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Epigenetic Analysis Citations

Table of Contents
Selected Citations Testimonials
Published Methods

Selected Citations

The Role of Acyl-CoA Synthetase 4 in Aggressive Breast Cancer

Castillo, et al. used the mRNA-Seq service from Zymo Research to investigate gene expression profilse in breast cancer cells overexpressing acyl-CoA synthetase 4 (ACSL4), since the expression of ACSL4 has been linked to aggressive breast cancer. Ingenuity Pathways Analysis performed on data obtained from mRNA-Seq showed that ACSL4 overexpression affected expression levels of genes involved in tumorigenesis-related biofunctions, with cell movement, migration, and proliferation being the top three. Furthermore, the results of the reverse phase protein array showed that cells overexpressing ACSL4 have a distinct protein signature. Based on DAVID analysis, the researchers concluded that ACSL4 can stimulate different signaling pathways, which mediate aggressiveness of breast cancer cells, for example, pathways activated by growth factors, cytokines, and nutrients. They suggested that ACSL4 could potentially be used to predict an aggressive breast cancer phenotype in patients and could be a target for cancer therapy.

Choosy DNA Methyltransferases Choose CpG-Dense Regions

While changes in DNA methylation have been implicated in a variety of diseases, little is known about the cellular mechanisms resulting in DNA methylation. In a recent paper, Baubec et al. investigated the de novo methyltransferase activity of DNMT3A and DNMT3B in mouse embryonic stem cells. Using the mass spectrometry services from Zymo Research, the group found reproducible and detectable rates of methylation following reintroduction of DNMT3A2 or DNMT3B1 to mouse Dnmt gene triple knockout (TKO) cells. They further validated and confirmed the findings of the mass spectrometry analysis by using a whole genome bisulfite sequencing analysis. Further analysis showed that the recruitment of DNMT3A and DNMT3B to CpG dense regions is independent of existing methylation states.

ADHD Epigenetics in Peripheral Tissue: Nothing to Spit at!

Wilmot, et al. used the Targeted Bisulfite Sequencing service from Zymo Research to validate differentially methylated regions in childhood ADHD. The researchers extracted DNA from saliva in order to analyze methylation patterns in a sample source peripheral to brain tissue. Following genome-wide differentially methylated region analysis using bead arrays, study authors identified VIPR2 as a gene of interest for ADHD. Targeted Bisulfite Sequencing confirmed the hypomethylation of 43 sites in ADHD subjects, including VIPR2, thus providing further confidence in the use of peripheral tissue as a potential sample source for methylation analysis in psychiatric disorders.

Understanding the effects and characteristics of TET oxidation in HEK293 cells on a genome-wide scale

To understand the DNA methylation (5mC) and hydroxymethylation (5hmC) changes that occur when TET expression levels are altered, researchers used a doxycycline-inducible HEK293 cell line to overexpress TET1 proteins and siRNA to knockdown all three TET proteins. TET1 overexpression resulted in increased global 5hmC levels and DNA demethylation of promoters, gene bodies, and CpG islands. TET triple knockdown resulted in lower 5hmC levels and slight hypermethylation in those same regions. Using Reduced Representation Hydroxymethylation Profiling (RRHP) to profile 5hmC sites in overexpressed and control cells, the authors found that TET oxidation was not biased towards any genetic elements or structures but was related to endogenous 5hmC levels. RRHP revealed de novo 5hmC sites as a result of TET1 overexpression, and motif analysis of the top de novo and 5hmC-gaining sites revealed enriched sequence motifs possibly involved in the TET oxidation pathway.

Mice Hustle to Gain More Muscle

Study authors investigated DNA methylation changes in mouse skeletal muscle following a 4 week regimen of regular exercise. Using Zymo Research’s Methyl-MiniSeq platform, Kanzleiter et al. detected 3692 differentially methylated CpGs distributed among 2762 promoter regions. When coupled with gene expression profiles, integrated analysis revealed 361 genes in which changes of DNA methylation were associated with changes in gene expression. These genes included those related to muscle growth and differentiation as well as metabolic regulation.

DNA Methylation Dynamics Following Liver Stem Cell Activation

Researchers sought to better understand epigenetic changes that occur in hepatic stellate cells (HSCs) upon activation. Götze, et al. stimulated HSC activation by altering in vitro culture conditions. The genome-wide DNA methylation patterns were then determined using the Methyl-MiniSeq service from Zymo Research. The data revealed approximately 400 differentially methylated regions and, according to DAVID pathway analysis, contained genes enriched for processes relevant during HSC activation. Furthermore, locus-specific validation tests revealed that DNA methylation changes correlated with gene expression changes of select genes. The researchers went on to show that the DNA methylation changes are likely the result of an active, as opposed to passive, demethylation mechanism.

Alcohol exposure influences gene expression and DNA methylation signatures in hESC model of pregnancy

Researchers investigated how alcohol affects the regulatory mechanisms needed for normal stem cell differentiation and embryonic development. Study authors utilized genome-wide DNA methylation analysis services provided by Zymo Research to help characterize epigenetic and gene expression changes in human embryonic stem cells (hESCs) exposed to low amounts of alcohol (Ethanol, EtOH). Genome-wide analysis revealed that alcohol exposure caused a significant amount of regional promoter hypermethylation, especially affecting segments of chromosomes 2, 16, and 18, and differential expression in undifferentiated hESCs versus controls. The study authors also found that EtOH exposure affected key pathways involved in normal cell function, such as metabolism, and reduced hESC pluripotency.

Epigenomic Effects of the Plasticizer DEHP

Researchers investigated the effects of di-(2-ethylhexyl) phthalate (DEHP), a ubiquitous plasticizer and a known endocrine disruptor, on DNA methylation changes in the adrenal gland of adult male rats exposed to DEHP in utero. Using the Methyl-MiniSeq™ service from Zymo Research, study authors found 972 differentially methylated CpGs out of 2,183,479 CpG sites surveyed throughout the genome. Most differentially methylated CpGs were found within CpG islands (40%). Interestingly, the second highest frequency of differentially methylated CpGs were found within shore/shelf regions (30%). By contrast, promoter regions did not contain a significant number of differentially methylated CpGs, suggesting that DEHP targets epigenomic regions that regulate tissue-specific expression. The researchers also identified several hotspots of differentially methylated CpGs that coincided with regions of differentially expressed genes. Two such hotspots at chr20p12 correlated with deregulation of gene expression. That region contains genes critical for the proper function of the immune system. The study’s findings support the notion that the typical amounts of DEHP present in the environment may influence a significant number of regions across the epigenome.

Evolutionary origins of placental Chr19 galectin gene cluster expression

Researchers investigating the mechanisms behind preeclampsia used the Targeted Bisulfite Sequencing service from Zymo Research to examine the DNA methylation status of three genes encoding for human galectins. Galectins found in a gene cluster on chromosome 19 are suspected to confer maternal immune tolerance to the fetus. Amplicons were designed to cover the highly methylated intragenic regions of the placental galectin genes LGALS13, LGALS14 and LGALS16. Using this strategy, several key CpG sites were found to be differentially methylated in association with preeclampsia. In conjunction with other assays, the findings suggest that differential methylation at these sites may impact or interfere with trophoblastic transcription of the cluster galectin genes.

Novel colorectal cancer methylation markers identified in African American patients

Researchers utilized Zymo Research’s Methyl MiniSeq and Targeted Bisulfite Sequencing services to identify and validate methylation markers linked to colorectal cancer (CRC) in African Americans. Genome-wide methylation analysis with Methyl MiniSeq revealed 355 differentially methylated CpG sites in 13 genes in addition to hypomethylated Long INterspersed Elements (LINEs) in CRC samples. Furthermore, six of these genes contained the 50 CpGs with the highest degrees of differential methylation and were then studied further using 42 samples and Targeted Bisulfite Sequencing. With the larger sample set and more focused sequencing, the authors were able to validate four genes, EID3, BMP3, GAS7, and GPR75, as novel CRC methylation markers in African American patients.

DNA methylation regulates transitions between cellular states

The Methyl-MiniSeq service was used by the authors to determine the role DNA methylation has in stabilizing gene expression of embryonic stem cells transitioning between cellular states. The expression of a pluripotent marker, Rex1, was found to be correlated with the expression of key methylation factors: Dnmt3b, Tet1, and Prdm14. Methyl-MiniSeq data showed that cells in the Rex1-low state had higher levels of promoter methylation compared to those in the Rex1-high state. Differential methylation occurred at the promoters of key ESC regulators such as Esrrb, Tet1, and Tcl2; these results correlated with the differential gene expression observed by single-molecular RNA-FISH (smFISH). Furthermore, cells with DNA methyltransferase knock-outs or those treated with a DNA methylation inhibitor had fewer cells in the Rex1-low state. These findings suggest that methylation is required for the transition between two cellular states and its maintenance.

Infection by Epstein-Barr virus induces genome-wide methylation changes

Researchers examining the epigenetic modifications in oral keratinocytes following Epstein-Barr virus (EBV) infection used an expanded and streamlined version of Reduced Representation Bisulfite Sequencing and bioinformatics analysis (Methyl-MiniSeq). The group found that EBV infection induced long-lasting DNA methylation changes at CpG islands, promoters, and within gene bodies when compared to uninfected cells. Furthermore, the DNA methylation changes correlated with gains and losses in expression of a subset of affected genes. The findings provide mechanistic insights as to how EBV infection contributes to DNA methylation changes commonly observed in EBV-associated carcinomas and hints at the potential for future epigenetics-based therapies.

Endothelial DNA Methylation Goes With the Flow

The Methyl-MiniSeq (expanded-RRBS) service from Zymo Research was used to study how disturbed blood flow (d-flow) epigenetically regulates endothelial gene expression and causes endothelial cells to initiate and sustain the development of plaque that contributes to atherosclerosis. Using the Methyl-MiniSeq platform, the researchers found that sections of carotid artery exposed to d-flow exhibited significantly higher levels of hypermethylated CpG sites compared to sections of carotid artery not exposed to d-flow. The method also revealed that treating mice with the DNA methyltransferase (DNMT)-inhibitor 5Aza reduced the number of hypermethylated CpG sites in endothelial sections exposed to d-flow. These findings suggest that DNMT is the mechanism by which d-flow induces genome-wide DNA methylation changes in endothelial cells.

Bcl6 acts in a “hit-and-run” manner during lymphomagenesis

Using the Methyl-MiniSeq service, study authors investigated how the BCL6 oncogene contributes to diffuse large B-cell lymphoma (DLBCL) development. The investigators used a mouse model in which Bcl6 expression was restricted to hematopoietic stem/progenitor cells (HSPCs) and found that BCL6 predisposed developing B-cells to a malignant fate. Data generated via the Methyl-MiniSeq service helped identify epigenetically reprogrammed DNA methylation patterns in HSPC genomes. The proposed “hit-and-run” model for BCL6 driven tumor development suggests a possible reason as to why certain treatment strategies fail in some lymphoma patients and not others and suggests new avenues for potential therapeutic development.

Maternal Diet Affects DNA Methylation Patterns in Offspring

The study’s authors used Zymo’s Methyl-MiniSeq service to identify genome-wide DNA methylation pattern changes in the brain tissues of mouse offspring whose pregnant mothers were fed a diet 10 times higher in folic acid concentration compared to low folic acid controls. The researchers found significant changes in the global patterns of DNA methylation between the two groups of mouse pups. Furthermore, the study showed significant changes in the levels of methylated cytosines in both CpG as well as CHG and CHH contexts in CpG islands, gene promoters, gene bodies, and non-coding regions at single-base resolution. In many cases the DNA methylation changes correlated with differential gene expression as measured by quantitative real-time PCR. The findings suggest that maternal folic acid supplementation may profoundly influence methylation and developmental patterns in children and has substantial implications for the observed recent increase in diseases such as Autism Spectrum Disorder.

5-hmC dynamics involved in arthritis disease progression

Researchers used the Reduced Representation Hydroxymethylation Profiling (RRHP) service from Zymo Research to investigate genome-wide changes in DNA hydroxymethylation (5-hmC) of primary human chondrocytes treated with the pro-inflammatory cytokines, IL-1ß & TNF-α. In comparison to untreated chondrocytes, the authors found that treated cells had reduced global 5-hmC levels. Cytokine treatment also resulted in lower levels of ten eleven translocation 1 (TET1), isocitrate dehydrogenases, and a-ketoglutarate, which are all key factors in the catalysis of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC). Using RRHP, the authors then found that IL-1ß treatment resulted in reduced hydroxymethylation at the promoter regions of several IL-1ß-regulated genes. These findings suggest IL-1ß interferes with key mechanisms to reduce hydroxymethylation, resulting in the up-regulation of targeted genes.

Epigenetic Differences Between Human Cancers and Mouse Models

The Methyl-MiniSeq™ (expanded RRBS) Service from Zymo Research was used to investigate genome-wide changes in DNA methylation in mouse medulloblastoma cancer models. The authors detected relatively few cancer-specific regions that exhibited DNA hypermethylation, which was different from what was previously observed for human cancers. The researchers next investigated genome-wide DNA methylation patterns in mouse models for additional types of human cancers, including breast cancer Burkitt’s lymphoma. They found different methylation patterns in the mouse models than their human counterparts, suggesting that some types of cancers require development of better animal models to use for studies related to cancer diagnostics and therapeutics.

Role for DNA Methylation in Rhabdomyosarcoma

Researchers used the Methyl-MiniSeq™ Service from Zymo Research to investigate the mechanisms responsible for establishment of rhabdomyosarcoma (RMS) tumors in children.  Rhabdomyosarcomas are pediatric skeletal muscle cancers that form due to incomplete differentiation of muscle cells.  MyoD is a cellular factor that generally drives the differentiation of normal skeletal muscle cells, but is not able to do so in RMS cells even though it is expressed to normal levels.  In this report, the authors used Methyl-MiniSeq™ to demonstrate that one of the MyoD target genes responsible for muscle differentiation, JDP2, is not expressed due to DNA hypermethylation in its promoter region, and that this epigenetic silencing likely contributes to the lack of differentiation in rhabdomyosarcoma cells. 

Epigenetic Reprogramming in Multiple Myeloma

Researchers used the Methyl-MiniSeq™ Service from Zymo Research to investigate the DNA methylation profiles of a mouse model for multiple myeloma. Compared to wild type controls, mouse hematopoietic stem/progenitor cells ectopically expressing the MafB oncogene underwent specific epigenetic reprogramming that predisposed activated plasma cells to a cancerous fate. The findings suggest a novel MafB-mediated “hands off” epigenetic molecular mechanism for multiple myeloma and associated plasma tumor cell initiation, and provide insight for innovative, targeted cancer therapy.


Published Methods

JBP1-seq: A fast and efficient method for genome-wide profiling of 5-hmC

Zymo Research scientists have developed an affinity based approach for genome wide 5-hmC profiling that combines the benefits of an improved recombinant JBP1 protein with Nextera-based NGS library construction. Recombinant JBP1 is biotinylated in vivo and conjugated to magnetic beads via biotin–streptavidin interactions. These modifications allow efficient and consistent pull-down of ß-glucosyl-5-hydroxymethylcytosine (ß-glu-5-hmC), and sequence-ready libraries can be generated within 4.5 h from DNA inputs as low as 50 ng. Comparison of technical duplicates and validations with alternative platforms demonstrate the method to be highly reproducible and reliable, thus providing a fast, efficient, and cost-effective method for accurate 5hmC genome-wide profiling.

RRHP: a tag-based approach for 5-hydroxymethylcytosine mapping at single-site Resolution

Current methods for genomic mapping of 5-hydroxymethylcytosine (5hmC) have been limited by either costly sequencing depth, high DNA input, or lack of single-base resolution. Zymo Research scientists present an approach called Reduced Representation 5-Hydroxymethylcytosine Profiling (RRHP) to map 5hmC sites at single-base resolution by exploiting the use of beta-glucosyltransferase to inhibit enzymatic digestion at the junction where adapters are ligated to a genomic library. By doing so, only library fragments presenting glucosylated 5hmC residues at the junction are sequenced. RRHP can detect sites with low 5hmC abundance, and when combined with RRBS data, 5-methylcytosine and 5-hydroxymethylcytosine can be compared at a specific site.


Testimonials

“I am a pediatric oncologist studying the role of aberrant DNA methylation in pediatric malignancies. I began using Zymo Research's genome-wide DNA methylation analysis services over 18 months ago and have been extremely pleased with the data I have received. In addition to carefully preparing RRBS libraries (Methyl-MiniSeq™) from precious patient samples, the bioinformatic analysis provided has been easy to interpret, accurate, and provided the necessary data to plan subsequent experiments. The customer service and technical support I have received has always been timely and helpful. I will continue to the services provided by Zymo Research, and I am excited to use some of their newly expanded epigenetic services such as Reduced Representation Hydroxymethylation Profiling.”

S.D.
Fred Hutchinson Cancer Research Center
Seattle Children's Hospital
University of Washington School of Medicine

"The Epigenetics Services from Zymo Research were exactly what I needed to take my research further. I shipped them my DNA samples, and a few weeks later I received the results. The best part is that their epigenetics team did all of the bioinformatics analysis for us! I'd highly recommend them!"

I.S.G.
CSIC
Universidad de Salamanca, Spain

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