Publications

The heritability of chromatin states through cell division is a potential contributor to the epigenetic maintenance of cellular memory of prior states. The macroH2A histone variant has properties of a regulator of epigenetic cell memory, including roles controlling gene silencing and cell differentiation. Its mechanisms of regional genomic targeting and maintenance through cell division are unknown. Here, we combined in vivo imaging with biochemical and genomic approaches to show that human macroH2A is incorporated into chromatin in the G1 phase of the cell cycle following DNA replication. The newly incorporated macroH2A retargets the same large heterochromatic domains where macroH2A was already enriched in the previous cell cycle. It remains heterotypic, targeting individual nucleosomes that do not already contain a macroH2A molecule. The pattern observed resembles that of a new deposition of centromeric histone variants during the cell cycle, indicating mechanistic similarities for macrodomain-scale regulation of epigenetic properties of the cell.

While human lymphoblastoid cell lines represent a valuable resource for population genetic studies, they have usually been regarded as difficult for CRISPR-mediated genomic editing because of very inefficient DNA transfection and retroviral or lentiviral transduction in these cells, which becomes a substantial problem when multiple constructs need to be co-expressed. Here we describe a protocol using a single-stranded donor oligonucleotide strategy for 'scarless' editing in lymphoblastoid cells, yielding 12/60 (20%) of clones with homology-directed recombination, when rates of <5-10% are frequently typical for many other cell types. The protocol does not require the use of lentiviruses or stable transfection, permitting lymphoblastoid cell lines to be used for CRISPR-mediated genomic targeting and screening in population genetic studies.

Functional variants in the genome are usually identified by their association with local gene expression, DNA methylation or chromatin states. DNA sequence motif analysis and chromatin immunoprecipitation studies have provided indirect support for the hypothesis that functional variants alter transcription factor binding to exert their effects. In this study, we provide direct evidence that functional variants can alter transcription factor binding. We identify a multifunctional variant within the TBC1D4 gene encoding a canonical NFκB binding site, and edited it using CRISPR-Cas9 to remove this site. We show that this editing reduces TBC1D4 expression, local chromatin accessibility and binding of the p65 component of NFκB. We then used CRISPR without genomic editing to guide p65 back to the edited locus, demonstrating that this re-targeting, occurring  182 kb from the gene promoter, is enough to restore the function of the locus, supporting the central role of transcription factors mediating the effects of functional variants.

Profound global loss of DNA methylation is a hallmark of many cancers. One potential consequence of this is the reactivation of transposable elements (TEs) which could stimulate the immune system via cell-intrinsic antiviral responses. Here, we develop REdiscoverTE, a computational method for quantifying genome-wide TE expression in RNA sequencing data. Using The Cancer Genome Atlas database, we observe increased expression of over 400 TE subfamilies, of which 262 appear to result from a proximal loss of DNA methylation. The most recurrent TEs are among the evolutionarily youngest in the genome, predominantly expressed from intergenic loci, and associated with antiviral or DNA damage responses. Treatment of glioblastoma cells with a demethylation agent results in both increased TE expression and de novo presentation of TE-derived peptides on MHC class I molecules. Therapeutic reactivation of tumor-specific TEs may synergize with immunotherapy by inducing inflammation and the display of potentially immunogenic neoantigens.

BACKGROUND: Pediatric obesity-related asthma is more severe and less responsive to medications than asthma in normal-weight children. Obese asthmatic children have nonatopic TH1-polarized systemic inflammation that correlates with pulmonary function deficits, but the pathways underlying TH1-polarized inflammation are not well understood.

OBJECTIVE: We compared the CD4+ T-cell transcriptome in obese children with asthma with that in normal-weight children with asthma to identify key differentially expressed genes associated with TH1-polarized inflammation.

METHODS: CD4+ T-cell transcriptome-wide differential gene expression was compared between 21 obese and 21 normal-weight children by using directional RNA sequencing. High-confidence differentially expressed genes were verified in the first cohort and validated in a second cohort of 20 children (10 obese and 10 normal-weight children) by using quantitative RT-PCR.

RESULTS: Transcriptome-wide differential gene expression among obese asthmatic children was enriched for genes, including VAV2, DOCK5, PAK3, PLD1, CDC42EP4, and CDC42PBB, which are associated with CDC42, a small guanosine triphosphate protein linked to T-cell activation. Upregulation of MLK3 and PLD1, genes downstream of CDC42 in the mitogen-activated protein kinase and mammalian target of rapamycin pathways and the inverse correlation of CDC42EP4 and DOCK5 transcript counts with FEV1/FVC ratio together support a role of CDC42 in the TH1 polarization and pulmonary function deficits found in patients with obesity-related asthma.

CONCLUSIONS: Our study identifies the CDC42 pathway as a novel target that is upregulated in TH cells of obese asthmatic children, suggesting its role in nonatopic TH1-polarized systemic inflammation and pulmonary function deficits found in patients with pediatric obesity-related asthma.

Current approaches to detect and characterize mosaic chromosomal aneuploidy are limited by sensitivity, efficiency, cost, or the need to culture cells. We describe the mosaic aneuploidy detection by massively parallel sequencing (MAD-seq) capture assay and the MADSEQ analytical approach that allow low (<10%) levels of mosaicism for chromosomal aneuploidy or regional loss of heterozygosity to be detected, assigned to a meiotic or mitotic origin, and quantified as a proportion of the cells in the sample. We show results from a multi-ethnic MAD-seq (meMAD-seq) capture design that works equally well in populations of diverse racial and ethnic origins and how the MADSEQ analytical approach can be applied to exome or whole-genome sequencing data, revealing previously unrecognized aneuploidy or copy number neutral loss of heterozygosity in samples studied by the 1000 Genomes Project, cell lines from public repositories, and one of the Illumina Platinum Genomes samples. We have made the meMAD-seq capture design and MADSEQ analytical software open for unrestricted use, with the goal that they can be applied in clinical samples to allow new insights into the unrecognized prevalence of mosaic chromosomal aneuploidy in humans and its phenotypic associations.

DNA methylation patterns in the genome both reflect and help to mediate transcriptional regulatory processes. The digital nature of DNA methylation, present or absent on each allele, makes this assay capable of quantifying events in subpopulations of cells, whereas genome-wide chromatin studies lack the same quantitative capacity. Testing DNA methylation throughout the genome is possible using whole-genome bisulfite sequencing (WGBS), but the high costs associated with the assay have made it impractical for studies involving more than limited numbers of samples. We have optimized a new transposase-based library preparation assay for the Illumina HiSeq X platform suitable for limited amounts of DNA and providing a major cost reduction for WGBS. By incorporating methylated cytosines during fragment end repair, we reveal an end-repair artifact affecting 1%-2% of reads that we can remove analytically. We show that the use of a high (G + C) content spike-in performs better than PhiX in terms of bisulfite sequencing quality. As expected, the loci with transposase-accessible chromatin are DNA hypomethylated and enriched in flanking regions by post-translational modifications of histones usually associated with positive effects on gene expression. Using these transposase-accessible loci to represent the cis-regulatory loci in the genome, we compared the representation of these loci between WGBS and other genome-wide DNA methylation assays, showing WGBS to outperform substantially all of the alternatives. We conclude that it is now technologically and financially feasible to perform WGBS in larger numbers of samples with greater accuracy than previously possible.

The predisposition of patients with Hepatitis C virus (HCV) infection to hepatocellular carcinoma (HCC) involves components of viral infection, inflammation and time. The development of multifocal, genetically distinct tumours is suggestive of a field defect affecting the entire liver. The molecular susceptibility mediating such a field defect is not understood. One potential mediator of long-term cellular reprogramming is heritable (epigenetic) regulation of transcription, exemplified by DNA methylation. We studied epigenetic and transcriptional changes in HCV-infected livers in comparison with control, uninfected livers and HCC, allowing us to identify pre-neoplastic epigenetic and transcriptional events. We find the HCV-infected liver to have a pattern of acquisition of DNA methylation targeted to candidate enhancers active in liver cells, enriched for the binding sites of the FOXA1, FOXA2 and HNF4A transcription factors. These enhancers can be subdivided into those proximal to genes implicated in liver cancer or to genes involved in stem cell development, the latter distinguished by increased CG dinucleotide density and polycomb-mediated repression, manifested by the additional acquisition of histone H3 lysine 27 trimethylation (H3K27me3). Transcriptional studies on our samples showed that the increased DNA methylation at enhancers was associated with decreased local gene expression, results validated in independent samples from The Cancer Genome Atlas. Pharmacological depletion of H3K27me3 using the EZH2 inhibitor GSK343 in HepG2 cells suppressed cell growth and also revealed that local acquired DNA methylation was not dependent upon the presence of polycomb-mediated repression. The results support a model of HCV infection influencing the binding of transcription factors to cognate sites in the genome, with consequent local acquisition of DNA methylation, and the added repressive influence of polycomb at a subset of CG-dense cis-regulatory sequences. These epigenetic events occur before neoplastic transformation, resulting in what may be a pharmacologically reversible epigenetic field defect in HCV-infected liver.

Studies on genetic-epigenetic interactions, including the mapping of methylation quantitative trait loci (mQTLs) and haplotype-dependent allele-specific DNA methylation (hap-ASM), have become a major focus in the post-genome-wide-association-study (GWAS) era. Such maps can nominate regulatory sequence variants that underlie GWAS signals for common diseases, ranging from neuropsychiatric disorders to cancers. Conversely, mQTLs need to be filtered out when searching for non-genetic effects in epigenome-wide association studies (EWAS). Sequence variants in CCCTC-binding factor (CTCF) and transcription factor binding sites have been mechanistically linked to mQTLs and hap-ASM. Identifying these sites can point to disease-associated transcriptional pathways, with implications for targeted treatment and prevention.