Martin J. Aryee

Epigenetic memory in induced pluripotent stem cells

Somatic cell nuclear transfer and transcription-factor-based reprogramming revert adult cells to an embryonic state, and yield pluripotent stem cells that can generate all tissues. Through different mechanisms and kinetics, these two reprogramming methods reset genomic methylation, an epigenetic modification of DNA that influences gene expression, leading us to hypothesize that the resulting pluripotent stem cells might have different properties. Here we observe that low-passage induced pluripotent stem cells (iPSCs) derived by factor-based reprogramming of adult murine tissues harbour residual DNA methylation signatures characteristic of their somatic tissue of origin, which favours their differentiation along lineages related to the donor cell, while restricting alternative cell fates. Such an ‘epigenetic memory’ of the donor tissue could be reset by differentiation and serial reprogramming, or by treatment of iPSCs with chromatin-modifying drugs. In contrast, the differentiation and methylation of nuclear-transfer-derived pluripotent stem cells were more similar to classical embryonic stem cells than were iPSCs. Our data indicate that nuclear transfer is more effective at establishing the ground state of pluripotency than factor-based reprogramming, which can leave an epigenetic memory of the tissue of origin that may influence efforts at directed differentiation for applications in disease modelling or treatment.

Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts

Induced pluripotent stem (iPS) cells are derived by epigenetic reprogramming, but their DNA methylation patterns have not yet been analyzed on a genome-wide scale. Here, we find substantial hypermethylation and hypomethylation of cytosine-phosphate-guanine (CpG) island shores in nine human iPS cell lines as compared to their parental fibroblasts. The differentially methylated regions (DMRs) in the reprogrammed cells (denoted R-DMRs) were significantly enriched in tissue-specific (T-DMRs; 2.6-fold, P < 10−4) and cancer-specific DMRs (C-DMRs; 3.6-fold, P < 10−4). Notably, even though the iPS cells are derived from fibroblasts, their R-DMRs can distinguish between normal brain, liver and spleen cells and between colon cancer and normal colon cells. Thus, many DMRs are broadly involved in tissue differentiation, epigenetic reprogramming and cancer. We observed colocalization of hypomethylated R-DMRs with hypermethylated C-DMRs and bivalent chromatin marks, and colocalization of hypermethylated R-DMRs with hypomethylated C-DMRs and the absence of bivalent marks, suggesting two mechanisms for epigenetic reprogramming in iPS cells and cancer.

Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing

Monomeric CRISPR-Cas9 nucleases are widely used for targeted genome editing but can induce unwanted off-target mutations with high frequencies. Here we describe dimeric RNA-guided FokI nucleases (RFNs) that can recognize extended sequences and edit endogenous genes with high efficiencies in human cells. RFN cleavage activity depends strictly on the binding of two guide RNAs (gRNAs) to DNA with a defined spacing and orientation substantially reducing the likelihood that a suitable target site will occur more than once in the genome and therefore improving specificities relative to wild-type Cas9 monomers. RFNs guided by a single gRNA generally induce lower levels of unwanted mutations than matched monomeric Cas9 nickases. In addition, we describe a simple method for expressing multiple gRNAs bearing any 5′ end nucleotide, which gives dimeric RFNs a broad targeting range. RFNs combine the ease of RNA-based targeting with the specificity enhancement inherent to dimerization and are likely to be useful in applications that require highly precise genome editing.

Engineered CRISPR-Cas9 nucleases with altered PAM specificities

Although CRISPR-Cas9 nucleases are widely used for genome editing, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM). As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome-editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis. In addition, we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also find that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.

Epigenome-wide association data implicate DNA methylation as an intermediary of genetic risk in rheumatoid arthritis

Epigenetic mechanisms integrate genetic and environmental causes of disease, but comprehensive genome-wide analyses of epigenetic modifications have not yet demonstrated robust association with common diseases. Using Illumina HumanMethylation450 arrays on 354 anti-citrullinated protein antibody–associated rheumatoid arthritis cases and 337 controls, we identified two clusters within the major histocompatibility complex (MHC) region whose differential methylation potentially mediates genetic risk for rheumatoid arthritis. To reduce confounding factors that have hampered previous epigenome-wide studies, we corrected for cellular heterogeneity by estimating and adjusting for cell-type proportions in our blood-derived DNA samples and used mediation analysis to filter out associations likely to be a consequence of disease. Four CpGs also showed an association between genotype and variance of methylation. The associations for both clusters replicated at least one CpG (P < 0.01), with the rest showing suggestive association, in monocyte cell fractions in an independent cohort of 12 cases and 12 controls. Thus, DNA methylation is a potential mediator of genetic risk.

Comprehensive methylome map of lineage commitment from haematopoietic progenitors.

Epigenetic modifications must underlie lineage-specific differentiation as terminally differentiated cells express tissue-specific genes, but their DNA sequence is unchanged. Haematopoiesis provides a well-defined model to study epigenetic modifications during cell-fate decisions, as multipotent progenitors (MPPs) differentiate into progressively restricted myeloid or lymphoid progenitors. Although DNA methylation is critical for myeloid versus lymphoid differentiation, as demonstrated by the myeloerythroid bias in Dnmt1 hypomorphs, a comprehensive DNA methylation map of haematopoietic progenitors, or of any multipotent/oligopotent lineage, does not exist. Here we examined 4.6 million CpG sites throughout the genome for MPPs, common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), and thymocyte progenitors (DN1, DN2, DN3). Marked epigenetic plasticity accompanied both lymphoid and myeloid restriction. Myeloid commitment involved less global DNA methylation than lymphoid commitment, supported functionally by myeloid skewing of progenitors following treatment with a DNA methyltransferase inhibitor. Differential DNA methylation correlated with gene expression more strongly at CpG island shores than CpG islands. Many examples of genes and pathways not previously known to be involved in choice between lymphoid/myeloid differentiation have been identified, such as Arl4c and Jdp2. Several transcription factors, including Meis1, were methylated and silenced during differentiation, indicating a role in maintaining an undifferentiated state. Additionally, epigenetic modification of modifiers of the epigenome seems to be important in haematopoietic differentiation. Our results directly demonstrate that modulation of DNA methylation occurs during lineage-specific differentiation and defines a comprehensive map of the methylation and transcriptional changes that accompany myeloid versus lymphoid fate decisions.

Androgen-induced TOP2B-mediated double-strand breaks and prostate cancer gene rearrangements

DNA double-strand breaks (DSBs) can lead to the development of genomic rearrangements, which are hallmarks of cancer. Fusions between TMPRSS2, encoding the transmembrane serine protease isoform 2, and ERG, encoding the v-ets erythroblastosis virus E26 oncogene homolog, are among the most common oncogenic rearrangements observed in human cancer. We show that androgen signaling promotes co-recruitment of androgen receptor and topoisomerase II beta (TOP2B) to sites of TMPRSS2-ERG genomic breakpoints, triggering recombinogenic TOP2B-mediated DSBs. Furthermore, androgen stimulation resulted in de novo production of TMPRSS2-ERG fusion transcripts in a process that required TOP2B and components of the DSB repair machinery. Finally, unlike normal prostate epithelium, prostatic intraepithelial neoplasia cells showed strong coexpression of androgen receptor and TOP2B. These findings implicate androgen-induced TOP2B-mediated DSBs in generating TMPRSS2-ERG rearrangements.

Personalized Epigenomic Signatures That Are Stable Over Time and Covary with Body Mass Index

A genome-scale, gene-specific analysis of DNA methylation in the same individuals over a decade apart identifies a personalized epigenomic signature that may correlate with a common genetic trait. The Writing is on the Genes: Can Epigenomics Predict Disease Risk? In the nature versus nurture debate about human traits, epigenomics holds a special place. Epigenetic changes are physical changes that happen to genes but do not change the gene (DNA) sequence itself—such as DNA methylation. The regulation of these changes is not yet well-understood, providing new ammunition to the age-old argument. It is possible that the methylation pattern is all “nature”, predetermined by a person’s genetic makeup, or alternatively methylation could be a result of “nurture”, reflecting the influence of regulatory signals outside the cell, that is, the environment. Having analyzed the detailed methylation patterns in several dozen individuals at two different time points, over a decade apart, Feinberg et al. present evidence that the answer may actually be both—a combination of genetic determinants and environmental regulation. In this study, the authors analyzed the full methylation pattern at 4.5 million sites genome-wide in 74 volunteers. The participants, who were on average 74 years old at the time of the first visit, provided blood samples again 11 to 14 years later, allowing for comparison of methylation patterns both between individuals, and in the same individuals across time. In doing this, the authors found 227 variably methylated regions (VMRs), which varied widely between the study participants. Of these, 119 VMRs remained stable within each individual over time, constituting an epigenetic fingerprint that may be genetically predetermined and differed between pairs of individual participants. The remaining VMRs were highly variable over time, suggesting that their pattern is affected by environmental influences. Four of the stable VMRs consistently correlated with study subjects’ body mass index in both visits. All four of these sites were located at or near genes that are known to be involved in the pathogenesis of diabetes or obesity, lending biological plausibility to the correlation between the methylation pattern and obesity risk. Through their analysis of the epigenome in a large pool of volunteer subjects, Feinberg et al. have demonstrated a unique signature of stable epigenetic changes within each individual. Several of these stable methylation sites were correlated with the patients’ body mass index. If these results are confirmed in younger individuals and consistent throughout the life span, tests for methylation might be used to screen patients in childhood and identify those at risk for obesity, allowing preventative treatment. In theory, similar testing for other common diseases that may have a stable epigenetic component, such as diabetes or asthma, could allow early intervention and prevention. The epigenome consists of non–sequence-based modifications, such as DNA methylation, that are heritable during cell division and that may affect normal phenotypes and predisposition to disease. Here, we have performed an unbiased genome-scale analysis of ~4 million CpG sites in 74 individuals with comprehensive array-based relative methylation (CHARM) analysis. We found 227 regions that showed extreme interindividual variability [variably methylated regions (VMRs)] across the genome, which are enriched for developmental genes based on Gene Ontology analysis. Furthermore, half of these VMRs were stable within individuals over an average of 11 years, and these VMRs defined a personalized epigenomic signature. Four of these VMRs showed covariation with body mass index consistently at two study visits and were located in or near genes previously implicated in regulating body weight or diabetes. This work suggests an epigenetic strategy for identifying patients at risk of common disease.

Diarrhea incidence in low- and middle-income countries in 1990 and 2010: a systematic review

BackgroundDiarrhea is recognized as a leading cause of morbidity and mortality among children under 5 years of age in low- and middle-income countries yet updated estimates of diarrhea incidence by age for these countries are greatly needed. We conducted a systematic literature review to identify cohort studies that sought to quantify diarrhea incidence among any age group of children 0-59 mo of age.MethodsWe used the Expectation-Maximization algorithm as a part of a two-stage regression model to handle diverse age data and overall incidence rate variation by study to generate country specific incidence rates for low- and middle-income countries for 1990 and 2010. We then calculated regional incidence rates and uncertainty ranges using the bootstrap method, and estimated the total number of episodes for children 0-59 mo of age in 1990 and 2010.ResultsWe estimate that incidence has declined from 3.4 episodes/child year in 1990 to 2.9 episodes/child year in 2010. As was the case previously, incidence rates are highest among infants 6-11 mo of age; 4.5 episodes/child year in 2010. Among these 139 countries there were nearly 1.9 billion episodes of childhood diarrhea in 1990 and nearly 1.7 billion episodes in 2010.ConclusionsAlthough our results indicate that diarrhea incidence rates may be declining slightly, the total burden on the health of each child due to multiple episodes per year is tremendous and additional funds are needed to improve both prevention and treatment practices in low- and middle-income countries.

Global Causes of Diarrheal Disease Mortality in Children <5 Years of Age: A Systematic Review

Estimation of pathogen-specific causes of child diarrhea deaths is needed to guide vaccine development and other prevention strategies. We did a systematic review of articles published between 1990 and 2011 reporting at least one of 13 pathogens in children <5 years of age hospitalized with diarrhea. We included 2011 rotavirus data from the Rotavirus Surveillance Network coordinated by WHO. We excluded studies conducted during diarrhea outbreaks that did not discriminate between inpatient and outpatient cases, reporting nosocomial infections, those conducted in special populations, not done with adequate methods, and rotavirus studies in countries where the rotavirus vaccine was used. Age-adjusted median proportions for each pathogen were calculated and applied to 712 000 deaths due to diarrhea in children under 5 years for 2011, assuming that those observed among children hospitalized for diarrhea represent those causing child diarrhea deaths. 163 articles and WHO studies done in 31 countries were selected representing 286 inpatient studies. Studies seeking only one pathogen found higher proportions for some pathogens than studies seeking multiple pathogens (e.g. 39% rotavirus in 180 single-pathogen studies vs. 20% in 24 studies with 5–13 pathogens, p<0·0001). The percentage of episodes for which no pathogen could be identified was estimated to be 34%; the total of all age-adjusted percentages for pathogens and no-pathogen cases was 138%. Adjusting all proportions, including unknowns, to add to 100%, we estimated that rotavirus caused 197 000 [Uncertainty range (UR) 110 000–295 000], enteropathogenic E. coli 79 000 (UR 31 000–146 000), calicivirus 71 000 (UR 39 000–113 000), and enterotoxigenic E. coli 42 000 (UR 20 000–76 000) deaths. Rotavirus, calicivirus, enteropathogenic and enterotoxigenic E. coli cause more than half of all diarrheal deaths in children <5 years in the world.