Tjadine M. Holling

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.

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.

A Role for EZH2 in Silencing of IFN-γ 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-γ 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-γ induction. Our data therefore reveal that EZH2 contributes to silencing of IFN-γ-inducible transcription of MHC2TA in uveal melanoma cells.

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‐κB, 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.

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.

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.