Chromatin is not an inert structure, but rather an instructive DNA scaffold that can respond to external cues to regulate the many uses of DNA. A principle component of chromatin that plays a key role in this regulation is the modification of histones. There is an ever-growing list of these modifications and the complexity of their action is only just beginning to be understood. However, it is clear that histone modifications play fundamental roles in most biological processes that are involved in the manipulation and expression of DNA. Here, we describe the known histone modifications, define where they are found genomically and discuss some of their functional consequences, concentrating mostly on transcription where the majority of characterisation has taken place.

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Regulation of chromatin by histone modifications

Polycomb group proteins maintain the gene-expression pattern of different cells that is set during early development by regulating chromatin structure. In mammals, two main Polycomb group complexes exist — Polycomb repressive complex 1 (PRC1) and 2 (PRC2). PRC1 compacts chromatin and catalyses the monoubiquitylation of histone H2A. PRC2 also contributes to chromatin compaction, and catalyses the methylation of histone H3 at lysine 27. PRC2 is involved in various biological processes, including differentiation, maintaining cell identity and proliferation, and stem-cell plasticity. Recent studies of PRC2 have expanded our perspectives on its function and regulation, and uncovered a role for non-coding RNA in the recruitment of PRC2 to target genes.

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The Polycomb complex PRC2 and its mark in life

Cancer epigenetics: from mechanism to therapy.

The past decade has highlighted the central role of epigenetic processes in cancer causation, progression and treatment. Next-generation sequencing is providing a window for visualizing the human epigenome and how it is altered in cancer. This view provides many surprises, including linking epigenetic abnormalities to mutations in genes that control DNA methylation, the packaging and the function of DNA in chromatin, and metabolism. Epigenetic alterations are leading candidates for the development of specific markers for cancer detection, diagnosis and prognosis. The enzymatic processes that control the epigenome present new opportunities for deriving therapeutic strategies designed to reverse transcriptional abnormalities that are inherent to the cancer epigenome.

http://sdjohnston.faculty.noctrl.edu/360/Cancer%20Epigenome.pdf

A decade of exploring the cancer epigenome — biological and translational implications

High-throughput DNA sequencing significantly contributed to diagnosis and prognostication in patients with myelodysplastic syndromes (MDS). We determined the biological and prognostic significance of genetic aberrations in MDS. In total, 944 patients with various MDS subtypes were screened for known/putative mutations/deletions in 104 genes using targeted deep sequencing and array-based genomic hybridization. In total, 845/944 patients (89.5%) harbored at least one mutation (median, 3 per patient; range, 0-12). Forty-seven genes were significantly mutated with TET2, SF3B1, ASXL1, SRSF2, DNMT3A, and RUNX1 mutated in >10% of cases. Many mutations were associated with higher risk groups and/or blast elevation. Survival was investigated in 875 patients. By univariate analysis, 25/48 genes (resulting from 47 genes tested significantly plus PRPF8) affected survival (P<0.05). The status of 14 genes combined with conventional factors revealed a novel prognostic model ('Model-1') separating patients into four risk groups ('low', 'intermediate', 'high', 'very high risk') with 3-year survival of 95.2, 69.3, 32.8, and 5.3% (P<0.001). Subsequently, a 'gene-only model' ('Model-2') was constructed based on 14 genes also yielding four significant risk groups (P<0.001). Both models were reproducible in the validation cohort (n=175 patients; P<0.001 each). Thus, large-scale genetic and molecular profiling of multiple target genes is invaluable for subclassification and prognostication in MDS patients.

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Landscape of genetic lesions in 944 patients with myelodysplastic syndromes

In myelodysplastic syndromes (MDS), deletions of chromosome 7 or 7q are common and correlate with a poor prognosis. The relevant genes on chromosome 7 are unknown. We report here that EZH2, located at 7q36.1, is frequently targeted in MDS. Analysis of EZH2 deletions, missense and frameshift mutations strongly suggests that EZH2 is a tumor suppressor. As EZH2 functions as a histone methyltransferase, abnormal histone modification may contribute to epigenetic deregulation in MDS.

/pdf/somatic-mutations-of-the-histone-methyltransferase-gene-ezh2-48ecnkb498.pdf

Somatic mutations of the histone methyltransferase gene EZH2 in myelodysplastic syndromes

High BRE expression predicts favorable outcome in adult acute myeloid leukemia, in particular among MLL-AF9 positive patients

Breast cancer is one of the leading causes of cancer mortality in women. Recent advances in gene expression profiling have indicated that breast cancer is a heterogeneous disease and the current prognostication using clinico-pathological features is not sufficient to fully predict therapy response and disease outcome. In this retrospective study, we show that expression levels of BRE, which encodes a member of the BRCA1 DNA damage repair complex, predicted disease-free survival (DFS) in non-familial breast cancer patients. The predictive value of BRE expression depended on whether patients received radiotherapy as a part of their primary treatment. In radiotherapy-treated patients, high BRE expression predicted a favorable DFS (hazard ratio (HR) = 0.47, 95 % confidence interval (CI) = 0.28–0.78, p = 0.004), while in non-treated patients, high BRE expression predicted an adverse prognosis (HR = 2.59, 95 % CI = 1.00–6.75, p = 0.05). Among radiotherapy-treated patients, the prognostic impact of BRE expression was confined to patients with smaller tumors (HR = 0.23, 95 % CI = 0.068–0.75, p = 0.015) and it remained an independent factor after correction for the other prognostic factors age, tumor size, lymph node involvement, and histological grade (HR = 0.50, CI = 0.27–0.90, p = 0.021). In addition, high BRE expression predicted a favorable relapse-free survival in a publicly available dataset of 2,324 breast cancer patients (HR = 0.59, CI = 0.51–0.68, p < 0.001). These data indicate that BRE is an interesting candidate for future functional studies aimed at developing targeted therapies.

/pdf/expression-of-the-brca1-complex-member-bre-predicts-disease-aelxm4feoa.pdf

Expression of the BRCA1 complex member BRE predicts disease free survival in breast cancer.

Screening for recurrent mutations in leukaemia is becoming increasingly important because many of them have an impact on disease outcome. Nowadays, mutational detection is mostly based on DNA sequencing of PCR products or QPCR using allele specific probes. With the increasing identification of recurrent mutations in cancer, the development of fast and efficient approaches for mutational screening is needed. An alternative technique for mutation detection is high resolution melting (HRM) analysis (Wittwer, 2009). HRM analysis is a fast ‘single-well-technique’ combining PCR using a fluorescent saturating dye that is intercalated in the DNA, and melt curve analysis afterwards. As mutations cause different melting properties, they are identified when compared to non-mutated samples. Positive samples in HRM analysis can be sequenced subsequently to determine exact nucleotide changes. Recently, mutations in the NADP-dependent isocitrate dehydrogenase genes IDH1 and IDH2 were shown in acute myeloid leukaemia (AML), myeloid dysplastic syndromes and myeloproliferative diseases (Mardis et al, 2009; Abbas et al, 2010; Chou et al, 2010; Green & Beer, 2010; Kosmider et al, 2010; Marcucci et al, 2010; Wagner et al, 2010). We study the application of HRM analysis and direct sequencing to screen for these mutations in a cohort of 168 AML patients, using the real-time PCR platform of Applied Biosystems (Carlsbad, CA, USA). Sequence analysis detected IDH1 or IDH2 mutations in 32 patients (19%) (Tables SI and SII). Using the methods as described in the Data S1, mutations could also be detected by HRM analysis (used primer combinations are indicated in Table SIII). Different primers with and without M13sequences were tested, showing differential results for IDH1 or IDH2 mutations (Fig 1). Using HRM analysis, no mutations were missed. For IDH1 R132, 12 mutated samples (7Æ1%) were found both by HRM and sequence analysis while an additional three positive samples (1Æ8%) were found only by HRM analysis. To elucidate whether the extra mutations found by HRM analysis were false positives, we compared the sensitivity of both techniques. For this, a dilution series of genomic DNA from a mutated sample was measured by HRM analysis followed by direct sequencing. Positivity in HRM analysis was lost at an allele frequency of approximately 9%, whereas the mutation could still be detected at a percentage of 4% by sequencing (Fig S1). As sequencing was more sensitive, this suggests that the extra positive samples found by HRM analysis, that did not contain any SNPs, represent false positive results. Nineteen samples were found positive for the IDH2 R140 mutation (11Æ3%) by sequence analysis. Initial HRM analysis for this mutation identified many false positive results. However, exclusion of samples with low PCR yields (Ct>26Æ8, eight samples) significantly improved the discrimination between mutated and wild type samples. After exclusion, all positive samples found by sequencing were confirmed by HRM analysis while two cases remained false positive. R172 IDH2 mutations were found in two patients (1Æ2%) both by HRM and sequence analysis and two false positive samples were found by HRM (1Æ2%) (Tables SI and SII). One patient showed both an IDH1 R132C and IDH2 R140Q mutation. Allele frequencies of the mutations were measured by pyrosequencing (primer combinations are specified in Table SIV). The allele frequency of the IDH1 R132C mutation was 15Æ5%, while the IDH2 R140Q frequency was even lower (data not shown). Because IDH1 and IDH2 mutations are typically heterozygous and the total allele frequencies of both mutations was below 50%, we cannot conclude whether the mutations represent one and the same or independent clones. One patient exhibited a homozygous IDH2 R140W mutation, identified by both sequence and HRM analysis (Fig S2). SNP array analysis showed that the homozygous mutation was caused by uniparental disomy (UPD) on a large part of chromosome 15, including the IDH2 locus (Fig S3). Of the 32 patients with IDH mutations, we found cooccurrences with other chromosomal aberrations confirming data of other studies (Chou et al, 2010; Kosmider et al, 2010; Marcucci et al, 2010; Paschka et al, 2010; Wagner et al, 2010). Furthermore, none of the IDH1 or IDH2 mutated patients showed overexpression of MECOM (also known as EVI1) (Table SI). Low frequencies of mutated alleles may be missed using sequencing or HRM analysis. To test whether IDH1 mutations occur at frequencies under the detection limit of HRM analysis, we designed an allele specific QPCR for the most frequently occurring IDH1 R132H mutation (for primer

Evelyn Tönnissen

Papers

Somatic mutations of the histone methyltransferase gene EZH2 in myelodysplastic syndromes

High BRE expression predicts favorable outcome in adult acute myeloid leukemia, in particular among MLL-AF9 positive patients

Expression of the BRCA1 complex member BRE predicts disease free survival in breast cancer.

Rapid identification of IDH1 and IDH2 mutations in acute myeloid leukaemia using high resolution melting curve analysis