Class II Transactivator Promoter Research Articles

CIITA promoter polymorphism impairs monocytes HLA-DR expression in patients with septic shock

SummaryLow monocyte (m)HLA-DR expression is associated with mortality in sepsis. G-286A∗rs3087456 polymorphism in promoter III of HLA class II transactivator (CIITA), the master regulator of HLA, has been associated with autoimmune diseases but its role in sepsis has never been demonstrated. In 203 patients in septic shock, GG genotype was associated with 28-day mortality and mHLA-DR remained low whereas it increased in patients with AA or AG genotype. In ex vivo cells, mHLA-DR failed to augment in GG in comparison with AG or AA genotype on exposure to IFN-γ. Promoter III transcript levels were similar in control monocytes regardless of genotype and exposure to IFN-γ. Promoter III activity was decreased in GG genotype in monocyte cell line but restored after stimulation with IFN-γ. Hereby, we demonstrated that G-286A∗rs3087456 significantly impact mHLA-DR expression in patients with septic shock in part through CIITA promoter III activity, that can be rescued using IFN-γ.

Chromatin-Remodeling Factor BRG1 Is a Negative Modulator of L. donovani in IFNγ Stimulated and Infected THP-1 Cells.

Intracellular pathogens manipulate the host cell for their own survival by contributing to modifications of host epigenome, and thus, altering expression of genes involved in the pathogenesis. Both ATP-dependent chromatin remodeling complex and histone modifications has been shown to be involved in the activation of IFNγ responsive genes. Leishmania donovani is an intracellular pathogen that causes visceral leishmaniasis. The strategies employed by Leishmania donovani to modulate the host epigenome in order to overcome the host defense for their persistence has been worked out in this study. We show that L. donovani negatively affects BRG1, a catalytic subunit of mammalian SWI/SNF chromatin remodeling complex, to alter IFNγ induced host responses. We observed that L. donovani infection downregulates BRG1 expression both at transcript and protein levels in cells stimulated with IFNγ. We also observed a significant decrease in IFNγ responsive gene, Class II transactivator (CIITA), as well as its downstream genes, MHC-II (HLA-DR and HLA-DM). Also, the occupancy of BRG1 at CIITA promoters I and IV was disrupted. A reversal in CIITA expression and decreased parasite load was observed with BRG1 overexpression, thus, suggesting BRG1 is a potential negative regulator for the survival of intracellular parasites in an early phase of infection. We also observed a decrease in H3 acetylation at the promoters of CIITA, post parasite infection. Silencing of HDAC1, resulted in increased CIITA expression, and further decreased parasite load. Taken together, we suggest that intracellular parasites in an early phase of infection negatively regulates BRG1 by using host HDAC1 for its survival inside the host.

Amelogenin Downregulates Interferon Gamma-Induced Major Histocompatibility Complex Class II Expression Through Suppression of Euchromatin Formation in the Class II Transactivator Promoter IV Region in Macrophages.

Enamel matrix derivatives (EMDs)-based periodontal tissue regenerative therapy is known to promote healing with minimal inflammatory response after periodontal surgery, i. e., it promotes wound healing with reduced pain and swelling. It has also been reported that macrophages stimulated with amelogenin, a major component of EMD, produce various anti-inflammatory cytokines and growth factors. We previously found that stimulation of monocytes with murine recombinant M180 (rM180) amelogenin suppresses major histocompatibility complex class II (MHC II) gene expression using microarray analysis. However, the detailed molecular mechanisms for this process remain unclear. In the present study, we demonstrated that rM180 amelogenin selectively downmodulates the interferon gamma (IFNγ)-induced cell surface expression of MHC II molecules in macrophages and this mechanism mediated by rM180 appeared to be widely conserved across species. Furthermore, rM180 accumulated in the nucleus of macrophages at 15 min after stimulation and inhibited the protein expression of class II transactivator (CIITA) which controls the transcription of MHC II by IFNγ. In addition, reduced MHC II expression on macrophages pretreated with rM180 impaired the expression of T cell activation markers CD25 and CD69, T cell proliferation ability, and IL-2 production by allogenic CD4+ T lymphocytes in mixed lymphocyte reaction assay. The chromatin immunoprecipitation assay showed that IFNγ stimulation increased the acetylation of histone H3 lysine 27, which is important for conversion to euchromatin, as well as the trimethylation of histone H3 lysine 4 levels in the CIITA promoter IV (p-IV) region, but both were suppressed in the group stimulated with IFNγ after rM180 treatment. In conclusion, the present study shows that amelogenin suppresses MHC II expression by altering chromatin structure and inhibiting CIITA p-IV transcription activity, and attenuates subsequent T cell activation. Clinically observed acceleration of wound healing after periodontal surgery by amelogenin may be partially mediated by the mechanism elucidated in this study. In addition, the use of recombinant amelogenin is safe because it is biologically derived protein. Therefore, amelogenin may also be used in future as an immunosuppressant with minimal side effects for organ transplantation or MHC II-linked autoimmune diseases such as type I diabetes, multiple sclerosis, and rheumatoid arthritis, among others.

Forkhead transcription factor FOXO3a mediates interferon-γ-induced MHC II transcription in macrophages.

Macrophages are professional antigen-presenting cells relying on the expression of class II major histocompatibility complex (MHC II) genes. Interferon-γ (IFN-γ) activates MHC II transcription via the assembly of an enhanceosome centred on class II trans-activator (CIITA). In the present study, we investigated the role of the forkhead transcription factor FOXO3a in IFN- γ-induced MHC II transcription in macrophages. Knockdown of FOXO3a, but not FOXO1 or FOXO4, diminished IFN-γ-induced MHC II expression in RAW cells. On the contrary, over-expression of FOXO3a, but neither FOXO1 nor FOXO4, enhanced CIITA-mediated trans-activation of the MHC II promoter. IFN-γ treatment promoted the recruitment of FOXO3a to the MHC II promoter. Co-immunoprecipitation and RE-ChIP assays showed that FOXO3a was a component of the MHC II enhanceosome forming interactions with CIITA, RFX5, RFXB and RFXAP. FOXO3a contributed to MHC II transcription by altering histone modifications surrounding the MHC II promoter. Of interest, FOXO3a was recruited to the type IV CIITA promoter and directly activated CIITA transcription by interacting with signal transducer of activation and transcription 1 in response to IFN-γ stimulation. In conclusion, our data unveil a novel role for FOXO3a in the regulation of MHC II transcription in macrophages.

Effects of triptolide on the expression of MHC II in microglia in kainic acid‑induced epilepsy.

The purpose of the present study was to determine whether triptolide (T10) had any effect on major histocompatibility complex class II (MHC II) expression in kainic acid (KA)‑activated microglia, and to investigate the underlying molecular mechanism. BV‑2 microglia were pretreated with T10 prior to activation with KA. The expression level of MHCII and classII transactivator (CIITA) mRNA was determined via reverse transcription‑polymerase chain reaction. The expression of MHCII, CIITA and the phosphorylation level of c‑Jun and proto‑oncogene c‑Fos (c‑Fos) was determined by western blotting. The protein expression level of MHCII was determined by immunocytochemistry. It was observed that the mRNA and protein levels of MHCII and CIITA were increased in KA‑activated BV‑2 microglia, and that this increase was almost completely eliminated by T10. AP‑1 is a family of homodimers or heterodimers, composed of Jun family and Fos family proteins. Sequence analysis revealed an AP‑1 DNA binding site in the promoter of CIITA. The phosphorylation of c‑Jun and c‑Fos was increased in KA‑activated microglia, while T10 was able to suppress the phosphorylation of c‑Jun and c‑Fos in KA‑activated microglia. These data suggested that T10 may exert suppressive effects on MHCII expression in KA‑activated microglia, and that the mechanism may involve the regulation of AP‑1 activity.

Mycobacterium tuberculosis EsxL inhibits MHC-II expression by promoting hypermethylation in class-II transactivator loci in macrophages

Mycobacterium tuberculosis is known to modulate the host immune responses to facilitate its persistence inside the host cells. One of the key mechanisms includes repression of class-II transactivator (CIITA) and MHC-II expression in infected macrophages. However, the precise mechanism of CIITA and MHC-II down-regulation is not well studied. M. tuberculosis 6-kDa early secretory antigenic target (ESAT-6) is a known potent virulence and antigenic determinant. The M. tuberculosis genome encodes 23 such ESAT-6 family proteins. We herein report that M. tuberculosis and M. bovis bacillus Calmette-Guérin infection down-regulated the expression of CIITA/MHC-II by inducing hypermethylation in histone H3 lysine 9 (H3K9me2/3). Further, we showed that M. tuberculosis ESAT-6 family protein EsxL, encoded by Rv1198, is responsible for the down-regulation of CIITA/MHC-II by inducing H3K9me2/3. We further report that M. tuberculosis esxL induced the expression of nitric-oxide synthase, NO production, and p38 MAPK pathway, which in turn was responsible for the increased H3K9me2/3 in CIITA via up-regulation of euchromatic histone-lysine N-methyltransferase 2 (G9a). In contrast, inhibition of nitric-oxide synthase, p38 MAPK, and G9a abrogated H3K9me2/3, resulting in increased CIITA expression. A chromatin immunoprecipitation assay confirmed that hypermethylation at the promoter IV region of CIITA is mainly responsible for CIITA down-regulation and subsequent antigen presentation. We found that co-culture of macrophages infected with esxL-expressing M. smegmatis and mouse splenocytes led to down-regulation of IL-2, a key cytokine involved in T-cell proliferation. In summary, we demonstrate that M. tuberculosis EsxL inhibits antigen presentation by enhancing H3K9me2/3 at the CIITA promoter, thereby repressing its expression through NO and p38 MAPK activation.

HIC1 epigenetically represses CIITA transcription in B lymphocytes

Differentiation of B lymphocytes into isotope-specific plasma cells represents a hallmark event in adaptive immunity. During B cell maturation, expression of the class II transactivator (CIITA) gene is down-regulated although the underlying epigenetic mechanism is not completely defined. Here we report that hypermethylated in cancer 1 (HIC1) was up-regulated in differentiating B lymphocytes paralleling CIITA repression. Over-expression of HIC1 directly repressed endogenous CIITA transcription in B cells. Reporter assay and chromatin immunoprecipitation (ChIP) assay confirmed that HIC1 bound to the proximal CIITA type III promoter (−545/−113); mutation of a conserved HIC1 site within this region abrogated CIITA trans-repression. More important, depletion of HIC1 with small interfering RNA (siRNA) restored CIITA expression in differentiating B cells. Mechanistically, HIC1 preferentially interacted with and recruited DNMT1 and DNMT3b to the CIITA promoter to synergistically repress CIITA transcription. On the contrary, silencing of DNMT1/DNMT3b or inhibition of DNMT activity with 5-aza-dC attenuated CIITA trans-repression. Therefore, our data identify HIC1 as a novel factor involved in B cell differentiation acting as an epigenetic repressor of CIITA transcription.

Polycomb recruitment at the Class II transactivator gene.

The Class II Transactivator (CIITA) is the master regulator of Major Histocompatibility Class II (MHC II) genes. Transcription of CIITA through the IFN-γ inducible CIITA promoter IV (CIITA pIV) during activation is characterized by a decrease in trimethylation of histone H3 lysine 27 (H3K27me3), catalyzed by the histone methyltransferase Enhancer of Zeste Homolog 2 (EZH2). While EZH2 is the known catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) and is present at the inactive CIITA pIV, the mechanism of PRC2 recruitment to mammalian promoters remains unknown. Here we identify two DNA-binding proteins, which interact with and regulate PRC2 recruitment to CIITA pIV. We demonstrate Yin Yang 1 (YY1) and Jumonji domain containing protein 2 (JARID2) are binding partners along with EZH2 in mammalian cells. Upon IFN-γ stimulation, YY1 dissociates from CIITA pIV while JARID2 binding to CIITA pIV increases, suggesting novel roles for these proteins in regulating expression of CIITA pIV. Knockdown of YY1 and JARID2 yields decreased binding of EZH2 and H3K27me3 at CIITA pIV, suggesting important roles for YY1 and JARID2 at CIITA pIV. JARID2 knockdown also results in significantly elevated levels of CIITA mRNA upon IFN-γ stimulation. This study is the first to identify novel roles of YY1 and JARID2 in the epigenetic regulation of the CIITA pIV by recruitment of PRC2. Our observations indicate the importance of JARID2 in CIITA pIV silencing, and also provide a novel YY1-JARID2-PRC2 regulatory complex as a possible explanation of differential PRC2 recruitment at inducible versus permanently silenced genes.

Epigenetic modification and preliminary investigation of the mechanism of the immune evasion of HL-60 cells.

The aim of the present study was to explore the effect of epigenetic modification of class II transactivator (CIITA) methylation on histocompatibility complex (MHC) class II expression and the immune evasion of leukemia HL-60 cells. HL-60 cells were treated with various concentrations of 5-aza-2′deoxycytidine (5-Aza-CdR) and 0.5 μmol/l suberoyl-anilide hydroxamic acid (SAHA) for 24 h and then stimulated by interferon γ (IFN-γ) for 48 h. The mRNA levels of MHC class I, II and co-stimulatory molecules were quantified by reverse transcription polymerase chain reaction (RT-PCR). The levels of CIITA protein were determined by western blot analysis, and the CpG island methylation ratios in the CIITA promoter IV (CIITApIV) were analyzed by bisulfite-sequencing PCR (BSP). MHC I as well as the co-stimulatory molecules CD40 and CD80 were significantly increased following treatment with 5-Aza-CdR + SAHA + IFN-γ (epigenetic groups) compared with those in the control group and IFN-γ group (P<0.05). The expression of MHC class II and CIITA was restored and increased in an 5-Aza-CdR concentration-dependent manner in the three epigenetic groups. The results of the BSP assay showed that the methylation rate of CIITApIV CpG sites decreased with the treatment of epigenetic modification and negatively correlated to the 5-Aza-CdR concentration. This demonstrated that the negative expression of CIITA protein was the key reason for the loss of MHC II expression in HL-60 cells. The results of the present study may help to illustrate the mechanism of immune evasion in HL-60 cells.

A2b adenosine signaling represses CIITA transcription via an epigenetic mechanism in vascular smooth muscle cells

Chronic inflammation plays a major role in the pathogenesis of atherosclerosis. Vascular smooth muscle cells (VSMC), by expressing and presenting major histocompatibility complex II (MHC II) molecules, help recruit T lymphocyte and initiate the inflammatory response within the vasculature. We have previously shown that VSMCs isolated from mice with deficient adenosine A2b receptor (A2b-null) exhibit higher expression of class II transactivator (CIITA), the master regulator of MHC II transcription, compared to wild type littermates. Here we report that activation of A2b adenosine signaling suppresses CIITA expression in human aortic smooth muscle cells. Down-regulation of CIITA expression was largely attributable to transcriptional repression of type III and IV promoters. Chromatin immunoprecipitation (ChIP) analyses revealed that A2b signaling repressed CIITA transcription by attenuating specific histone modifications on the CIITA promoters in a STAT1-dependent manner. STAT1 interacted with PCAF/GCN5, histone H3K9 acetyltransferases, and WDR5, a key component of the mammalian H3K4 methyltransferase complex, to activate CIITA transcription. A2b signaling prevented recruitment of PCAF/GCN5 and WDR5 to the CIITA promoters in a STAT1-dependent manner. In conclusion, our data suggest that adenosine A2b signaling represses CIITA transcription in VSMCs by manipulating the interaction between STAT1 and the epigenetic machinery.

Histone deacetylase inhibitors activate CIITA and MHC class II antigen expression in diffuse large B‐cell lymphoma

Diffuse large B-cell lymphoma (DLBCL), the most common form of non-Hodgkin's lymphoma (NHL) diagnosed in the USA, consists of at least two distinct subtypes: germinal centre B (GCB) and activated B-cell (ABC). Decreased MHC class II (MHCII) expression on the tumours in both DLBCL subtypes directly correlates with significant decreases in patient survival. One common mechanism accounting for MHCII down-regulation in DLBCL is reduced expression of the MHC class II transactivator (CIITA), the master regulator of MHCII transcription. Furthermore, reduced CIITA expression in ABC DLBCL correlates with the presence of the transcriptional repressor positive regulatory domain-I-binding factor-1 (PRDI-BF1). However, the mechanisms underlying down-regulation of CIITA in GCB DLBCL are currently unclear. In this study, we demonstrate that neither PRDI-BF1 nor CpG hypermethylation at the CIITA promoters are responsible for decreased CIITA in GCB DLBCL. In contrast, histone modifications associated with an open chromatin conformation and active transcription were significantly lower at the CIITA promoters in CIITA(-) GCB cells compared with CIITA(+) B cells, which suggests that epigenetic mechanisms contribute to repression of CIITA transcription. Treatment of CIITA(-) or CIITA(low) GCB cells with several different histone deacetylase inhibitors (HDACi) activated modest CIITA and MHCII expression. However, CIITA and MHCII levels were significantly higher in these cells after exposure to the HDAC-1-specific inhibitor MS-275. These results suggest that CIITA transcription is repressed in GCB DLBCL cells through epigenetic mechanisms involving HDACs, and that HDACi treatment can alleviate repression. These observations may have important implications for patient therapy.

MHC Class II Transactivator Is an In Vivo Regulator of Osteoclast Differentiation and Bone Homeostasis Co-opted From Adaptive Immunity

The molecular networks controlling bone homeostasis are not fully understood. The common evolution of bone and adaptive immunity encourages the investigation of shared regulatory circuits. MHC Class II Transactivator (CIITA) is a master transcriptional co-activator believed to be exclusively dedicated for antigen presentation. CIITA is expressed in osteoclast precursors, and its expression is accentuated in osteoporotic mice. We thus asked whether CIITA plays a role in bone biology. To this aim, we fully characterized the bone phenotype of two mouse models of CIITA overexpression, respectively systemic and restricted to the monocyte-osteoclast lineage. Both CIITA-overexpressing mouse models revealed severe spontaneous osteoporosis, as assessed by micro-computed tomography and histomorphometry, associated with increased osteoclast numbers and enhanced in vivo bone resorption, whereas osteoblast numbers and in vivo bone-forming activity were unaffected. To understand the underlying cellular and molecular bases, we investigated ex vivo the differentiation of mutant bone marrow monocytes into osteoclasts and immune effectors, as well as osteoclastogenic signaling pathways. CIITA-overexpressing monocytes differentiated normally into effector macrophages or dendritic cells but showed enhanced osteoclastogenesis, whereas CIITA ablation suppressed osteoclast differentiation. Increased c-fms and receptor activator of NF-κB (RANK) signaling underlay enhanced osteoclast differentiation from CIITA-overexpressing precursors. Moreover, by extending selected phenotypic and cellular analyses to additional genetic mouse models, namely MHC Class II deficient mice and a transgenic mouse line lacking a specific CIITA promoter and re-expressing CIITA in the thymus, we excluded MHC Class II expression and T cells from contributing to the observed skeletal phenotype. Altogether, our study provides compelling genetic evidence that CIITA, the molecular switch of antigen presentation, plays a novel, unexpected function in skeletal homeostasis, independent of MHC Class II expression and T cells, by exerting a selective and intrinsic control of osteoclast differentiation and bone resorption in vivo.

Nitric Oxide and KLF4 Protein Epigenetically Modify Class II Transactivator to Repress Major Histocompatibility Complex II Expression during Mycobacterium bovis Bacillus Calmette-Guérin Infection

Pathogenic mycobacteria employ several immune evasion strategies such as inhibition of class II transactivator (CIITA) and MHC-II expression, to survive and persist in host macrophages. However, precise roles for specific signaling components executing down-regulation of CIITA/MHC-II have not been adequately addressed. Here, we demonstrate that Mycobacterium bovis bacillus Calmette-Guérin (BCG)-mediated TLR2 signaling-induced iNOS/NO expression is obligatory for the suppression of IFN-γ-induced CIITA/MHC-II functions. Significantly, NOTCH/PKC/MAPK-triggered signaling cross-talk was found critical for iNOS/NO production. NO responsive recruitment of a bifunctional transcription factor, KLF4, to the promoter of CIITA during M. bovis BCG infection of macrophages was essential to orchestrate the epigenetic modifications mediated by histone methyltransferase EZH2 or miR-150 and thus calibrate CIITA/MHC-II expression. NO-dependent KLF4 regulated the processing and presentation of ovalbumin by infected macrophages to reactive T cells. Altogether, our study delineates a novel role for iNOS/NO/KLF4 in dictating the mycobacterial capacity to inhibit CIITA/MHC-II-mediated antigen presentation by infected macrophages and thereby elude immune surveillance.

Adenosine signaling inhibits CIITA‐mediated MHC class II transactivation in lung fibroblast cells

Efficient antigen presentation by major histocompatibility complex (MHC) molecules represents a critical process in adaptive immunity. Class II transactivator (CIITA) is considered the master regulator of MHC class II (MHC II) transcription. Previously, we have shown that CIITA expression is upregulated in smooth muscle cells deficient in A2b adenosine receptor. Here, we report that treatment with the adenosine receptor agonist adenosine-5'N-ethylcarboxamide (NECA) attenuated MHC II transcription in lung fibro-blast cells as a result of CIITA repression. Further analysis revealed that NECA preferentially abrogated CIITA transcription through promoters III and IV. Blockade with a selective A2b receptor antagonist MRS-1754 restored CIITA-dependent MHC II transactivation. Forskolin, an adenylyl cyclase activator, achieved the same effect as NECA. A2b signaling repressed CIITA transcription by altering histone modifications and recruitment of key factors on the CIITA promoters in a STAT1-dependent manner. MRS-1754 blocked the antagonism of transforming growth factor beta (TGF-β) in CIITA induction by interferon gamma (IFN-γ), alluding to a potential dialogue between TGF-β and adenosine signaling pathways. Finally, A2b signaling attenuated STAT1 phosphorylation and stimulated TGF-β synthesis. In conclusion, we have identified an adenosine-A2b receptor-adenylyl cyclase axis that influences CIITA-mediated MHC II transactivation in lung fibroblast cells and as such have provided invaluable insights into the development of novel immune-modulatory strategies.

Dysregulated Recruitment of the Histone Methyltransferase EZH2 to the Class II Transactivator (CIITA) Promoter IV in Breast Cancer Cells

One mechanism frequently utilized by tumor cells to escape immune system recognition and elimination is suppression of cell surface expression of Major Histocompatibility Class II (MHC II) molecules. Expression of MHC II is regulated primarily at the level of transcription by the Class II Transactivator, CIITA, and decreased CIITA expression is observed in multiple tumor types. We investigate here contributions of epigenetic modifications to transcriptional silencing of CIITA in variants of the human breast cancer cell line MDA MB 435. Significant increases in histone H3 lysine 27 trimethylation upon IFN-γ stimulation correlate with reductions in transcription factor recruitment to the interferon-γ inducible CIITA promoter, CIITApIV, and with significantly increased CIITApIV occupancy by the histone methyltransferase enhancer of zeste homolog 2 (EZH2). Most compelling is evidence that decreased expression of EZH2 in MDA MB 435 variants results in significant increases in CIITA and HLA-DRA mRNA expression, even in the absence of interferon-γ stimulation, as well as increased cell surface expression of MHC II. Together, these data add mechanistic insight to prior observations of increased EZH2 expression and decreased CIITA expression in multiple tumor types.

Epigenetic Regulation of the Class II Transactivator (CIITA) by the Polycomb Repressive Complex 2 (PRC2)

Major Histocompatibility Class II (MHC II) molecules present extracellular peptides to T helper cells and are thus responsible for initiating, regulating, and terminating immune responses to foreign agents and tumors. Expression of MHC II is induced by the inflammatory cytokine interferon gamma (IFN‐γ) which induces transcription of the Class II Transactivator (CIITA), the master regulator of MHC II genes. Transcription of CIITA through the IFN‐γ inducible CIITA promoter IV is frequently suppressed in tumor cells through specific trimethylation of histone H3 lysine 27 (H3K27me3) by the histone methyltransferase Enhancer of Zeste Homolog 2 (EZH2). EZH2 is the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2); however the mechanisms of recruitment of PRC2 to mammalian promoters remains unknown as proteins in the PRC2 complex contain no DNA binding domains. We identify here two EZH2 binding partners which bind DNA and regulate PRC2 recruitment to CIITApIV. We demonstrate that Yin Yang 1 (YY1), the homolog of the Drosophila PRC2 recruiting protein Pleiohomoeotic (Pho), and Jumonji domain containing protein 2 (JARID2), a member of histone demethylase family, both interact with EZH2. Upon IFN‐γ stimulation, YY1 dissociates from CIITA pIV while Jarid2 CIITA pIV binding increases, suggesting novel roles for each of these proteins in regulating inducible gene promoter dynamics. Research supported by grants from the American Cancer Society, the Georgia Cancer Coalition, and the Georgia Research Alliance (to S.F. Greer).

CIITA promoter I CARD-deficient mice express functional MHC class II genes in myeloid and lymphoid compartments

Three distinct promoters control the master regulator of MHC class II expression, CIITA, in a cell type specific manner. Promoter I (pI) CIITA, expressed primarily by dendritic cells and macrophages, expresses a unique isoform that contains a caspase recruitment domain. The activity and function of this isoform is not understood but has been thought to enhance the function of CIITA in antigen presenting cells. To determine if isoform I of CIITA has specific functions, CIITA mutant mice were created in which isoform I was replaced with isoform III sequences. Mice in which pI and the CARD encoding exon were deleted were also created. No defect in the formation of CD4 T cells, the ability to respond to a model antigen, or bacterial or viral challenge was observed in mice lacking CIITA isoform I. Although CIITA and MHC-II expression was decreased in splenic DC, the pI knockout animals expressed CIITA from downstream promoters, suggesting that control of pI activity is mediated by unknown s II distal elements that could act at the pIII, the B cell promoter. Thus, no critical function is linked to the CARD domain of CIITA isoform I with respect to basic immune system development, function and challenge.

Abstract 804: Class II transactivator (CIITA) transcription is reduced by the ER-E2 signaling pathway in breast cancer cells

Abstract The immune response is regulated by the interaction of CD4+T-cells with human leukocyte antigen class II (HLA-II). HLA-II genes code for HLA-DR that display peptides from antigens, which have undergone proteolysis in the endocytic pathway. Peptide loading of HLA-DR molecules also requires the co-chaperones; Invariant chain (Ii) and HLA-DM. Regulation of HLA-II occurs mainly at the transcription level, through interaction of transcription factors with the class II transactivator (CIITA). We previously reported that discordant expression of HLA-II in breast cancer associated with decreased estrogen receptor (ER) levels and younger age at diagnosis, suggesting that the estrogen-ER signaling pathway is implicated in regulating HLA-II in breast carcinoma cells. Moreover, using an experimental model of ER+ and ER− breast cancer cell lines, we found that estradiol (E2) augmented expression in the ER− cell line but decreased expression in the ER+ transfectant. To address whether ER and E2 interfere with CIITA expression, we performed Western blotting and quantitative real-time PCR in the same experimental cell system. CIITA expression was markedly reduced in ER+, but not in ER− cells; furthermore, CIITA inversely correlated with ER levels. CIITA promoter activity was analysed using a luciferase reporter gene construct in cell lines, treated or not with E2 and/or ICI 182,780 (ER antagonist). CIITA luciferase activity was reduced in ER+ cells and was further inhibited by E2 and was restored by ICI. This suggests that ER may be recruited to the CIITA promoter and indirectly interfere with transcription of HLA-II genes. We further analyzed HLA-II in a wild type ER+ line, MCF-7, which was stimulated with IFN-γ or stably transfected with CIITA. HLA-DR surface expression, ascertained by flow cytometry, was upregulated in cells grown in E2-depleted medium. Western blotting confirmed that ER degradation by ICI in MCF-7 correlated with up regulation of CIITA and HLA-II. In aggregate, these results indicate that the E2-ER pathway interferes with CIITA and HLA-II expression. Investigations such as this should improve our understanding of how ER and estrogen modulate the antigen presentation capabilities and immune recognition of breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 804. doi:10.1158/1538-7445.AM2011-804

Notch1-mediated Signaling Induces MHC Class II Expression through Activation of Class II Transactivator Promoter III in Mast Cells

Mast cells constitutively express Notch1 and Notch2 on the cell surface. Notch ligand Dll1 (Delta-like 1) stimulation induces MHC class II expression in mast cells and renders them as antigen-presenting cells. However, nothing is known about the mechanism by which Notch signaling induces MHC class II expression in mast cells. MHC class II genes are regulated by the class II transactivator (CIITA). In mice, transcription of the CIITA gene is controlled by three cell type-specific promoters (pI, pIII, and pIV). Here, we show that CIITA expression induced by Dll1 stimulation in mouse bone marrow-derived mast cells (BMMCs) depends critically on the signal mediated by Notch1 and that the most dominant promoter in Notch signaling-mediated CIITA expression in BMMCs is pIII, which is a lymphoid lineage-specific promoter. ChIP assays indicated that Notch signaling increased the binding of the transcription factor PU.1 to CIITA pIII in BMMCs. The knockdown of PU.1 expression using a specific siRNA suppressed Notch signaling-mediated CIITA expression, suggesting that PU.1 contributes to the expression of MHC class II induced by Notch signaling in mast cells. Furthermore, we show that a portion of freshly isolated splenic mast cells express MHC class II and that the most dominant promoter of CIITA in mast cells is pIII. These findings indicate that activation of CIITA pIII plays an important role in MHC class II expression in mast cells.

Early epigenetic events regulate the adaptive immune response gene CIITA

Precise regulation of Major Histocompatibility class II (MHC II) genes plays important roles in initiation, propagation and termination of adaptive immune responses by controlling antigen presentation to CD4+ T cells. MHC II genes are constitutively expressed in only a few cell types and are inducibly expressed by the inflammatory response cytokine interferon gamma (IFNγ) in all nucleated cells. The regulation of MHC II is tightly controlled by a Master Regulator, the class II transactivator (CIITA), which is a general regulator of both constitutive and inducible MHC II expression. Although much is known about the transcription factors necessary for CIITA expression, less is known about the epigenetic modifications and the requisite enzymes needed to provide these transcription factors access to DNA. We show here that multiple epigenetic changes occur at the IFNγ inducible CIITA promoter within 20’ of IFNγ stimulation and that these changes correlate with the opening of the promoter and the initiation of transcription. Our study links these rapidly occurring epigenetic events at the inducible CIITA promoter to decreased promoter binding of the histone methyltransferase EZH2, and shows that decreased promoter binding of EZH2 transforms this previously tightly regulated and cytokine inducible promoter into a constitutively active and dysregulated gene.