes in the absence of CIITA. Nearly all CIITA-bound sites were within regions containing accessible chromatin, and CIITA's presence at these sites was associated with increased histone H3K27 acetylation, suggesting that CIITA's role at these non-regulated loci may be to poise the region for subsequent regulation. Computational genome-wide modeling of the CIITA bound XY box motifs provided constraints for sequences associated with CIITA-mediated gene regulation versus binding. These data therefore define the CIITA regulome in B cells and establish sequence specificities that predict activity for an essential regulator of the adaptive immune response.

STAT3, STAT4, NFATc1, and CTCF regulate PD-1 through multiple novel regulatory regions in murine T cells.

Programmed 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 nonbiased DNase I hypersensitivity assay, four novel regulatory regions within the Pdcd1 locus were identified. Two of these elements flanked the locus, bound the transcriptional insulator protein CCCTC-binding factor, 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 IL-6 and IL-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.

B cell differentiation is associated with reprogramming the CCCTC binding factor-dependent chromatin architecture of the murine MHC class II locus.

The transcriptional insulator CCCTC binding factor (CTCF) was shown previously to be critical for human MHC class II (MHC-II) gene expression. Whether the mechanisms used by CTCF in humans were similar to that of the mouse and whether the three-dimensional chromatin architecture created was specific to B cells were not defined. Genome-wide CTCF occupancy was defined for murine B cells and LPS-derived plasmablasts by chromatin immunoprecipitation sequencing. Fifteen CTCF sites within the murine MHC-II locus were associated with high CTCF binding in B cells. Only one-third of these sites displayed significant CTCF occupancy in plasmablasts. CTCF was required for maximal MHC-II gene expression in mouse B cells. In B cells, a subset of the CTCF regions interacted with each other, creating a three-dimensional architecture for the locus. Additional interactions occurred between MHC-II promoters and the CTCF sites. In contrast, a novel configuration occurred in plasma cells, which do not express MHC-II genes. Ectopic CIITA expression in plasma cells to induce MHC-II expression resulted in high levels of MHC-II proteins, but did not alter the plasma cell architecture completely. These data suggest that reorganizing the three-dimensional chromatin architecture is an epigenetic mechanism that accompanies the silencing of MHC-II genes as part of the cell fate commitment of plasma cells.

ZBTB32 is an early repressor of the CIITA and MHC class II gene expression during B cell differentiation to plasma cells.

CIITA and MHC class II expression is silenced during the differentiation of B cells to plasma cells. When B cell differentiation is carried out ex vivo, CIITA silencing occurs rapidly, but the factors contributing to this event are not known. ZBTB32, also known as repressor of GATA3, was identified as an early repressor of CIITA in an ex vivo plasma cell differentiation model. ZBTB32 activity occurred at a time when B lymphocyte-induced maturation protein-1 (Blimp-1), the regulator of plasma cell fate and suppressor of CIITA, was minimally induced. Ectopic expression of ZBTB32 suppressed CIITA and I-A gene expression in B cells. Short hairpin RNA depletion of ZBTB32 in a plasma cell line resulted in re-expression of CIITA and I-A. Compared with conditional Blimp-1 knockout and wild-type B cells, B cells from ZBTB32/ROG-knockout mice displayed delayed kinetics in silencing CIITA during ex vivo plasma cell differentiation. ZBTB32 was found to bind to the CIITA gene, suggesting that ZBTB32 directly regulates CIITA. Lastly, ZBTB32 and Blimp-1 coimmunoprecipitated, suggesting that the two repressors may ultimately function together to silence CIITA expression. These results introduce ZBTB32 as a novel regulator of MHC-II gene expression and a potential regulatory partner of Blimp-1 in repressing gene expression.

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.

Regulation of major histocompatibility complex class II genes.

The major histocompatibility complex class II (MHC-II) genes are regulated at the level of transcription. Recent studies have shown that chromatin modification is critical for efficient transcription of these genes, and a number of chromatin modifying complexes recruited to MHC-II genes have been described. The MHC-II genes are segregated from each other by a series of chromatin elements, termed MHC-II insulators. Interactions between MHC-insulators and the promoters of MHC-II genes are mediated by the insulator factor CCCTC-binding factor and are critical for efficient expression. This regulatory mechanism provides a novel view of how the entire MHC-II locus is assembled architecturally and can be coordinately controlled.

CTCF controls expression and chromatin architecture of the human major histocompatibility complex class II locus.

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.

Diverse transcription influences can be insulated by the Drosophila SF1 chromatin boundary.

Chromatin boundaries regulate gene expression by modulating enhancer-promoter interactions and insulating transcriptional influences from organized chromatin. However, mechanistic distinctions between these two aspects of boundary function are not well understood. Here we show that SF1, a chromatin boundary located in the Drosophila Antennapedia complex (ANT-C), can insulate the transgenic miniwhite reporter from both enhancing and silencing effects of surrounding genome, a phenomenon known as chromosomal position effect or CPE. We found that the CPE-blocking activity associates with different SF1 sub-regions from a previously characterized insulator that blocks enhancers in transgenic embryos, and is independent of GAF-binding sites essential for the embryonic insulator activity. We further provide evidence that the CPE-blocking activity cannot be attributed to an enhancer-blocking activity in the developing eye. Our results suggest that SF1 contains multiple non-overlapping activities that block diverse transcriptional influences from embryonic or adult enhancers, and from positive and negative chromatin structure. Such diverse insulating capabilities are consistent with the proposed roles of SF1 to functionally separate fushi tarazu (ftz), a non-Hox gene, from the enhancers and the organized chromatin of the neighboring Hox genes.

Yin yang 1 regulates the expression of snail through a distal enhancer.

Expression of the Snail gene is required for the epithelial-mesenchymal transitions that accompany mammalian gastrulation, neural crest migration, and organ formation. Pathologic expression of Snail contributes to the migratory capacity of invasive tumors, including melanomas. To investigate the mechanism of Snail up-regulation in human melanoma cells, a conserved enhancer located 3' of the Snail gene was analyzed. An overlapping Ets and yin yang 1 (YY1) consensus sequence, in addition to a SOX consensus sequence, was required for full enhancer activity. Proteins specifically binding these sequences were detected by electrophoretic mobility shift assay. The Ets/YY1 binding activity was purified by DNA-affinity chromatography and identified as YY1. Although ubiquitously expressed, YY1 was bound at the Snail 3' enhancer in vivo in Snail-expressing cells but not in cells that did not express Snail. Knockdown of YY1 in A375 cells led to decreased Snail expression. These results identify a role for YY1 in regulating transcription of Snail in melanoma cells through binding to the Snail 3' enhancer.

The insulator factor CTCF controls MHC class II gene expression and is required for the formation of long-distance chromatin interactions.

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.

A 3' enhancer controls snail expression in melanoma cells.

The snail gene encodes a transcriptional repressor that functions during animal development and in cancer progression to promote epithelial-mesenchymal transitions. Strict spatial and temporal boundaries of Snail expression in development imply precise transcriptional control, which becomes inappropriately activated in many cancer subtypes. To gain insight into the molecular mechanism(s) governing transcriptional control of Snail, we analyze chromatin structural changes associated with Snail transcription in melanoma cells. Regardless of transcriptional status, the Snail promoter displays three constitutive DNase hypersensitive sites (HS) and a moderate level of histone H3 Lys(4) dimethylation. A robust HS is found in the 3' region of A375 melanoma cells, in which Snail is highly expressed, but is absent in cells not expressing Snail. This element is conserved throughout the mammalian lineage and strongly activates expression of a reporter in A375 and Colo829 melanoma cells, but not in keratinocytes or primary melanocytes. Activity of this enhancer is associated with enrichment of H3 Lys(4) dimethylation and H3 acetylation at both the enhancer and the promoter. Additionally, enhancer activity is associated with H3 Lys(4) trimethylation at the promoter. A physical interaction between the 3' enhancer and promoter was observed in Snail-expressing cells, demonstrating a direct role for the enhancer in Snail expression. These results suggest a model in which the Snail promoter is constitutively packaged in a poised chromatin structure that can be activated in melanoma cells by a tissue-specific enhancer, which physically contacts the promoter.

The human major histocompatibility complex class II HLA-DRB1 and HLA-DQA1 genes are separated by a CTCF-binding enhancer-blocking element.

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 approximately 44 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.

Conserved residues of the bare lymphocyte syndrome transcription factor RFXAP determine coordinate MHC class II expression.

RFXAP is required for the transcriptional regulation of MHC-II genes. Mutations in RFXAP are the genetic basis for complementation group D cases of the bare lymphocyte syndrome (BLS) immunodeficiency. Comparative genomic sequence analysis was conducted and found that only the C-terminal half of the protein is conserved among vertebrates. The C-terminal third of RFXAP, which contained an extensive glutamine-rich tract, could rescue HLA-DR, but not HLA-DQ or HLA-DP expression in a BLS cell line. To understand this phenomenon, a detailed analysis of the role of specific sequences in the C-terminal third of RFXAP with respect to MHC-II regulation was undertaken. Surprisingly, mutation of the conserved glutamine residues had no effect on activity, whereas mutation of hydrophobic and other conserved residues resulted in discoordinate MHC-II isotype expression. Moreover, mutation of potential phosphorylation sites abolished RFXAP activity. The ability of RFXAP mutants to rescue one isotype, but not another was investigated by their ability to form RFX complexes, bind DNA in vivo, recruit CIITA to promoters and to activate a series of chimeric reporter genes. The results suggest that certain RFXAP mutants exaggerate isotype promoter-specific differences and form transcriptionally inefficient activation complexes with factors at the neighboring cis-acting elements. These results show a distinction in factor recognition that is associated with specific MHC-II isotypes and may explain the basis of allele-specific expression differences.

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.

A novel boundary element may facilitate independent gene regulation in the Antennapedia complex of Drosophila.

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 functional analysis of insulator interactions in the Drosophila embryo.

Chromatin boundaries or insulators modulate enhancer-promoter interactions in complex genetic loci. However, the mechanism underlying insulator activity is not known. Previous studies showed that the activity of the Drosophila suHw insulator is abolished by the tandem arrangement (pairing) of the insulator elements, suggesting that interactions between insulators or like elements may be involved in their enhancer-blocking mechanism. To test whether such phenomenon reflects a general property of chromatin insulators, we tested the effect of pairing on enhancer-blocking activity of 11 homologous and heterologous insulator combinations using suHw, scs, or SF1 insulators. We found that, unlike the homologous pairing of suHw, the heterologous combinations of suHw with other insulators do not reduce their enhancer-blocking activity. Rather, paired insulators exhibit a higher level of enhancer-blocking activity than either single insulator alone, suggesting that they can function independently or additively. Furthermore, the analyses of two additional chromatin boundaries, scs and SF1, in homologous or heterologous pairing with other boundary elements, also showed no reduction but rather enhancement of insulator activity. We propose that diverse mechanisms may underlie insulator activity, and selective interactions among insulators could influence their function as well as the formations of independent chromatin domains.