A role for KMT1c in monocyte to dendritic cell differentiation: Epigenetic regulation of monocyte differentiation.

Monocytes play a key role in immune system function. Chromatin remodeling is crucial for various differentiation and gene regulation processes and is rather well studied in T cells. However, for monocytes not much is known regarding how the epigenetic machinery influences the differentiation into various effector cell types. In the work presented here, we explore the epigenetic underpinnings of monocyte differentiation. By transcriptional profiling we show that transcription of lysine methyltransferases (KMTs) and in particular KMT1c is markedly up regulated after differentiation of monocytes into immature dendritic cells (iDCs). Specifically inhibiting KMT1c function, using the small-molecule inhibitor BIX-01294, changes the transcription levels of the DC marker DC-SIGN, but does not affect surface protein expression. Blocking global KMT activity, using DZNep, does influence monocyte differentiation into iDCs, indicated by a loss of DC-SIGN surface expression. When BIX-01294 and DZNep treatment was combined DC-SIGN expression was almost lost completely. This work shows that the activities of KMTs are required for successful differentiation of monocyte-derived dendritic cells. Furthermore it shows the importance of KMT inhibitors in the field of epigenetic immune therapy, which is still much focused around HDAC inhibitors.

Global histone H3 lysine 27 triple methylation levels are reduced in vessels with advanced atherosclerotic plaques.

AIMS: Alterations in epigenetic processes are frequently noted in human disease. These epigenetic processes involve methylation of DNA and post-translational modifications of histones. It is well established that in particular histone methylation plays a key role in gene transcription. In this study, we have investigated the relationship between triple methylation of lysine 27 in histone H3 (H3K27Me3) modifications and atherosclerotic plaque stage. MATERIALS AND METHODS: 28 peri-renal aortic tissue patches covering the entire spectrum of atherosclerotic plaque development were evaluated by immunohistochemistry for the levels of H3K27Me3, EZH2, JMJD3 and BMI1. KEY FINDINGS: The results of our studies are in support of a reduction in global levels of the H3K27Me3 modification in vessels with advanced atherosclerotic plaques. This reduction in H3K27Me3 levels is not accompanied by alterations in global levels of the corresponding histone methyltransferase EZH2, the catalytic subunit of the polycomb repressive complex 2 (PRC2). Likewise no alterations in global levels of BMI1, a component of the PRC1 complex, which binds to H3K27Me3-modified histones or the global expression levels of the histone demethylase JMJD3, which removes the methyl marks on H3K27, were observed. SIGNIFICANCE: Together, our data show that in atherosclerosis development alterations in global levels of H3K27Me3 occur. The reduction in the number of nuclei in the tunica media that display the repressive H3K27Me3 mark in vessels with advanced atherosclerosis plaques therefore could be a reflection of the dynamic pattern of smooth muscle cell differentiation and proliferation associated with atherosclerotic disease.

Genetic and Epigenetic Regulation of CCR5 Transcription.

The chemokine receptor CCR5 regulates trafficking of immune cells of the lymphoid and the myeloid lineage (such as monocytes, macrophages and immature dendritic cells) and microglia. Because of this, there is an increasing recognition of the important role of CCR5 in the pathology of (neuro-) inflammatory diseases such as atherosclerosis and multiple sclerosis. Expression of CCR5 is under the control of a complexly organized promoter region upstream of the gene. The transcription factor cAMP-responsive element binding protein 1 (CREB-1) transactivates the CCR5 P1 promoter. The cell-specific expression of CCR5 however is realized by using various epigenetic marks providing a multivalent chromatin state particularly in monocytes. Here we discuss the transcriptional regulation of CCR5 with a focus on the epigenetic peculiarities of CCR5 transcription.

Epigenetic control of CCR5 transcript levels in immune cells and modulation by small molecules inhibitors.

Previously, we have shown that CCR5 transcription is regulated by CREB-1. However, the ubiquitous pattern of CREB-1 expression suggests the involvement of an additional level of transcriptional control in the cell type-specific expression of CCR5. In this study, we show that epigenetic changes (i.e. DNA methylation and histone modifications) within the context of the CCR5 P1 promoter region correlate with transcript levels of CCR5 in healthy and in malignant CD4(+) T lymphocytes as well as in CD14(+) monocytes. In normal naïve T cells and CD14(+) monocytes the CCR5 P1 promoter resembles a bivalent chromatin state, with both repressive and permissive histone methylation and acetylation marks. The CCR5-expressing CD14(+) monocytes however show much higher levels of acetylated histone H3 (AcH3) compared to the non-CCR5-expressing naïve T cells. Combined with a highly methylated promoter in CD14(+) monocytes, this indicates a dominant role for AcH3 in CCR5 transcription. We also show that pharmacological interference in the epigenetic repressive mechanisms that account for the lack of CCR5 transcription in T leukaemic cell lines results in an increase in CREB-1 association with CCR5 P1 chromatin. Furthermore, RNA polymerase II was also recruited into CCR5 P1 chromatin resulting in CCR5 re-expression. Together, these data indicate that epigenetic modifications of DNA, and of histones, contribute to the control of CCR5 transcription in immune effector cells.

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.

Epigenetic control in immune function.

This chapter describes recent advances in our understanding how epigenetic events control immune functions with emphasis on transcriptional regulation of major histocompatibility complex ClassI (MIC-I) and Class II (MHC-II) genes. MHC-I and MHC-II molecules play an essential role in the adaptive immune response by virtue of their ability to present peptides, respectively to CD8+ and CD4+ T cells. Central to the onset of an adequate immune response to pathogens is the presentation of pathogen-derived peptides in the context of MHC-II molecules by antigen presenting cells (APCs) to CD4+ T cells of the immune system. In particular dendritic cells are highly specialized APCs that are capable to activate naïve T cells. Given their central role in adaptive immunity, MHC-I and MHC-II genes are regulated in a tight fashion at the transcriptional level to meet with local requirements of an effective antigen-specific immune response. In these regulatory processes the MHC2TA encoded Class II transactivator (CIITA) plays a crucial role. CIITA is essential for transcriptional activation of all MHC-I genes, whereas it plays an ancillary function in the transcriptional control of MHC-I genes. The focus of this chapter therefore will be on the transcription factors that interact with conserved cis-acting promoter elements and epigenetic mechanisms that modulate cell type-specific regulation of MHC-I, MHC-I, and MHC2TA genes. Furthermore, we will also briefly discuss how genetic and epigenetic mechanisms contribute to T helper cell differentiation.

Genetic variation in PCAF, a key mediator in epigenetics, is associated with reduced vascular morbidity and mortality: evidence for a new concept from three independent prospective studies.

AIMS: This study was designed to investigate the counterbalancing influence of genetic variation in the promoter of the gene encoding P300/CBP associated factor (PCAF), a lysine acetyltransferase (KAT), on coronary heart disease (CHD) and mortality. METHODS AND RESULTS: The association of genetic variation in the PCAF-gene with CHD, restenosis and mortality was investigated in three large cohorts. The results were combined to examine overall effects on CHD mortality and on restenosis risk. Compared with the homozygous -2481G allele in the PCAF promoter, a significant reduction in CHD mortality risk with the homozygous -2481C PCAF promoter allele was observed. A combined risk reduction for CHD death for the three studies was 21% (15-26%; p=8.1×10(-4)). In elderly patients (>58 years) the effects were stronger. Furthermore, this PCAF allele was significantly associated with all-cause mortality (p=0.001). Functional analysis showed that nuclear factors interact in vitro with the oligonucleotides encompassing the -2481G/C polymorphism and that this interaction might be influenced by this polymorphism in the PCAF promoter. Moreover, modulation of PCAF gene expression was detectable upon cuff-placement in an animal model of reactive stenosis. CONCLUSION: We showed in three large prospective studies that the -2481C allele in the PCAF promoter is associated with a significant survival advantage in elderly patients. Our observations promote the concept that epigenetic processes are under genetic control and that, other than environment, variation in genes encoding KATs may also determine susceptibility to CHD outcomes and mortality.

Epigenetics in atherosclerosis and inflammation.

Atherosclerosis is a multifactorial disease with a severe burden on western society. Recent insights into the pathogenesis of atherosclerosis underscore the importance of chronic inflammation in both the initiation and progression of vascular remodelling. Expression of immunoregulatory molecules by vascular wall components within the atherosclerotic lesions is accordingly thought to contribute to the ongoing inflammatory process. Besides gene regulatory proteins (transcription factors), epigenetic mechanisms also play an essential and fundamental role in the transcriptional control of gene expression. These epigenetic mechanisms change the accessibility of chromatin by DNA methylation and histone modifications. Epigenetic modulators are thus critically involved in the regulation of vascular, immune and tissue-specific gene expression within the atherosclerotic lesion. Importantly, epigenetic processes are reversible and may provide an excellent therapeutic target. The concept of epigenetic regulation is gradually being recognized as an important factor in the pathogenesis of atherosclerosis. Recent research provides an essential link between inflammation and reprogramming of the epigenome. In this review we therefore discuss the basis of epigenetic regulation - and the contribution thereof in the regulation of inflammatory processes in general and during atherosclerosis in particular. Moreover we highlight potential therapeutic interventions based on epigenetic mechanisms.

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.

Low-pH-induced membrane fusion mediated by human metapneumovirus F protein is a rare, strain-dependent phenomenon.

Membrane fusion promoted by human metapneumovirus (HMPV) fusion (F) protein was suggested to require low pH (R. M. Schowalter, S. E. Smith, and R. E. Dutch, J. Virol. 80:10931-10941, 2006). Using prototype F proteins representing the four HMPV genetic lineages, we detected low-pH-dependent fusion only with some lineage A proteins and not with lineage B proteins. A glycine at position 294 was found responsible for the low-pH requirement in lineage A proteins. Only 6% of all HMPV lineage A F sequences have 294G, and none of the lineage B sequences have 294G. Thus, acidic pH is not a general trigger of HMPV F proteins for activity.