FOXP1 is a regulator of quiescence in healthy human CD4+ T cells and is constitutively repressed in T cells from patients with lymphoproliferative disorders.

The forkhead box P1 (FOXP1) transcription factor has been shown to regulate the generation and maintenance of quiescent naïve murine T cells. In humans, FOXP1 expression has been correlated with overall survival in patients with peripheral T-cell lymphoma (PTCL), although its regulatory role in T-cell function is currently unknown. We found that FOXP1 is normally expressed in all human leukocyte subpopulations. Focusing on primary human CD4+ T cells, we show that nuclear expression of FOXP1 predominates in naïve cells with significant downregulation detected in memory cells from blood and tonsils. FOXP1 is repressed following in vitro T-cell activation of naïve T cells, and later re-established in memory CD4+ T cells, albeit at lower levels. DNA methylation analysis revealed that epigenetic mechanisms participate in regulating the human FOXP1 gene. ShRNA-mediated FOXP1 repression induces CD4+ T cells to enter the cell cycle, acquire memory-like markers and upregulate helper T-cell differentiation genes. In patients with lymphoproliferative disorders, FOXP1 expression is constitutionally repressed in the clonal T cells in parallel with overexpression of helper T-cell differentiation genes. Collectively, these data identify FOXP1 as an essential transcriptional regulator for primary human CD4+ T cells and suggest its potential important role in the development of PTCL.

DNA methylation profiling identifies epigenetic dysregulation in pancreatic islets from type 2 diabetic patients.

In addition to genetic predisposition, environmental and lifestyle factors contribute to the pathogenesis of type 2 diabetes (T2D). Epigenetic changes may provide the link for translating environmental exposures into pathological mechanisms. In this study, we performed the first comprehensive DNA methylation profiling in pancreatic islets from T2D and non-diabetic donors. We uncovered 276 CpG loci affiliated to promoters of 254 genes displaying significant differential DNA methylation in diabetic islets. These methylation changes were not present in blood cells from T2D individuals nor were they experimentally induced in non-diabetic islets by exposure to high glucose. For a subgroup of the differentially methylated genes, concordant transcriptional changes were present. Functional annotation of the aberrantly methylated genes and RNAi experiments highlighted pathways implicated in ß-cell survival and function; some are implicated in cellular dysfunction while others facilitate adaptation to stressors. Together, our findings offer new insights into the intricate mechanisms of T2D pathogenesis, underscore the important involvement of epigenetic dysregulation in diabetic islets and may advance our understanding of T2D aetiology.

Recommendations for the design and analysis of epigenome-wide association studies.

Epigenome-wide association studies (EWAS) hold promise for the detection of new regulatory mechanisms that may be susceptible to modification by environmental and lifestyle factors affecting susceptibility to disease. Epigenome-wide screening methods cover an increasing number of CpG sites, but the complexity of the data poses a challenge to separating robust signals from noise. Appropriate study design, a detailed a priori analysis plan and validation of results are essential to minimize the danger of false positive results and contribute to a unified approach. Epigenome-wide mapping studies in homogenous cell populations will inform our understanding of normal variation in the methylome that is not associated with disease or aging. Here we review concepts for conducting a stringent and powerful EWAS, including the choice of analyzed tissue, sources of variability and systematic biases, outline analytical solutions to EWAS-specific problems and highlight caveats in interpretation of data generated from samples with cellular heterogeneity.

A comprehensive overview of Infinium HumanMethylation450 data processing.

Infinium HumanMethylation450 beadarray is a popular technology to explore DNA methylomes in health and disease, and there is a current explosion in the use of this technique. Despite experience acquired from gene expression microarrays, analyzing Infinium Methylation arrays appeared more complex than initially thought and several difficulties have been encountered, as those arrays display specific features that need to be taken into consideration during data processing. Here, we review several issues that have been highlighted by the scientific community, and we present an overview of the general data processing scheme and an evaluation of the different normalization methods available to date to guide the 450K users in their analysis and data interpretation.

The histone demethylase Kdm3a is essential to progression through differentiation.

Histone demethylation has important roles in regulating gene expression and forms part of the epigenetic memory system that regulates cell fate and identity by still poorly understood mechanisms. Here, we examined the role of histone demethylase Kdm3a during cell differentiation, showing that Kdm3a is essential for differentiation into parietal endoderm-like (PE) cells in the F9 mouse embryonal carcinoma model. We identified a number of target genes regulated by Kdm3a during endoderm differentiation; among the most dysregulated were the three developmental master regulators Dab2, Pdlim4 and FoxQ1. We show that dysregulation of the expression of these genes correlates with Kdm3a H3K9me2 demethylase activity. We further demonstrate that either Dab2 depletion or Kdm3a depletion prevents F9 cells from fully differentiating into PE cells, but that ectopic expression of Dab2 cannot compensate for Kdm3a knockdown; Dab2 is thus necessary, but insufficient on its own, to promote complete terminal differentiation. We conclude that Kdm3a plays a crucial role in progression through PE differentiation by regulating expression of a set of endoderm differentiation master genes. The emergence of Kdm3a as a key modulator of cell fate decision strengthens the view that histone demethylases are essential to cell differentiation.

Natural selection of adaptive mutations in non-structural genes increases trans-encapsidation of hepatitis C virus replicons lacking envelope protein genes.

A trans-packaging system for hepatitis C virus (HCV) replicons lacking envelope glycoproteins was developed. The replicons were efficiently encapsidated into infectious particles after expression in trans of homologous HCV envelope proteins under the control of an adenoviral vector. Interestingly, expression in trans of core or core, p7 and NS2 with envelope proteins did not enhance trans-encapsidation. Expression of heterologous envelope proteins, in the presence or absence of heterologous core, p7 and NS2, did not rescue single-round infectious particle production. To increase the titre of homologous, single-round infectious particles in our system, successive cycles of trans-encapsidation and infection were performed. Four cycles resulted in a 100-fold increase in the yield of particles. Sequence analysis revealed a total of 16 potential adaptive mutations in two independent experiments. Except for a core mutation in one experiment, all the mutations were located in non-structural regions mainly in NS5A (four in domain III and two near the junction with the NS5B gene). Reverse genetics studies suggested that D2437A and S2443T adaptive mutations, which are located at the NS5A-B cleavage site did not affect viral replication, but enhanced the single-round infectious particles assembly only in trans-encapsidation model. In conclusion, our trans-encapsidation system enables the production of HCV single-round infectious particles. This system is adaptable and can positively select variants. The adapted variants promote trans-encapsidation and should constitute a valuable tool in the development of replicon-based HCV vaccines.

Unravelling the epigenomic dimension of breast cancers.

PURPOSE OF REVIEW: Breast cancer remains the first cause of cancer-related mortality in women. This can be explained by the high histological and molecular heterogeneity of the disease, making it hard to choose a therapy adapted to each patient. Over this last year, several groups have evaluated the epigenetic component of breast cancer, as epigenetics appears to be important in carcinogenesis. Results suggest that assessing the epigenetic aspects of breast tumours could strongly improve our understanding of the biology and heterogeneity of breast cancers. RECENT FINDINGS: The heterogeneity of breast tumours described at the histological and transcriptional levels exists also at the epigenetic level. DNA methylation profiles both confirm previous observations based on histological and transcriptomic analyses and add new levels of complexity to the picture. Several methylation signatures have been evidenced, that can stratify patients in terms of prognosis. DNA methylation profiles could also be a marker of lineage restriction, reflecting the cell type from which a tumour originates. SUMMARY: While focusing mainly on gene promoters and CpG islands, the genome-scale DNA methylation profiling studies, conducted last year, highlighted the need to evaluate the epigenetic component in order to gain better knowledge of breast cancer biology, thereby, to improve patient management. Clearly, however, the epigenomic exploration of breast cancers has only just begun, recent studies having revealed the importance of DNA methylation in regions such as gene bodies and intergenic regions that still need to be explored.

Immunity drives TET1 regulation in cancer through NF-κB.

Ten-eleven translocation enzymes (TET1, TET2, and TET3), which induce DNA demethylation and gene regulation by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), are often down-regulated in cancer. We uncover, in basal-like breast cancer (BLBC), genome-wide 5hmC changes related to TET1 regulation. We further demonstrate that TET1 repression is associated with high expression of immune markers and high infiltration by immune cells. We identify in BLBC tissues an anticorrelation between TET1 expression and the major immunoregulator family nuclear factor κB (NF-κB). In vitro and in mice, TET1 is down-regulated in breast cancer cells upon NF-κB activation through binding of p65 to its consensus sequence in the TET1 promoter. We lastly show that these findings extend to other cancer types, including melanoma, lung, and thyroid cancers. Together, our data suggest a novel mode of regulation for TET1 in cancer and highlight a new paradigm in which the immune system can influence cancer cell epigenetics.

A focus reduction neutralization assay for hepatitis C virus neutralizing antibodies.

BACKGROUND/AIM: The role of humoral immunity in hepatitis C virus (HCV) infection is poorly understood. Nevertheless, there is increasing interest in characterizing the neutralizing antibodies in the serum of HCV-infected patients. Focus reduction assays have been widely used to evaluate neutralizing antibody responses against a range of non-cytopathic viruses. Based on the recent development of a HCV cell culture system using the genotype 2 JFH-1-strain, we developed a focus reduction assay for HCV-neutralizing antibodies. METHODS: The focus reduction assay was based on a standard microneutralization assay in which immunostained foci on tissue culture plates are counted. The neutralizing anti-HCV antibodies titers of purified serum immunoglobulin samples from seventy-seven individuals were determined using a 50% focus reduction neutralization assay. Each titer was determined as the log value of the reciprocal antibody dilution that reduced the number of viral foci by 50%. IgG antibodies were first purified from each serum in order to avoid the facilitating effect of HDL on HCV entry. RESULTS: The assay's cut-off using an ELISA and RNA HCV-negative samples was found to be 1.25 log, corresponding to a dilution of 1:18. The assay was compared with a commercial HCV ELISA and exhibited specificity and sensitivity values of 100% and 96.5%, respectively, and good reproducibility (with intra-assay and inter-assay coefficients of variation of 6.7% and 12.6%, respectively). The assay did not show any cross-reactivity with anti-HIV, anti-HBs or heterophile antibody-positive samples. The neutralizing antibodies titers were 2.13 log (1:134) for homologous samples from HCV genotype 2 infected patients harboring the same genotype as JFH-1 and 1.93 log (1:85) for heterologous samples from patients infected by genotypes other than type 2. These results confirm the presence of broadly cross-neutralizing antibodies already reported using the HCV pseudoparticles system. CONCLUSION: This study presents a simple, specific and reproducible cell culture-based assay for determination of HCV-neutralizing antibodies in human sera. The assay should be an important tool for gauging the relationship between the neutralizing antibodies response and viral load kinetics in acutely or chronically infected patients and for investigating the possible eradication or prevention of HCV infection by neutralizing antibodies.

DNA methylation-based immune response signature improves patient diagnosis in multiple cancers.

BACKGROUND: The tumor immune response is increasingly associated with better clinical outcomes in breast and other cancers. However, the evaluation of tumor-infiltrating lymphocytes (TILs) relies on histopathological measurements with limited accuracy and reproducibility. Here, we profiled DNA methylation markers to identify a methylation of TIL (MeTIL) signature that recapitulates TIL evaluations and their prognostic value for long-term outcomes in breast cancer (BC). METHODS: MeTIL signature scores were correlated with clinical endpoints reflecting overall or disease-free survival and a pathologic complete response to preoperative anthracycline therapy in 3 BC cohorts from the Jules Bordet Institute in Brussels and in other cancer types from The Cancer Genome Atlas. RESULTS: The MeTIL signature measured TIL distributions in a sensitive manner and predicted survival and response to chemotherapy in BC better than did histopathological assessment of TILs or gene expression-based immune markers, respectively. The MeTIL signature also improved the prediction of survival in other malignancies, including melanoma and lung cancer. Furthermore, the MeTIL signature predicted differences in survival for malignancies in which TILs were not known to have a prognostic value. Finally, we showed that MeTIL markers can be determined by bisulfite pyrosequencing of small amounts of DNA from formalin-fixed, paraffin-embedded tumor tissue, supporting clinical applications for this methodology. CONCLUSIONS: This study highlights the power of DNA methylation to evaluate tumor immune responses and the potential of this approach to improve the diagnosis and treatment of breast and other cancers. FUNDING: This work was funded by the Fonds National de la Recherche Scientifique (FNRS) and Télévie, the INNOVIRIS Brussels Region BRUBREAST Project, the IUAP P7/03 program, the Belgian "Foundation against Cancer," the Breast Cancer Research Foundation (BCRF), and the Fonds Gaston Ithier.

The interplay between the lysine demethylase KDM1A and DNA methyltransferases in cancer cells is cell cycle dependent.

DNA methylation and histone modifications are key epigenetic regulators of gene expression, and tight connections are known between the two. DNA methyltransferases are upregulated in several tumors and aberrant DNA methylation profiles are a cancer hallmark. On the other hand, histone demethylases are upregulated in cancer cells. Previous work on ES cells has shown that the lysine demethylase KDM1A binds to DNMT1, thereby affecting DNA methylation. In cancer cells, the occurrence of this interaction has not been explored. Here we demonstrate in several tumor cell lines an interaction between KDM1A and both DNMT1 and DNMT3B. Intriguingly and in contrast to what is observed in ES cells, KDM1A depletion in cancer cells was found not to trigger any reduction in the DNMT1 or DNMT3B protein level or any change in DNA methylation. In the S-phase, furthermore, KDM1A and DNMT1 were found, to co-localize within the heterochromatin. Using P-LISA, we revealed substantially increased binding of KDM1A to DNMT1 during the S-phase. Together, our findings propose a mechanistic link between KDM1A and DNA methyltransferases in cancer cells and suggest that the KDM1A/DNMT1 interaction may play a role during replication. Our work also strengthens the idea that DNMTs can exert functions unrelated to act on DNA methylation.

MicroRNAs regulate KDM5 histone demethylases in breast cancer cells.

MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression. Alteration of miRNA levels is common in tumors and contributes to the pathogenesis of human malignancies. In the present study we examined the role played by miR-137 in breast tumorigenesis. We found miR-137 levels to be lower in breast cancer cells than in their non-tumorigenic counterparts and observed reduced proliferation and migration of breast cancer cells overexpressing miR-137. We further identified KDM5B, a histone demethylase known to be involved in breast cancer tumorigenesis, as a target of miR-137. As the involvement of histone demethylases in cancer is still poorly understood and as the role of miRNAs in controlling epigenetic mechanisms in cancer is emerging, we broadened our study to the whole KDM5 histone demethylase family to see if the genes coding for these epigenetic enzymes might be regulated by miRNAs in cancer cells. We discovered that KDM5C is overexpressed in breast cancer cells, providing evidence that miR-138 regulates its expression. We found miR-138 overexpression to affect breast cancer cell proliferation. Altogether, our findings suggest that miRNAs may regulate KDM5 histone demethylase levels in breast cancer and thereby control breast cancer cell proliferation and migration.

Dietary flavanols modulate the transcription of genes associated with cardiovascular pathology without changes in their DNA methylation state.

BACKGROUND: In a recent intervention study, the daily supplementation with 200 mg monomeric and oligomeric flavanols (MOF) from grape seeds for 8 weeks revealed a vascular health benefit in male smokers. The objective of the present study was to determine the impact of MOF consumption on the gene expression profile of leukocytes and to assess changes in DNA methylation. METHODOLOGY/PRINCIPAL FINDINGS: Gene expression profiles were determined using whole genome microarrays (Agilent) and DNA methylation was assessed using HumanMethylation450 BeadChips (Illumina). MOF significantly modulated the expression of 864 genes. The majority of the affected genes are involved in chemotaxis, cell adhesion, cell infiltration or cytoskeleton organisation, suggesting lower immune cell adhesion to endothelial cells. This was corroborated by in vitro experiments showing that MOF exposure of monocytes attenuates their adhesion to TNF-α-stimulated endothelial cells. Nuclear factor kappa B (NF-κB) reporter gene assays confirmed that MOF decrease the activity of NF-κB. Strong inter-individual variability in the leukocytes' DNA methylation was observed. As a consequence, on group level, changes due to MOF supplementation could not be found. CONCLUSION: Our study revealed that an 8 week daily supplementation with 200 mg MOF modulates the expression of genes associated with cardiovascular disease pathways without major changes of their DNA methylation state. However, strong inter-individual variation in leukocyte DNA methylation may obscure the subtle epigenetic response to dietary flavanols. Despite the lack of significant changes in DNA methylation, the modulation of gene expression appears to contribute to the observed vascular health effect of MOF in humans.

Regulation of DNA methylation patterns by CK2-mediated phosphorylation of Dnmt3a.

DNA methylation is a central epigenetic modification that is established by de novo DNA methyltransferases. The mechanisms underlying the generation of genomic methylation patterns are still poorly understood. Using mass spectrometry and a phosphospecific Dnmt3a antibody, we demonstrate that CK2 phosphorylates endogenous Dnmt3a at two key residues located near its PWWP domain, thereby downregulating the ability of Dnmt3a to methylate DNA. Genome-wide DNA methylation analysis shows that CK2 primarily modulates CpG methylation of several repeats, most notably of Alu SINEs. This modulation can be directly attributed to CK2-mediated phosphorylation of Dnmt3a. We also find that CK2-mediated phosphorylation is required for localization of Dnmt3a to heterochromatin. By revealing phosphorylation as a mode of regulation of de novo DNA methyltransferase function and by uncovering a mechanism for the regulation of methylation at repetitive elements, our results shed light on the origin of DNA methylation patterns.

DNA methylation markers for breast cancer prognosis: Unmasking the immune component.

Currently, most of the prognostic and predictive gene expression signatures emerging for breast cancer concern the tumor component. In Dedeurwaerder et al. we show that DNA methylation profiling of breast tumors is a particularly sensitive means of capturing features of the immune component of breast tumors. Most importantly, correlation is observed between T-cell marker genes and breast cancer clinical outcome.

Evaluation of the Infinium Methylation 450K technology.

AIMS: Studies of DNA methylomes hold enormous promise for biomedicine but are hampered by the technological challenges of analyzing many samples cost-effectively. Recently, a major extension of the previous Infinium HumanMethylation27 BeadChip® (Illumina, Inc. CA, USA), called Infinium HumanMethylation450 (Infinium Methylation 450K; Illumina, Inc. CA, USA) was developed. This upgraded technology is a hybrid of two different chemical assays, the Infinium I and Infinium II assays, allowing (for 12 samples in parallel) assessment of the methylation status of more than 480,000 cytosines distributed over the whole genome. In this article, we evaluate Infinium Methylation 450K on cell lines and tissue samples, highlighting some of its advantages but also some of its limitations. In particular, we compare the methylation values of the Infinium I and Infinium II assays. MATERIALS & METHODS: We used Infinium Methylation 450K to profile: first, the well-characterized HCT116 wild-type and double-knockout cell lines and then, 16 breast tissue samples (including eight normal and eight primary tumor samples). Absolute methylation values (ß-values) were extracted with the GenomeStudio™ software and then subjected to detailed analysis. RESULTS: While this technology appeared highly robust as previously shown, we noticed a divergence between the ß-values retrieved from the type I and type II Infinium assays. Specifically, the ß-values obtained from Infinium II probes were less accurate and reproducible than those obtained from Infinium I probes. This suggests that data from the type I and type II assays should be considered separately in any downstream bioinformatic analysis. To be able to deal with the Infinium I and Infinium II data together, we developed and tested a new correction technique, which we called 'peak-based correction'. The idea was to rescale the Infinium II data on the basis of the Infinium I data. While this technique should be viewed as an approximation method, it significantly improves the quality of Infinium II data. CONCLUSION: Infinium 450K is a powerful technique in terms of reagent costs, time of labor, sample throughput and coverage. It holds great promise for the better understanding of the epigenetic component in health and disease. Yet, due to the nature of its design comprising two different chemical assays, analysis of the whole set of data is not as easy as initially anticipated. Correction strategies, such as the peak-based approach proposed here, are a step towards adequate output data analysis.

DNA methylation profiling reveals a predominant immune component in breast cancers.

Breast cancer is a molecularly, biologically and clinically heterogeneous group of disorders. Understanding this diversity is essential to improving diagnosis and optimizing treatment. Both genetic and acquired epigenetic abnormalities participate in cancer, but the involvement of the epigenome in breast cancer and its contribution to the complexity of the disease are still poorly understood. By means of DNA methylation profiling of 248 breast tissues, we have highlighted the existence of previously unrecognized breast cancer groups that go beyond the currently known 'expression subtypes'. Interestingly, we showed that DNA methylation profiling can reflect the cell type composition of the tumour microenvironment, and in particular a T lymphocyte infiltration of the tumours. Further, we highlighted a set of immune genes having high prognostic value in specific tumour categories. The immune component uncovered here by DNA methylation profiles provides a new perspective for the importance of the microenvironment in breast cancer, holding implications for better management of breast cancer patients.

Activity of an atypical Arabidopsis thaliana pectin methylesterase.

An Arabidopsis thaliana pectin methylesterase that was not predicted to contain any signaling sequence was produced in E. coli and purified using a His tag added at its N-terminus. The enzyme demethylesterified Citrus pectin with a Km of 0.86 mg/ml. The enzyme did not require salt for activity and was found to be relatively temperature-sensitive. The precipitation of enzyme-treated pectin by CaCl2 suggested that the enzyme had a blockwise mode of pectin demethylesterification. A purified kiwi (Actinidia chinensis) pectin methylesterase inhibitor had no effect on the activity of the enzyme whereas it strongly inhibited a flax pectin methylesterase. A model of the protein structure revealed that an extra amino acid sequence in this particular Arabidopsis pectin methylesterase could form a ss-strand outside the core structure, which might be preventing the inhibitor from binding the protein.