Single-cell DNA methylome analysis of circulating tumor cells

Objective: Previous investigations of circulating tumor cells(CTCs) have mainly focused on their genomic or transcriptomic features, leaving their epigenetic landscape relatively uncharacterized. Here, we investigated the genome-wide DNA methylome of CTCs with a view to understanding the epigenetic regulatory mechanisms underlying cancer metastasis.Methods: We evaluated single-cell DNA methylome and copy number alteration(CNA) in 196 single cells,including 107 CTCs collected from 17 cancer patients covering six different cancer types. Our single-cell bisulfite sequencing(sc BS-seq) covered on average 11.78% of all Cp G dinucleotides and accurately deduced the CNA patterns at 500 kb resolution.Results: We report distinct subclonal structures and different evolutionary histories of CTCs inferred from CNA and DNA methylation profiles. Furthermore, we demonstrate potential tumor origin classification based on the tissue-specific DNA methylation profiles of CTCs.Conclusions: Our work provides a comprehensive survey of genome-wide DNA methylome in single CTCs and reveals 5-methylcytosine(5-m C) heterogeneity in CTCs, addressing the potential epigenetic regulatory mechanisms underlying cancer metastasis and facilitating the future clinical application of CTCs.

DMF-scMT-seq linking methylome and transcriptome within single cells with digital microfluidics

Single-cell joint analysis of methylome and transcriptome reveals how the methylation regulates the transcriptional activity.However,traditional bench-top protocols for single-cell DNA methylation and RNA transcription co-detection are laborintensive,cost-ineffective and contaminant-prone.Herein,we establish the DMF-sc MT-seq,a highly-efficient and cost-effective method to simultaneously analyze single-cell DNA methylation and transcriptional activity based on digital microfluidics.DMFsc MT-seq automates the workflow of single-cell isolation,cellular hypotonic lysis,nucleic acid separation and methylome/transcriptome library construction in a contactless and addressable way.The system ensures high accuracy(R>0.85),high gene detection ability(14,697 genes per cell at 4 million sequencing depth),and high CpG coverage(677,198 CpG sites per cell at 1million sequencing depth).By using DMF-sc MT-seq,the relationship of DNA methylation and RNA transcription under different genomic contexts is resolved.We further apply DMF-sc MT-seq to study the dynamics of transcription regulation with methylation-inhibiting anti-tumor Decitabine,and identify the methylated promoter/gene body driven genes in response to Decitabine treatment.DMF-sc MT-seq facilitates the construction of the correlation of DNA methylation and transcriptional activity at the single-cell level in a flexible,sensitive and accurate way,which is anticipated to be a powerful tool in studying single-cell biological systems.

Genome wide abnormal DNA methylome of human blastocyst in assisted reproductive technology

Proper reprogramming of parental DNA methylomes is essential for mammalian embryonic development. However, it is unknown whether abnormal methylome reprogramming occurs and is associated with the failure of embryonic development. Here we analyzed the DNA methylomes of 57 blastocysts and 29 trophectoderm samples with different morphological grades during assisted reproductive technology （ART） practices. Our data reveal that the global methylation levels of high-quality blastocysts are similar （0.30 ± 0.02, mean ± SD）, while the methylation levels of low-quality blastocysts are divergent and away from those of high-quality blastocysts. The proportion of blastocysts with a methylation level falling within the range of 0.30± 0.02 in different grades correlates with the live birth rate for that grade. Moreover, abnormal methylated regions are associated with the failure of embryonic development. Furthermore, we can use the methylation data of cells biopsied from trophectoderm to predict the blastocyst methylation level as well as to detect the aneuploidy of the blastocysts. Our data indicate that global abnormal methylome reprogramming often occurs in human embryos, and suggest that DNA methylome is a potential biomarker in blastocyst selection in ART.

DNA methylome study of human cerebellar tissues identified genes and pathways possibly involved in essential tremor

Background:Essential tremor(ET)is a neurological syndrome of unknown origin with poorly understood etiology and pathogenesis.It is suggested that the cerebellum and its tracts may be involved in the pathophysiology of ET.DNA methylome interrogation of cerebellar tissue may help shine some light on the understanding of the mechanism of the development of ET.Our study used postmortem human cerebellum tissue samples collected from 12 ET patients and 11 matched non-ET controls for DNA methylome study to identify differentially methylated genes in ET.Results:Using Nugen’s Ovation reduced representation bisulfite sequencing(RRBS),we identified 753 genes encompassing 938 CpG sites with significant differences in DNA methylation between the ET and the control group.Identified genes were further analyzed with Ingenuity Pathway Analysis(IPA)by which we identified certain significant pathways,upstream regulators,diseases and functions,and networks associated with ET.Conclusions:Our study provides evidence that there are significant differences in DNA methylation patterns between the ET and control samples,suggesting that the methylation alteration of certain genes in the cerebellum may be associated with ET pathogenesis.The identified genes allude to the GABAergic hypothesis which supports the notation that ET is a neurodegenerative disease,particularly involving the cerebellum.

Spaceflight decelerates the epigenetic clock orchestrated with a global alteration in DNA methylome and transcriptome in the mouse retina

Astronauts exhibit an assortment of clinical abnormalities in their eyes during long-duration spaceflight.The purpose of this studywas to determinewhether spaceflight induces epigenomic and transcriptomic reprogramming in the retina or alters the epigenetic clock.The mice were flown for 37 days in animal enclosure modules on the International Space Station;ground-based control animals weremaintained under similar housing conditions.Mouse retinas were isolated and both DNA methylome and transcriptome were determined by deep sequencing.We found that a large number of genes were differentially methylated with spaceflight,whereas there were fewer differentially expressed genes at the transcriptome level.Several biological pathways involved in retinal diseases such as macular degeneration were significantly altered.Our results indicated that spaceflight decelerated the retinal epigenetic clock.This study demonstrates that spaceflight impacts the retina at the epigenomic and transcriptomic levels,and such changes could be involved in the etiology of eye-related disorders among astronauts.

MBRidge: an accurate and cost-effective method for profiling DNA methylome at single-base resolution

Organisms and cells,in response to environmental influences or during development,undergo considerable changes in DNA methylation on a genome-wide scale,which are linked to a variety of biological processes.Using MethylC-seq to decipher DNA methylome at single-base resolution is prohibitively costly.In this study,we develop a novel approach,named MBRidge,to detect the methylation levels of repertoire CpGs,by innovatively introducing C-hydroxylmethylated adapters and bisulfate treatment into the MeDIP-seq protocol and employing ridge regression in data analysis.A systematic evaluation of DNA methylome in a human ovarian cell line T29 showed that MBRidge achieved high correlation(R>0.90)with much less cost(∼10%)in comparison with MethylC-seq.We further applied MBRidge to profiling DNA methylome in T29H,an oncogenic counterpart of T29’s.By comparing methylomes of T29H and T29,we identified 131790 differential methylation regions(DMRs),which are mainly enriched in carcinogenesis-related pathways.These are substantially different from7567 DMRs that were obtained by RRBS and related with cell development or differentiation.The integrated analysis ofDMRsin the promoterand expression of DMR-corresponding genes revealed thatDNAmethylation enforced reverse regulation of gene expression,depending on the distance fromthe proximalDMRto transcription starting sites in both mRNA and lncRNA.Taken together,our results demonstrate that MBRidge is an efficient and cost-effective method that can be widely applied to profiling DNA methylomes.

Whole‑genome transcriptome and DNA methylation dynamics of pre‑implantation embryos reveal progression of embryonic genome activation in buffaloes

Background During mammalian pre-implantation embryonic development(PED),the process of maternal-to-zygote transition(MZT)is well orchestrated by epigenetic modification and gene sequential expression,and it is related to the embryonic genome activation(EGA).During MZT,the embryos are sensitive to the environment and easy to arrest at this stage in vitro.However,the timing and regulation mechanism of EGA in buffaloes remain obscure.Results Buffalo pre-implantation embryos were subjected to trace cell based RNA-seq and whole-genome bisulfite sequencing(WGBS)to draw landscapes of transcription and DNA-methylation.Four typical developmental steps were classified during buffalo PED.Buffalo major EGA was identified at the 16-cell stage by the comprehensive analy-sis of gene expression and DNA methylation dynamics.By weighted gene co-expression network analysis,stage-spe-cific modules were identified during buffalo maternal-to-zygotic transition,and key signaling pathways and biological process events were further revealed.Programmed and continuous activation of these pathways was necessary for success of buffalo EGA.In addition,the hub gene,CDK1,was identified to play a critical role in buffalo EGA.Conclusions Our study provides a landscape of transcription and DNA methylation in buffalo PED and reveals deeply the molecular mechanism of the buffalo EGA and genetic programming during buffalo MZT.It will lay a foundation for improving the in vitro development of buffalo embryos.

Identification of lncRNAs Associated withβ-conglycininα-null Allele Based on a Genome-wide Transcriptome Analysis ofα-null-type Hypoallergenic Soybean(Glycine max)Near-isogenic Lines

Soybean mutants withα-nullβ-conglycinin are associated with high nutritional value and low allergenic risk.Although long noncoding RNAs(lncRNAs)are increasingly recognized as functional regulatory components affecting eukaryotic gene expression,little is known about lnc RNA profiles inα-null-type hypoallergenic soybeans.In this study,a genome-wide integrative analysis of lncRNAs,m RNAs and epigenomic data in the soybean cgy-2(confirmedα-null)near-isogenic line(NIL)and its recurrent parent Dongnong47(DN47)was conducted.Nineteen novel lncRNAs that were differentially expressed(DE)only in the NIL at 18 days after flowering(i.e.,α-null-associated DE lncRNAs)were delected.Sixteen putative soybean stress-responsive lncRNAs were identified,and observed to regulate 257 stress-related genes DE in the NIL.This result indicated that theα-null allele might represent an intrinsic defect stress that altered the expression of various stress-related genes inα-null-type hypoallergenic soybean.Additionally,25 epigenetic-related lncRNAs regulated 831 DE epigenetic-related genes and simultaneously initiated multiple epigenetic activities,including ubiquitination,methylation and acetylation.Kyoto encyclopedia of genes and genomes(KEGG)analysis indicated that the biosynthesis of amino acids pathway was enriched with 83 DE genes regulated by nine DE lncRNAs.Changes in the expression of these lncRNAs and genes might be the reason for the altered amino acid composition in the NIL.Among all detected DE lncRNAs,MSTRG.12518 was the most conspicuousα-null-specific cis/trans-lnc RNA that played an efficient,versatile and vital role in the NIL.The data indicated that the lnc RNA profile differed between the NIL and DN47.Variations in lncRNAs,gene expression levels and DNA methylation states likely contributed to the intrinsic defect stress response mechanism inα-null-type hypoallergenic soybeans.

Integration of genome scale data for identifying newplayers in colorectal cancer

Colorectal cancers(CRCs) display a wide variety of genomic aberrations that may be either causally linked to their development and progression, or might serve as biomarkers for their presence. Recent advances in rapid high-throughput genetic and genomic analysis have helped to identify a plethora of alterations that can potentially serve as new cancer biomarkers, and thus help to improve CRC diagnosis, prognosis, and treatment. Each distinct data type(copy number variations, gene and micro RNAs expression, Cp G island methylation) provides an investigator with a different, partially independent, and complementary view of the entire genome. However, elucidation of gene function will require more information than can be provided by analyzing a single type of data. The integration of knowledge obtained from different sources is becoming increasingly essential for obtaining an interdisciplinary view of large amounts of information, and also for cross-validating experimental results. The integration of numerous types of genetic and genomic data derived from public sources, and via the use of ad-hoc bioinformatics tools and statistical methods facilitates the discovery and validation of novel, informative biomarkers. This combinatory approach will also enable researchers to more accurately and comprehensively understand the associations between different biologic pathways, mechanisms, and phenomena, and gain new insights into the etiology of CRC.

DNA methylation modification in heterosis initiation through analyzing rice hybrid contemporary seeds

Heterosis is an important biological phenomenon and widely applied in agriculture.Although many studies have been performed by using vegetative organs of F_(1) hybrid plants,how heterosis (or hybrid vigor) is initiated and formed,particularly the underlying molecular mechanism,remain elusive.Hybrid contemporary seeds of rice indica varieties 9311 and PA64 were innovatively used and analysis of DNA methylome of embryo and endosperm at early developing stages revealed the globally decreased DNA methylation.Genes,especially those relate to hormones function and transcriptional regulation present non-additive methylation.Previously identified heterosis-related superior genes are non-additively methylated in early developing hybrid contemporary seeds,suggesting that key genes/loci responsible for heterosis are epigenetically modified even in early developing hybrid seeds and hypomethylation of hybrid seeds after cross-pollination finally result in the long-term transcriptional change of F_(1) hybrid vegetative tissues after germination,demonstrating that altered DNA methylation in hybrid seeds is essential for initiation regulation and maintenance of heterosis exhibiting in F_(1) hybrid plants.Notably,a large number of genes show non-additive methylation in the endosperm of reciprocal hybrids,suggesting that endosperm might also contribute to heterosis.

N6-Methyladenine DNA Methylation in Japonica and Indica Rice Genomes and Its Association with Gene Expression,Plant Development, and Stress Responses

N6-Methyladenine (6mA)DNA methylation has recently been implicated as a potential new epigenetic marker in eukaryotes,including the dioot modelArabidopsis thaliana.However,the conservation and divergence of 6mA distribution patterns and functions in plants remain elusive.Here we report high-quality 6mA methylomes at single-nucleotide resolution in rice based on substantially improved genome sequences of two rice cultivars,Nipponbare (Nip;Japonica)and 93-11 (Indica).Analysis of 6mA genomic distribution and its association with transcription suggest that 6mA distribution and function is rather conserved between rice and Arabidopsis.We found that 6mA levels are positively correlated with the expression of key stressrelated genes,which may be responsible for the difference in stress tolerance between Nip and 93-11. Moreover,we showed that mutations in DDM1 cause defects in plant growth and decreased 6mA level. Our results reveal that 6mA is a conserved DNA modification that is positively associated with gene expression and contributes to key agronomic traits in plants.

BRIF-Seq: Bisulfite-Converted Randomly Integrated Fragments Sequencing at the Single-Cell Level

Single-cell bisulfite sequencing (scBS-seq) was developed to assess DNA methylation heterogeneity in human and mouse. However, the reads are under-represented in regions with high DNA methylation, because these regions are usually fragmented into long segments and are seldom sequenced on the lllumina plat. form. To reduce the read distribution bias and maximize the use of these long segments, we developed bisulfite-converted randomly integrated fragments sequencing (BRIF-seq), a method with high rates of read mapping and genome coverage. Single microspore of maize, which has a highly methylated and repetitive genome, was used to perform BRIF.seq. High coverage of the haploid genome was obtained to evaluate the methylation states of CG, CHG, and CHH (H = A, C, or T). Compared with scBS-seq, BRIF-seq produced reads that were distributed more evenly across the genome, including regions with high DNA methylation. Surprisingly, the methylation rates among the four microspores within one tetrad were similar, but differed significantly among tetrads, suggesting that non-simultaneous methylation reprogramming could occur among tetrads. Similar levels of heterogeneity, which often occur in lowcopy regions, were detected in different genetic backgrounds. These results suggest that BRIF-seq can be applied for single-cell methylome analysis of any species with diverse genetic backgrounds.

Independent variations in genome-wide expression, alternative splicing, and DNA methylation in brain tissues among castes of the buff-tailed bumblebee, Bombus terrestris

Caste differentiation in social hymenopterans is an intriguing example of phenotypic plasticity. However, the co-ordination among gene regulatory factors to mediate caste differentiation remains inconclusive. In this study, we determined the role of gene regulation and related epigenetic processes in pre-imaginal caste differentiation in the primitively eusocial bumblebee Bombus terrestris. By combining RNA-Seq data from Illumina and Pac Bio and accurately quantifying methylation at whole-genomic base pair resolution, we found that queens, workers, and drones mainly differentiate in gene expression but not in alternative splicing and DNA methylation. Gynes are the most distinct with the lowest global level of whole-genomic methylation and with the largest number of caste-specific transcripts and alternative splicing events. By contrast, workers exhibit few uniquely expressed or alternatively spliced genes. Moreover, several genes involved in hormone and neurotransmitter metabolism are related to caste differentiation, whereas several neuropeptides are linked with sex differentiation. Despite little genome-wide association among differential gene expression, splicing, and differential DNA methylation, the overlapped gene ontology(GO) terms point to nutrition-related activity. Therefore, variations in gene regulation correlate with the behavioral differences among castes and highlight the specialization of toolkit genes in bumblebee gynes at the beginning of the adult stage.

Genome-wide antagonism between 5-hydroxymethylcytosine and DNA methylation in the adult mouse brain

Mounting evidence points to critical roles for DNA modifications, including 5-methylcytosine （5mC） and its oxidized forms, in the development, plasticity and disorders of the mammalian nervous system. The novel DNA base 5- hydroxymethylcytosine （5hmC） is known to be capable of initiating passive or active DNA demethylation, but whether and how extensively 5hmC functions in shaping the post-mitotic neuronal DNA methylome is unclear. Here we report the genome-wide distribution of 5hmC in dentate granule neurons from adult mouse hippocampus in vivo. 5hmC in the neuronal genome is highly enriched in gene bodies, especially in exons, and correlates with gene expression. Direct genome-wide comparison of 5hmC distribution between embryonic stem cells and neurons reveals extensive differences, reflecting the functional disparity between these two cell types. Importantly, integrative analysis of 5hmC, overall DNA methylation and gene expression profiles of dentate granule neurons in vivo reveals the genome-wide antagonism between these two states of cytosine modifications, supporting a role for 5hmC in shaping the neuronal DNA methylome by promoting active DNA demethylation.

The progress on the estimation of DNA methylation level and the detection of abnormal methylation

Background:DNA methylation is a key heritable epigenetic modification that plays a crucial role in transcriptional regulation and therefore a broad range of biological processes.The complex patterns of DNA methylation highlight the significance of the profiling the DNA methylation landscape.Results:In this review,the main high-throughput detection technologies are summarized,and then the three trends of computational estimation of DNA methylation levels were analyzed,especially the expanding of the methylation data with lower coverage.Furthermore,the detection methods of differential methylation patterns for sequencing and array data were presented.Conclusions:More and more research indicated the great importance of DNA methylation changes across different diseases,such as cancers.Although a lot of enormous progress has been made in understanding the role of DNA methylation,only few methylated genes or functional elements serve as clinically relevant cancer biomarkers.The bottleneck in DNA methylation advances has shifted from data generation to data analysis.Therefore,it is meaningful to develop machine learning models for computational estimation of methylation profiling and identify the potential biomarkers.