Epigenetic regulation of CIITA expression in human T-cells.

In humans, T-cells accomplish expression of MHC-II molecules through induction of CIITA upon activation. Here we show that CIITA promoter accessibility in T-cells is epigenetically regulated. In unstimulated T-cells, CIITA-PIII chromatin displays relative high levels of repressive histone methylation marks (3Me-K27-H3 and 3Me-K20-H4) and low levels of acetylated histones H3 (Ac-H3) and H4 (Ac-H4). These repressive histone marks are replaced by histone methylation marks associated with transcriptional active genes (3Me-K4-H3) and high levels of Ac-H3 and Ac-H4 in activated T-cells. This is associated with concomitant recruitment of RNA polymerase II. In T-leukemia cells, devoid of CIITA expression, similar repressive histone methylation marks and low levels of acetylated histone H3 correlated with lack of CIITA expression. This in contrast to CIITA expressing T-lymphoma cells, which display high levels of Ac-H3 and 3Me-K4-H3, and relative low levels of the 3Me-K27-H3 and 3Me-K20-H4 marks. Of interest was the observation that the levels of histone acetylation and methylation modifications in histones H3 and H4 were also noted in chromatin of the downstream CIITA-PIV promoter as well as the upstream CIITA-PI and CIITA-PII promoters both in normal T-cells and in malignant T-cells. Together our data show that CIITA chromatin in T-cells expressing CIITA display similar histone acetylation and methylation characteristics associated with an open chromatin structure. The opposite is true for T-cells lacking CIITA expression, which display histone modifications characteristic of condensed chromatin.

Systemic administration of PRO051 in Duchenne's muscular dystrophy.

BACKGROUND: Local intramuscular administration of the antisense oligonucleotide PRO051 in patients with Duchenne's muscular dystrophy with relevant mutations was previously reported to induce the skipping of exon 51 during pre-messenger RNA splicing of the dystrophin gene and to facilitate new dystrophin expression in muscle-fiber membranes. The present phase 1-2a study aimed to assess the safety, pharmacokinetics, and molecular and clinical effects of systemically administered PRO051. METHODS: We administered weekly abdominal subcutaneous injections of PRO051 for 5 weeks in 12 patients, with each of four possible doses (0.5, 2.0, 4.0, and 6.0 mg per kilogram of body weight) given to 3 patients. Changes in RNA splicing and protein levels in the tibialis anterior muscle were assessed at two time points. All patients subsequently entered a 12-week open-label extension phase, during which they all received PRO051 at a dose of 6.0 mg per kilogram per week. Safety, pharmacokinetics, serum creatine kinase levels, and muscle strength and function were assessed. RESULTS: The most common adverse events were irritation at the administration site and, during the extension phase, mild and variable proteinuria and increased urinary α(1)-microglobulin levels; there were no serious adverse events. The mean terminal half-life of PRO051 in the circulation was 29 days. PRO051 induced detectable, specific exon-51 skipping at doses of 2.0 mg or more per kilogram. New dystrophin expression was observed between approximately 60% and 100% of muscle fibers in 10 of the 12 patients, as measured on post-treatment biopsy, which increased in a dose-dependent manner to up to 15.6% of the expression in healthy muscle. After the 12-week extension phase, there was a mean (±SD) improvement of 35.2±28.7 m (from the baseline of 384±121 m) on the 6-minute walk test. CONCLUSIONS: Systemically administered PRO051 showed dose-dependent molecular efficacy in patients with Duchenne's muscular dystrophy, with a modest improvement in the 6-minute walk test after 12 weeks of extended treatment. (Funded by Prosensa Therapeutics; Netherlands National Trial Register number, NTR1241.).

Genetic and epigenetic control of the major histocompatibility complex class Ib gene HLA-G in trophoblast cell lines.

The transcriptional regulation of the major histocompatibility complex class (MHC) Ib gene HLA-G differs from the classical MHC class I genes. The cis-acting regulatory elements typical for classical MHC class I promoters are divergent in the promoter of HLA-G, rendering this gene unresponsive to NF-kappaB, IRF-1, and class II transactivator (CIITA)-mediated activation pathways. However, as we have previously shown, transactivation of HLA-G is regulated by CREB-1. Because CREB-1 is ubiquitously expressed, this observation does not explain the tissue-restricted expression of HLA-G in extravillous cytotrophoblasts. Using HLA-G-expressing JEG-3 cells and HLA-G-deficient JAR trophoblast-derived choriocarcinoma cells as a model, we have investigated the contribution of DNA methylation and histone acetylation in the transcriptional activation of HLA-G. Despite similar levels of DNA methylation both in JEG3 and JAR cells, we found the levels of histone acetylation in HLA-G promoter chromatin to be significantly enhanced in JEG3 cells coinciding with HLA-G expression.

A role for EZH2 in silencing of IFN-gamma inducible MHC2TA transcription in uveal melanoma.

We investigated the contribution of epigenetic mechanisms in MHC2TA transcriptional silencing in uveal melanoma. Although no correlation was observed between impaired CIITA transcript levels after IFN-gamma induction and DNA methylation of MHC2TA promoter IV (CIITA-PIV), an association was found with high levels of trimethylated histone H3-lysine 27 (3Me-K27-H3) in CIITA-PIV chromatin. The 3Me-K27-H3 modification correlated with a strong reduction in RNA polymerase II-recruitment to CIITA-PIV. Interestingly, we observed that none of these epigenetic modifications affected recruitment of activating transcription factors to this promoter. Subsequently, we demonstrated the presence of the histone methyltransferase EZH2 in CIITA-PIV chromatin, which is known to be a component of the Polycomb repressive complex 2 and able to triple methylate histone H3-lysine 27. RNA interference-mediated down-regulation of EZH2 expression resulted in an increase in CIITA transcript levels after IFN-gamma induction. Our data therefore reveal that EZH2 contributes to silencing of IFN-gamma-inducible transcription of MHC2TA in uveal melanoma cells.

Constitutive and IFNgamma-induced activation of MHC2TA promoter type III in human melanoma cell lines is governed by separate regulatory elements within the PIII upstream regulatory region.

Cell lines established from tumor tissue of cutaneous melanoma biopsies often display constitutive and IFNgamma-inducible expression of MHC class II molecules. The expression of MHC class II molecules in melanoma is associated with an overall poor prognosis and unfavorable clinical outcome. We have analyzed the DNA elements and interacting transcription factors that control the constitutive and IFNgamma-inducible expression of the class II transactivator (CIITA), a co-activator essential for transcription of all MHC class II genes. Our studies reveal the activation of multiple CIITA promoter regions (CIITA-PII, -PIII and -PIV) in melanoma cell lines for both the constitutive and IFNgamma-inducible expression of MHC class II molecules. Furthermore, we show that constitutive and IFNgamma-inducible expression of the CIITA-PIII isoform is governed by separate regulatory elements within the PIII upstream regulatory region (PURR). Similarly constitutive activation in melanoma of CIITA-PII, CIITA-PIII, and CIITA-PIV does not require components of the IFNgamma signaling pathway. However, these components are readily recruited to the PURR and CIITA-PIV after exposure of cells to IFNgamma and account for the IFNgamma-induced expression of CIITA. Together, our data reveal the contribution of distinct elements and factors in the constitutive and IFNgamma-inducible expression of CIITA in melanoma cell lines of the skin.

Epigenetic silencing of MHC2TA transcription in cancer.

Lack of expression of major histocompatibility complex (MHC) molecules of both classes is frequently noted on tumour cells . It is thought that in this way tumour cells escape immunosurveillance. The genes encoding both classes of MHC molecules are localized on the distal part of chromosome 6 (6p21.3). The class II transactivator (CIITA), encoded by the MHC2TA gene, is essential for transcriptional activation of all MHC-II genes, while it has a helper function in the transcriptional regulation of MHC-I genes (with the exception of human leukocyte antigen (HLA)-G) and of the gene encoding beta2-microglobulin (beta2m) . Here we discuss our current knowledge on the expression characteristics of MHC2TA and argue for an important role of epigenetic factors and mechanisms in the transcriptional silencing of MHC2TA in cancer cells.

Induction of apoptosis in hematopoietic cells with an antibody against tomoregulin-1.

Tomoregulin-1, a type-I transmembrane protein with two follistatin modules, a unique epidermal growth factor (EGF) domain and a short, highly conserved cytoplasmic tail, was studied. A number of hematopoietic cell lines (L1210, CEM, Jurkat, U937, K562, JY, THP-1 and T2) express tomoregulin-1 endogenously. In these cells, apoptosis was induced by an antiserum (C29) and purified IgG against the follistatin modules, but not by antisera against the EGF-domain or the cytoplasmic tail. Furthermore, C29 induced apoptosis in tomoregulin-1-, but not in mock-transfected cells. Apoptosis was monitored through genomic DNA fragmentation, annexin-V staining and caspase-3 activation. Treatment of the cells with C29 in the presence of H89 (a SerlThr kinase inhibitor) or 8'-bromo-cyclicAMP revealed that apoptosis was mediated by a cAMP-dependent Ser/Thr kinase. Moreover, C29 increased [cAMP]i over 5-fold. Together, these data suggest that the C29 antiserum against tomoregulin-1 induces apoptosis of hematopoietic cells.

Lack of MHC-II expression in activated mouse T cells correlates with DNA methylation at the CIITA-PIII region.

In contrast to activated human T cells, activated mouse T cells fail to express MHC class II molecules (MHC-II) at their cell surface. This is because mouse T cells hardly produce mRNA encoding the MHC-II molecules I-A and I-E, due to severely impaired expression levels upon T-cell activation of the mhc2ta gene, encoding the class II transactivator (CIITA). In humans, activated T cells express exclusively the CIITA promoter III (CIITA-PIII) isoform, which results in cell surface expression of all MHC-II isotypes (HLA-DR, -DP and -DQ). In this study, we demonstrate that methylation of CIITA-PIII contributes to the failure of mouse T cells to transcribe the mhc2ta and the resulting I-A/E genes, explaining the lack of I-A/E molecule expression at the cell surface following activation.

Function and regulation of MHC class II molecules in T-lymphocytes: of mice and men.

The main function of major histocompatibility complex (MHC) class II molecules is to present processed antigens, which are derived primarily from exogenous sources, to CD4(+) T-lymphocytes. MHC class II molecules thereby are critical for the initiation of the antigen-specific immune response. Besides antigen presentation, growing evidence is showing that ligation of MHC class II molecules also activates intracellular signaling pathways, frequently leading to apoptosis. Constitutive expression of MHC class II molecules is confined to professional antigen-presenting cells (APC) of the immune system, and in nonprofessional APCs MHC class II molecules can be induced by a variety of immune regulators. Interestingly, activated T cells from many species, with the exception of mice, synthesize and express MHC class II molecules at their cell surface. In this review, we discuss our current knowledge on the transcriptional regulation of MHC class II expression in activated human and mouse T cells, and the contribution of DNA methylation of the T-cell employed class II transactivator promoter III to the MHC class II deficiency of mouse T cells. We also discuss the proposed functions of the activated T cell synthesized and expressed MHC class II molecules, including antigen presentation, T-T cell interactions, and MHC class II-mediated intracellular signaling.

Transcriptional regulation of antigen presentation.

MHC class I and class II molecules play essential roles in the adaptive immune response by virtue of their ability to present peptides to T lymphocytes. Given their central role in adaptive immunity, the genes encoding these peptide-presenting molecules are regulated in a tight fashion to meet with local requirements for an adequate immune response. In contrast to MHC class I gene products, which are expressed on almost all nucleated cells, constitutive expression of MHC class II molecules is found only in specialized antigen-presenting cells of the immune system. Expression of both classes of MHC molecules can be induced by immune regulators and upon cell activation. A set of conserved cis-acting regulatory promoter elements mediate the transcription of MHC class I and beta2-microglobulin genes. Of these regulatory elements, the promoters of MHC class II and accessory genes also have the SXY module. The MHC class II transactivator (CIITA) is essential for the activation of MHC class II promoters, and it functions through protein-protein interactions with regulatory factors bound to the SXY module. Given the central role of CIITA in these regulatory processes, it is of interest to identify the DNA-binding factors and co-activators that assemble on CIITA promoters in a cell-type-specific fashion. Accordingly, recent studies include investigations into chromatin remodeling and epigenetic control mechanisms that modulate cell-type-specific transcriptional regulation of genes involved in antigen presentation.

Regulation of MHC class II expression in human T-cell malignancies.

Expression of major histocompatibility complex (MHC) class II molecules in human activated T cells is under normal circumstances regulated exclusively by the CIITA-PIII subtype of the class II transactivator (CIITA). In this study, we show that the absence of MHC class II expression in leukemic T cells was due to a lack of expression of CIITA, whereas in T-lymphoma cells, expression of CIITA correlated with expression of MHC class II. Interestingly, activation of a CIITA-promoter (P)III-reporter construct was not affected in leukemic T cells. This revealed that the absence of endogenous CIITA expression was not caused by a lack of transcription factors critical for CIITA-PIII activation but suggests the involvement of an epigenetic silencing mechanism. Subsequent analysis showed that the lack of human leukocyte antigen-DR (HLA-DR) expression correlated with hypermethylation of CIITA-PIII in leukemic T-cell lines and in primary T-cell acute lymphoblastic leukemia (T-ALL) and a T-cell prolymphocytic leukemia (T-PLL). Treatment of leukemic T-cell lines with a demethylation agent showed re-expression of CIITA-PIII and HLA-DRA. Furthermore, in vitro methylation of CIITA-PIII and subsequent assessment of CIITA-PIII activity in Jurkat leukemic T cells resulted in reduction of constitutive and CREB-1 (cyclic adenosine monophosphate [cAMP]-response element binding protein 1)-induced promoter activity. Together, these results argue for an important role of DNA hyper-methylation in the control of CIITA expression in leukemic T cells.

DNA methylation and expression of major histocompatibility complex class I and class II transactivator genes in human developmental tumor cells and in T cell malignancies.

Major histocompatibility complex (MHC) class I and class II molecules play essential roles in the immune response by virtue of their ability to present peptides to T lymphocytes. Given their central role in adaptive immunity, the genes encoding these peptide-presenting molecules are regulated in a tight fashion to meet with local requirements for an adequate immune response. In contrast to MHC class I gene products, which are expressed on almost all nucleated cells, constitutive expression of MHC class II molecules is found in specialized antigen presenting cells of the immune system only. Transcription of both MHC class I and class II genes can be induced by immune regulators and upon cell activation. Transcription of MHC class I genes is mediated by a set of conserved cis acting regulatory elements in their promoters. Of these regulatory elements, MHC class II promoters share the SXY-module. Essential for activation of MHC class II promoters is the class II transactivator (CIITA), which acts through protein/protein interactions with regulatory factors bound to the SXY module. In this review, we discuss the role of DNA methylation in relation to altered expression of MHC class I and CIITA genes as observed in malignancies and in development.

Transgene-driven protein expression specific to the intermediate pituitary melanotrope cells of Xenopus laevis.

In the present study, we examined the amphibian Xenopus laevis as a model for stable transgenesis and in particular targeted transgene protein expression to the melanotrope cells in the intermediate pituitary. For this purpose, we have fused a Xenopus proopiomelanocortin (POMC) gene promoter fragment to the gene encoding the reporter green fluorescent protein (GFP). The transgene was integrated into the Xenopus genome as short concatemers at one to six different integration sites and at a total of one to approximately 20 copies. During early development the POMC gene promoter fragment gave rise to GFP expression in the total prosencephalon, whereas during further development expression became more restricted. In free-swimming stage 40 embryos, GFP was found to be primarily expressed in the melanotrope cells of the intermediate pituitary. Immunohistochemical analysis of cryosections of brains/pituitaries from juvenile transgenic frogs revealed the nearly exclusive expression of GFP in the intermediate pituitary. Metabolic labelling of intermediate and anterior pituitaries showed newly synthesized GFP protein to be indeed primarily expressed in the intermediate pituitary cells. Hence, stable Xenopus transgenesis with the POMC gene promoter is a powerful tool to study the physiological role of proteins in a well-defined neuroendocrine system and close to the in vivo situation.

Activated human T cells accomplish MHC class II expression through T cell-specific occupation of class II transactivator promoter III.

Activated human T cells express HLA-DR, HLA-DQ, and HLA-DP on their surface, but the regulation and functioning of MHC class II molecules in T lymphocytes are poorly understood. Because the MHC class II transactivator (CIITA) is essential for MHC class II expression, we have investigated transcriptional activation of CIITA in activated T cells. In this study, we show that in human activated CD4(+) T cells, CIITA promoter III (CIITA-PIII) drives the expression of CIITA. The in vivo genomic footprint analysis revealed activated T cell-specific occupation of CIITA-PIII. Subsequent EMSA analysis of several promoter regions showed differences in banding pattern among activated T cells, naive T cells, primary B cells, and Raji B cells. Activating response element (ARE)-1 is shown to interact with the acute myeloid leukemia 2 transcription factor in nuclear extracts derived from both T and B cells. Interestingly, the acute myeloid leukemia 3 transcription factor was bound in nuclear extracts of T cells only. The ARE-2 sequence is able to bind CREB/activating transcription factor family members in both T and B cells. In addition, a yet unidentified Ets family member was found to interact with site C in activated T cells, whereas in B cells site C was bound by PU.1 and Pip/IFN regulatory factor 4/IFN consensus sequence binding protein for activated T cells. In Jurkat T cells, both ARE-1 and ARE-2 are crucial for CIITA-PIII activity, similar to Raji B cells. The differential banding pattern in in vivo genomic footprinting and transcription factor binding at the ARE-1 and site C between T cells and B cells probably reflects differences in CIITA-PIII activation pathways employed by these cell types.