A distinct DNA methylation signature defines pediatric pre-B cell acute lymphoblastic leukemia

Pre-B cell acute lymphoblastic leukemia (ALL) is the most prevalent childhood malignancy and remains one of the highest causes of childhood mortality. Despite this, the mechanisms leading to disease remain poorly understood. We asked if recurrent aberrant DNA methylation plays a role in childhood ALL and have defined a genome-scale DNA methylation profile associated with the ETV6-RUNX1 subtype of pediatric ALL. Archival bone marrow smears from 19 children collected at diagnosis and remission were used to derive a disease specific DNA methylation profile. The gene signature was confirmed in an independent cohort of 86 patients. A further 163 patients were analyzed for DNA methylation of a three gene signature. We found that the DNA methylation signature at diagnosis was unique from remission. Fifteen loci were sufficient to discriminate leukemia from disease-free samples and purified CD34+ cells. DNA methylation of these loci was recurrent irrespective of cytogenetic subtype of pre-B cell ALL. We show that recurrent aberrant genomic methylation is a common feature of pre-B ALL, suggesting a shared pathway for disease development. By revealing new DNA methylation markers associated with disease, this study has identified putative targets for development of novel epigenetic-based therapies.

Folic acid supplementation in postpolypectomy patients in a randomized controlled trial increases tissue folate concentrations and reduces aberrant DNA biomarkers in colonic tissues adjacent to the former polyp site

BACKGROUND: Low folate status is associated with an increased risk of colorectal carcinogenesis. Optimal folate status may be genoprotective by preventing uracil misincorporation into DNA and DNA hypomethylation. Adenomatous polyps have low folate status compared with normal colonic mucosa, and they are surrounded by histologically normal mucosa that also is of low folate status. OBJECTIVE: In a randomized controlled trial conducted at a single Dublin hospital between April 2002 and March 2004, we assessed the effect of folic acid supplementation on tissue folate, uracil misincorporation into DNA, and global DNA hypomethylation in colonocytes isolated from sites of adenomatous polyps and from histologically normal tissue adjacent and 10-15 cm distal to them. METHODS: Twenty patients with adenomatous polyps on initial colonoscopy and polypectomy were randomly assigned to receive either 600 μg folic acid/d [n = 12, 38% men, mean age 64.3 y, and body mass index (BMI, in kg/m(2)) 26.6] or placebo (n = 8, 50% men, mean age 68.4 y, and BMI 27.2) for 6 mo, and then repeat the colonoscopy. Blood and colonocyte tissue folate concentrations were measured with the use of a microbiological assay. Uracil misincorporation and global DNA hypomethylation were measured in colonocytes with the use of modified comet assays. RESULTS: Over time, folic acid supplementation, compared with placebo, increased tissue folate (mean ± SEM) from 15.6 ± 2.62 pg/10(5) cells to 18.1 ± 2.12 pg/10(5) cells (P < 0.001) and decreased the global DNA hypomethylation ratio from 1.7 ± 0.1 to 1.0 ± 0.1 (P < 0.001). The uracil misincorporation ratio decreased by 0.5 ± 0.1 for the site adjacent to the polyp over time (P = 0.05). CONCLUSION: A response to folic acid supplementation, which increased colonocyte folate and improved folate-related DNA biomarkers of cancer risk, was seen in the participants studied. Exploratory analysis points toward the area formerly adjacent to polyps as possibly driving the response. That these areas persist after polypectomy in the absence of folate supplementation is consistent with a potentially carcinogenic field's causing the appearance of the polyp.

Extensible motif and aberrant nucleotide extraction in genomic sequences

The project focusses on the discovery of conserved DNA sequences in bacterial genomes and comparative analysis of bacterial genomes to elicit evolutionary trends. The outcomes have produced novel techniques for modelling motifs in DNA and the characterisation of evolutionary processes in medically significant bacterial pathogens.

Functions for S. cerevisiae Swd2p in 3' end formation of specific mRNAs and snoRNAs and global histone 3 lysine 4 methylation

The Saccharomyces cerevisiae WD-40 repeat protein Swd2p associates with two functionally distinct multiprotein complexes: the cleavage and polyadenylation factor (CPF) that is involved in pre-mRNA and snoRNA 3′ end formation and the SET1 complex (SET1C) that methylates histone 3 lysine 4. Based on bioinformatic analysis we predict a seven-bladed β-propeller structure for Swd2p proteins. Northern, transcriptional run-on and in vitro 3′ end cleavage analyses suggest that temperature sensitive swd2 strains were defective in 3′ end formation of specific mRNAs and snoRNAs. Protein–protein interaction studies support a role for Swd2p in the assembly of 3′ end formation complexes. Furthermore, histone 3 lysine 4 di-and tri-methylation were adversely affected and telomeres were shortened in swd2 mutants. Underaccumulation of the Set1p methyltransferase accounts for the observed loss of SET1C activity and suggests a requirement for Swd2p for the stability or assembly of this complex. We also provide evidence that the roles of Swd2p as component of CPF and SET1C are functionally independent. Taken together, our results establish a dual requirement for Swd2p in 3′ end formation and histone tail modification.

Protein interactions within the SET1 complex and their roles in the regulation of histone 3 lysine 4 methylation

Set1 is the catalytic subunit and the central component of the evolutionarily conserved Set1 complex (Set1C) that methylates histone 3 lysine 4 (H3K4). Here we have determined protein/protein interactions within the complex and related the substructure to function. The loss of individual Set1C subunits differentially affects Set1 stability, complex integrity, global H3K4 methylation, and distribution of H3K4 methylation along active genes. The complex requires Set1, Swd1, and Swd3 for integrity, and Set1 amount is greatly reduced in the absence of the Swd1-Swd3 heterodimer. Bre2 and Sdc1 also form a heteromeric subunit, which requires the SET domain for interaction with the complex, and Sdc1 strongly interacts with itself. Inactivation of either Bre2 or Sdc1 has very similar effects. Neither is required for complex integrity, and their removal results in an increase of H3K4 mono- and dimethylation and a severe decrease of trimethylation at the 5′ end of active coding regions but a decrease of H3K4 dimethylation at the 3′ end of coding regions. Cells lacking Spp1 have a reduced amount of Set1 and retain a fraction of trimethylated H3K4, whereas cells lacking Shg1 show slightly elevated levels of both di- and trimethylation. Set1C associates with both serine 5- and serine 2-phosphorylated forms of polymerase II, indicating that the association persists to the 3′ end of transcribed genes. Taken together, our results suggest that Set1C subunits stimulate Set1 catalytic activity all along active genes.

The COMPASS subunit Spp1 links histone methylation to initiation of meiotic recombination

During meiosis, combinatorial associations of genetic traits arise from homologous recombination between parental chromosomes. Histone H3 lysine 4 trimethylation marks meiotic recombination hotspots in yeast and mammals, but how this ubiquitous chromatin modification relates to the initiation of double-strand breaks (DSBs) dependent on Spo11 remains unknown. Here, we show that the tethering of a PHD-containing protein, Spp1 (a component of the COMPASS complex), to recombinationally cold regions is sufficient to induce DSB formation. Furthermore, we found that Spp1 physically interacts with Mer2, a key protein of the differentiated chromosomal axis required for DSB formation. Thus, by interacting with H3K4me3 and Mer2, Spp1 promotes recruitment of potential meiotic DSB sites to the chromosomal axis, allowing Spo11 cleavage at nearby nucleosome-depleted regions.

Aberrant cytokine signaling in ovarian cancer

The studies described in this thesis suggest that IL-6R family members have the potential to significantly impact on ovarian cancer progression. These results suggest that assessment of IL-6R family expression represents a potential prognostic tool in determining appropriate therapy for ovarian cancer patients.

Validation of DNA methylation biomarkers for diagnosis of acute lymphoblastic leukemia

DNA methylation biomarkers capable of diagnosis and subtyping have been found for many cancers. Fifteen such markers have previously been identified for pediatric acute lymphoblastic leukemia (ALL). Validation of these markers is necessary to assess their clinical utility for molecular diagnostics. Substantial efficiencies could be achieved with these DNA methylation markers for disease tracking with potential to replace patient-specific genetic testing.

Paediatric leukaemia DNA methylation profiling using MBD enrichment and SOLiD sequencing on archival bone marrow smears

BACKGROUND: Acute Lymphoblastic Leukaemia (ALL) is the most common cancer in children. Over the past four decades, research has advanced the treatment of this cancer from a less than 60% chance of survival to over 85% today. The causal molecular mechanisms remain unclear. Here, we performed sequencing-based genomic DNA methylation profiling of eight paediatric ALL patients using archived bone marrow smear microscope slides. FINDINGS: SOLiD™ sequencing data was collected from Methyl-Binding Domain (MBD) enriched fractions of genomic DNA. The primary tumour and remission bone marrow sample was analysed from eight patients. Four patients relapsed and the relapsed tumour was analysed. Input and MBD-enriched DNA from each sample was sequenced, aligned to the hg19 reference genome and analysed for enrichment peaks using MACS (Model-based Analysis for ChIP-Seq) and HOMER (Hypergeometric Optimization of Motif EnRichment). In total, 3.67 gigabases (Gb) were sequenced, 2.74 Gb were aligned to the reference genome (average 74.66% alignment efficiency). This dataset enables the interrogation of differential DNA methylation associated with paediatric ALL. Preliminary results reveal concordant regions of enrichment indicative of a DNA methylation signature. CONCLUSION: Our dataset represents one of the first SOLiD™MBD-Seq studies performed on paediatric ALL and is the first to utilise archival bone marrow smears. Differential DNA methylation between cancer and equivalent disease-free tissue can be identified and correlated with existing and published genomic studies. Given the rarity of paediatric haematopoietic malignancies, relative to adult counterparts, our demonstration of the utility of archived bone marrow smear samples to high-throughput methylation sequencing approaches offers tremendous potential to explore the role of DNA methylation in the aetiology of cancer.