Tracy Ballinger

Conserved role of intragenic DNA methylation in regulating alternative promoters.

Although it is known that the methylation of DNA in 5′ promoters suppresses gene expression, the role of DNA methylation in gene bodies is unclear. In mammals, tissue- and cell type-specific methylation is present in a small percentage of 5′ CpG island (CGI) promoters, whereas a far greater proportion occurs across gene bodies, coinciding with highly conserved sequences. Tissue-specific intragenic methylation might reduce, or, paradoxically, enhance transcription elongation efficiency. Capped analysis of gene expression (CAGE) experiments also indicate that transcription commonly initiates within and between genes. To investigate the role of intragenic methylation, we generated a map of DNA methylation from the human brain encompassing 24.7 million of the 28 million CpG sites. From the dense, high-resolution coverage of CpG islands, the majority of methylated CpG islands were shown to be in intragenic and intergenic regions, whereas less than 3% of CpG islands in 5′ promoters were methylated. The CpG islands in all three locations overlapped with RNA markers of transcription initiation, and unmethylated CpG islands also overlapped significantly with trimethylation of H3K4, a histone modification enriched at promoters. The general and CpG-island-specific patterns of methylation are conserved in mouse tissues. An in-depth investigation of the human SHANK3 locus and its mouse homologue demonstrated that this tissue-specific DNA methylation regulates intragenic promoter activity in vitro and in vivo. These methylation-regulated, alternative transcripts are expressed in a tissue- and cell type-specific manner, and are expressed differentially within a single cell type from distinct brain regions. These results support a major role for intragenic methylation in regulating cell context-specific alternative promoters in gene bodies.

Genome-wide analysis of Arabidopsis thaliana DNA methylation uncovers an interdependence between methylation and transcription

Cytosine methylation, a common form of DNA modification that antagonizes transcription, is found at transposons and repeats in vertebrates, plants and fungi. Here we have mapped DNA methylation in the entire Arabidopsis thaliana genome at high resolution. DNA methylation covers transposons and is present within a large fraction of A. thaliana genes. Methylation within genes is conspicuously biased away from gene ends, suggesting a dependence on RNA polymerase transit. Genic methylation is strongly influenced by transcription: moderately transcribed genes are most likely to be methylated, whereas genes at either extreme are least likely. In turn, transcription is influenced by methylation: short methylated genes are poorly expressed, and loss of methylation in the body of a gene leads to enhanced transcription. Our results indicate that genic transcription and DNA methylation are closely interwoven processes.

Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications

Analysis of DNA methylation patterns relies increasingly on sequencing-based profiling methods. The four most frequently used sequencing-based technologies are the bisulfite-based methods MethylC-seq and reduced representation bisulfite sequencing (RRBS), and the enrichment-based techniques methylated DNA immunoprecipitation sequencing (MeDIP-seq) and methylated DNA binding domain sequencing (MBD-seq). We applied all four methods to biological replicates of human embryonic stem cells to assess their genome-wide CpG coverage, resolution, cost, concordance and the influence of CpG density and genomic context. The methylation levels assessed by the two bisulfite methods were concordant (their difference did not exceed a given threshold) for 82% for CpGs and 99% of the non-CpG cytosines. Using binary methylation calls, the two enrichment methods were 99% concordant and regions assessed by all four methods were 97% concordant. We combined MeDIP-seq with methylation-sensitive restriction enzyme (MRE-seq) sequencing for comprehensive methylome coverage at lower cost. This, along with RNA-seq and ChIP-seq of the ES cells enabled us to detect regions with allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression.

Histone H2A.Z and DNA methylation are mutually antagonistic chromatin marks

Eukaryotic chromatin is separated into functional domains differentiated by post-translational histone modifications, histone variants and DNA methylation. Methylation is associated with repression of transcriptional initiation in plants and animals, and is frequently found in transposable elements. Proper methylation patterns are crucial for eukaryotic development, and aberrant methylation-induced silencing of tumour suppressor genes is a common feature of human cancer. In contrast to methylation, the histone variant H2A.Z is preferentially deposited by the Swr1 ATPase complex near 5′ ends of genes where it promotes transcriptional competence. How DNA methylation and H2A.Z influence transcription remains largely unknown. Here we show that in the plant Arabidopsis thaliana regions of DNA methylation are quantitatively deficient in H2A.Z. Exclusion of H2A.Z is seen at sites of DNA methylation in the bodies of actively transcribed genes and in methylated transposons. Mutation of the MET1 DNA methyltransferase, which causes both losses and gains of DNA methylation, engenders opposite changes (gains and losses) in H2A.Z deposition, whereas mutation of the PIE1 subunit of the Swr1 complex that deposits H2A.Z leads to genome-wide hypermethylation. Our findings indicate that DNA methylation can influence chromatin structure and effect gene silencing by excluding H2A.Z, and that H2A.Z protects genes from DNA methylation.

DNA demethylation in the Arabidopsis genome

Cytosine DNA methylation is considered to be a stable epigenetic mark, but active demethylation has been observed in both plants and animals. In Arabidopsis thaliana, DNA glycosylases of the DEMETER (DME) family remove methylcytosines from DNA. Demethylation by DME is necessary for genomic imprinting, and demethylation by a related protein, REPRESSOR OF SILENCING1, prevents gene silencing in a transgenic background. However, the extent and function of demethylation by DEMETER-LIKE (DML) proteins in WT plants is not known. Using genome-tiling microarrays, we mapped DNA methylation in mutant and WT plants and identified 179 loci actively demethylated by DML enzymes. Mutations in DML genes lead to locus-specific DNA hypermethylation. Reintroducing WT DML genes restores most loci to the normal pattern of methylation, although at some loci, hypermethylated epialleles persist. Of loci demethylated by DML enzymes, >80% are near or overlap genes. Genic demethylation by DML enzymes primarily occurs at the 5′ and 3′ ends, a pattern opposite to the overall distribution of WT DNA methylation. Our results show that demethylation by DML DNA glycosylases edits the patterns of DNA methylation within the Arabidopsis genome to protect genes from potentially deleterious methylation.

Retrotransposition of gene transcripts leads to structural variation in mammalian genomes.

BackgroundRetroposed processed gene transcripts are an important source of material for new gene formation on evolutionary timescales. Most prior work on gene retrocopy discovery compared copies in reference genome assemblies to their source genes. Here, we explore gene retrocopy insertion polymorphisms (GRIPs) that are present in the germlines of individual humans, mice, and chimpanzees, and we identify novel gene retrocopy insertions in cancerous somatic tissues that are absent from patient-matched non-cancer genomes.ResultsThrough analysis of whole-genome sequence data, we found evidence for 48 GRIPs in the genomes of one or more humans sequenced as part of the 1,000 Genomes Project and The Cancer Genome Atlas, but which were not in the human reference assembly. Similarly, we found evidence for 755 GRIPs at distinct locations in one or more of 17 inbred mouse strains but which were not in the mouse reference assembly, and 19 GRIPs across a cohort of 10 chimpanzee genomes, which were not in the chimpanzee reference genome assembly. Many of these insertions are new members of existing gene families whose source genes are highly and widely expressed, and the majority have detectable hallmarks of processed gene retrocopy formation. We estimate the rate of novel gene retrocopy insertions in humans and chimps at roughly one new gene retrocopy insertion for every 6,000 individuals.ConclusionsWe find that gene retrocopy polymorphisms are a widespread phenomenon, present a multi-species analysis of these events, and provide a method for their ascertainment.

A Role for Mitochondria in the Establishment and Maintenance of the Maize Root Quiescent Center

Mitochondria in the oxidizing environment of the maize (Zea mays) root quiescent center (QC) are altered in function, but otherwise structurally normal. Compared to mitochondria in the adjacent, rapidly dividing cells of the proximal root tissues, mitochondria in the QC show marked reductions in the activities of tricarboxylic acid cycle enzymes. Pyruvate dehydrogenase activity was not detected in the QC. Use of several mitochondrial membrane potential (ΔΨm) sensing probes indicated a depolarization of the mitochondrial membrane in the QC, which suggests a reduction in the capacity of QC mitochondria to generate ATP and NADH. We postulate that modifications of mitochondrial function are central to the establishment and maintenance of the QC.

Abstract B1-11: Application of the CN-AVG method to reconstruct the evolutionary history of glioblastoma multiforme

We have constructed evolutionary histories of cancer genomes using a new method, the Copy Number Ancestral Variation Graph (CN-AVG). Unlike previous cancer evolutionary studies which infer the clonal structure and evolutionary history of a tumor using single nucleotide variant frequencies, the CN-AVG method predicts the ordering of structural rearrangements, such as inversions, duplications, and deletions, using copy number changes and breakpoints derived from whole genome sequencing data. Because finding the most parsimonious ordering of genomic rearrangements is an NP hard problem, we build a consensus history by merging a large number of possible evolutionary histories generated from MCMC sampling. We tested the CN-AVG method on simulated rearranged genomes and achieved an average accuracy of 62%. We also found that accuracy decreased with increasing complexity of the simulated rearrangements, as expected. We then applied the method to TCGA Glioblastoma Multiforme genomes to search for evolutionary patterns in this disease. The CN-AVG method may distinguish driver mutations as the early rearrangement events and passenger or secondary mutations as the later events in the reconstructed evolutionary history of the tumor, and will be powerful for both the clinic and for research. Citation Format: Tracy J. Ballinger, Daniel Zerbino, Benedict Paten, David Haussler. Application of the CN-AVG method to reconstruct the evolutionary history of glioblastoma multiforme. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B1-11.

Abstract A29: Searching for retrotransposon insertions from paired end sequencing of tumors

The question of how active transposons are in tumors is a salient one because tumors are hypermethylated and stem-cell-like, two conditions under which transposons are active, and because there are many known cases of germline retrotransposon insertion polymorphisms leading to cancer [1]. While studies have shown that some retroelements are transcribed at higher levels in certain types of tumors [2], there are currently very few studies that have looked at somatic retrotransposon insertions in tumor genomes. Reactivation of transposable elements could be an alternate pathway to genomic instability, one of the defining properties of cancer, by increasing the rate of mutation via retrotransposon insertions. Indeed, Collier et. al. have proven this principle in mice using the Sleeping Beauty transposon, a reactivated ancient DNA transposon [3]. The characterization of somatic as well as germline polymorphic insertions will be important in the study of cancer, as it will provide insight into how active transposable elements are in tumors and what role they play in tumor formation. The Cancer Genome Atlas project has generated paired-end whole-genome sequencing data of tumors and normal tissue from hundreds of cancer patients in order to detect mutations and, ultimately, develop personalized treatment for patients. We have applied a pipeline to detect insertion polymorphisms of LINE-1 and Alu elements in both patients9 samples. The first step in the detection method is to filter out all discordant reads and map them to a library of consensus sequences for a specific retrotransposon family. Next, read pairs of reads that match a repeat element are mapped back to the reference genome. Lastly, the mapped reads are clustered based on their location in the reference genome, and insertion calls are made. Using this method we have found over four hundred new candidates for polymorphic insertions in seven patients, including some found only in the tumor samples. While this study is in the preliminary stages, it will address the question of whether or to what degree retrotransposons are active in tumors by being able to detect an increase in the number of retrotransposons found in the tumor sample as compared to the normal tissue of the same patient. Furthermore, heritable polymorphic insertions can be linked to cancer if there are certain ones that are over-represented in cancer cohorts as compared to a normal population, such as those found in the 1000 Genomes project data. Including insertion mutations in the characterization of cancer genomes will contribute to better individualized treatment for patients. 1. Belancio et al. Genome Res. 2008;18(3):343–58. 2. Schulz. J Biomed Biotechnol. 2006;(1):836–72. 3. Collier et al. Nature. 2005;436(7048):272–76. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A29.