Life Sciences Research for Lifelong Health

Publications gavin-kelsey

Title / Authors / Details Open Access Download

Genome-Scale Oscillations in DNA Methylation during Exit from Pluripotency.
Rulands S, Lee HJ, Clark SJ, Angermueller C, Smallwood SA, Krueger F, Mohammed H, Dean W, Nichols J, Rugg-Gunn P, Kelsey G, Stegle O, Simons BD, Reik W

Pluripotency is accompanied by the erasure of parental epigenetic memory, with naïve pluripotent cells exhibiting global DNA hypomethylation both in vitro and in vivo. Exit from pluripotency and priming for differentiation into somatic lineages is associated with genome-wide de novo DNA methylation. We show that during this phase, co-expression of enzymes required for DNA methylation turnover, DNMT3s and TETs, promotes cell-to-cell variability in this epigenetic mark. Using a combination of single-cell sequencing and quantitative biophysical modeling, we show that this variability is associated with coherent, genome-scale oscillations in DNA methylation with an amplitude dependent on CpG density. Analysis of parallel single-cell transcriptional and epigenetic profiling provides evidence for oscillatory dynamics both in vitro and in vivo. These observations provide insights into the emergence of epigenetic heterogeneity during early embryo development, indicating that dynamic changes in DNA methylation might influence early cell fate decisions.

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Cell systems, , 2405-4712, , 2018

PMID: 30031774

Open Access

Epigenetic regulation in development: is the mouse a good model for the human?
Hanna CW, Demond H, Kelsey G

Over the past few years, advances in molecular technologies have allowed unprecedented mapping of epigenetic modifications in gametes and during early embryonic development. This work is allowing a detailed genomic analysis, which for the first time can answer long-standing questions about epigenetic regulation and reprogramming, and highlights differences between mouse and human, the implications of which are only beginning to be explored.

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Human reproduction update, , 1460-2369, , 2018

PMID: 29992283

scNMT-seq enables joint profiling of chromatin accessibility DNA methylation and transcription in single cells.
Clark SJ, Argelaguet R, Kapourani CA, Stubbs TM, Lee HJ, Alda-Catalinas C, Krueger F, Sanguinetti G, Kelsey G, Marioni JC, Stegle O, Reik W

Parallel single-cell sequencing protocols represent powerful methods for investigating regulatory relationships, including epigenome-transcriptome interactions. Here, we report a single-cell method for parallel chromatin accessibility, DNA methylation and transcriptome profiling. scNMT-seq (single-cell nucleosome, methylation and transcription sequencing) uses a GpC methyltransferase to label open chromatin followed by bisulfite and RNA sequencing. We validate scNMT-seq by applying it to differentiating mouse embryonic stem cells, finding links between all three molecular layers and revealing dynamic coupling between epigenomic layers during differentiation.

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Nature communications, 9, 2041-1723, 781, 2018

PMID: 29472610

MLL2 conveys transcription-independent H3K4 trimethylation in oocytes.
Hanna CW, Taudt A, Huang J, Gahurova L, Kranz A, Andrews S, Dean W, Stewart AF, Colomé-Tatché M, Kelsey G

Histone 3 K4 trimethylation (depositing H3K4me3 marks) is typically associated with active promoters yet paradoxically occurs at untranscribed domains. Research to delineate the mechanisms of targeting H3K4 methyltransferases is ongoing. The oocyte provides an attractive system to investigate these mechanisms, because extensive H3K4me3 acquisition occurs in nondividing cells. We developed low-input chromatin immunoprecipitation to interrogate H3K4me3, H3K27ac and H3K27me3 marks throughout oogenesis. In nongrowing oocytes, H3K4me3 was restricted to active promoters, but as oogenesis progressed, H3K4me3 accumulated in a transcription-independent manner and was targeted to intergenic regions, putative enhancers and silent H3K27me3-marked promoters. Ablation of the H3K4 methyltransferase gene Mll2 resulted in loss of transcription-independent H3K4 trimethylation but had limited effects on transcription-coupled H3K4 trimethylation or gene expression. Deletion of Dnmt3a and Dnmt3b showed that DNA methylation protects regions from acquiring H3K4me3. Our findings reveal two independent mechanisms of targeting H3K4me3 to genomic elements, with MLL2 recruited to unmethylated CpG-rich regions independently of transcription.

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Nature structural & molecular biology, 25, 1545-9985, 73-82, 2018

PMID: 29323282

Cultured bovine embryo biopsy conserves methylation marks from original embryo.
Fonseca Balvís N, Garcia-Martinez S, Pérez-Cerezales S, Ivanova E, Gomez-Redondo I, Hamdi M, Rizos D, Coy P, Kelsey G, Gutierrez-Adan A

A major limitation of embryo epigenotyping by chromatin immunoprecipitation analysis is the reduced amount of sample available from an embryo biopsy. We developed an in vitro system to expand trophectoderm cells from an embryo biopsy to overcome this limitation. This work analyzes whether expanded trophectoderm (EX) is representative of the trophectoderm (TE) methylation or adaptation to culture has altered its epigenome. We took a small biopsy from the trophectoderm (30-40 cells) of in vitro produced bovine-hatched blastocysts and cultured it on fibronectin-treated plates until we obtained ∼4 × 104 cells. The rest of the embryo was allowed to recover its spherical shape and, subsequently, TE and inner cell mass were separated. We examined whether there were DNA methylation differences between TE and EX of three bovine embryos using whole-genome bisulfite sequencing. As a consequence of adaptation to culture, global methylation, including transposable elements, was higher in EX, with 5.3% of quantified regions showing significant methylation differences between TE and EX. Analysis of individual embryos indicated that TE methylation is more similar to its EX counterpart than to TE from other embryos. Interestingly, these similarly methylated regions are enriched in CpG islands, promoters and transcription units near genes involved in biological processes important for embryo development. Our results indicate that EX is representative of the embryo in terms of DNA methylation, thus providing an informative proxy for embryo epigenotyping.

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Biology of reproduction, 97, 1529-7268, 189-196, 2017

PMID: 29044423

Single-cell epigenomics: Recording the past and predicting the future.
Kelsey G, Stegle O, Reik W

Single-cell multi-omics has recently emerged as a powerful technology by which different layers of genomic output-and hence cell identity and function-can be recorded simultaneously. Integrating various components of the epigenome into multi-omics measurements allows for studying cellular heterogeneity at different time scales and for discovering new layers of molecular connectivity between the genome and its functional output. Measurements that are increasingly available range from those that identify transcription factor occupancy and initiation of transcription to long-lasting and heritable epigenetic marks such as DNA methylation. Together with techniques in which cell lineage is recorded, this multilayered information will provide insights into a cell's past history and its future potential. This will allow new levels of understanding of cell fate decisions, identity, and function in normal development, physiology, and disease.

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Science (New York, N.Y.), 358, 1095-9203, 69-75, 2017

PMID: 28983045

DNA Methylation in Embryo Development: Epigenetic Impact of ART (Assisted Reproductive Technologies).
Canovas S, Ross PJ, Kelsey G, Coy P

DNA methylation can be considered a component of epigenetic memory with a critical role during embryo development, and which undergoes dramatic reprogramming after fertilization. Though it has been a focus of research for many years, the reprogramming mechanism is still not fully understood. Recent results suggest that absence of maintenance at DNA replication is a major factor, and that there is an unexpected role for TET3-mediated oxidation of 5mC to 5hmC in guarding against de novo methylation. Base-resolution and genome-wide profiling methods are enabling more comprehensive assessments of the extent to which ART might impair DNA methylation reprogramming, and which sequence elements are most vulnerable. Indeed, as we also review here, studies showing the effect of culture media, ovarian stimulation or embryo transfer on the methylation pattern of embryos emphasize the need to face ART-associated defects and search for strategies to mitigate adverse effects on the health of ART-derived children.

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BioEssays : news and reviews in molecular, cellular and developmental biology, , 1521-1878, , 2017

PMID: 28940661

Genomic imprinting beyond DNA methylation: a role for maternal histones.
Hanna CW, Kelsey G

Inheritance of DNA methylation states from gametes determines genomic imprinting in mammals. A new study shows that repressive chromatin in oocytes can also confer imprinting.

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Genome biology, 18, 1474-760X, 177, 2017

PMID: 28927436

Open Access

The histone 3 lysine 4 methyltransferase Setd1b is a maternal effect gene required for the oogenic gene expression program.
Brici D, Zhang Q, Reinhardt S, Dahl A, Hartmann H, Schmidt K, Goveas N, Huang J, Gahurova L, Kelsey G, Anastassiadis K, Stewart AF, Kranz A

Germ cell development involves major reprogramming of the epigenome to prime the zygote for totipotency. Histone 3 lysine 4 (H3K4) methylations are universal epigenetic marks mediated in mammals by six H3K4 methyltransferases related to fly Trithorax, including two yeast Set1 orthologs: Setd1a and Setd1b. Whereas Setd1a plays no role in oogenesis, we report that Setd1b deficiency causes female sterility. Oocyte specific Gdf9iCre conditional knockout (Setd1b(Gdf9) cKO) ovaries develop through all stages however follicular loss accumulated with age and unfertilized metaphase II (MII) oocytes exhibited irregularities of the zona pellucida and meiotic spindle. Most Setd1b(Gdf9) cKO zygotes remained in the pronuclear stage and displayed polyspermy in the perivitelline space. Expression profiling of Setd1b(Gdf9) cKO MII oocytes revealed (i) that Setd1b promotes the expression of the major oocyte transcription factors including Obox1, 2, 5, 7, Meis2 and Sall4; and (ii) two-times more up- than downregulated mRNAs suggesting that Setd1b also promotes the expression of negative regulators of oocyte development with multiple Zfp-KRAB factors implicated. Together, these findings indicate that Setd1b serves as maternal effect gene through regulation of the oocyte gene expression program.

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Development (Cambridge, England), , 1477-9129, , 2017

PMID: 28619824

Transcription and chromatin determinants of de novo DNA methylation timing in oocytes.
Gahurova L, Tomizawa SI, Smallwood SA, Stewart-Morgan KR, Saadeh H, Kim J, Andrews SR, Chen T, Kelsey G

Gametogenesis in mammals entails profound re-patterning of the epigenome. In the female germline, DNA methylation is acquired late in oogenesis from an essentially unmethylated baseline and is established largely as a consequence of transcription events. Molecular and functional studies have shown that imprinted genes become methylated at different times during oocyte growth; however, little is known about the kinetics of methylation gain genome wide and the reasons for asynchrony in methylation at imprinted loci.

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Epigenetics & chromatin, 10, 1756-8935, 25, 2017

PMID: 28507606

Open Access

Genome-wide base-resolution mapping of DNA methylation in single cells using single-cell bisulfite sequencing (scBS-seq).
Clark SJ, Smallwood SA, Lee HJ, Krueger F, Reik W, Kelsey G

DNA methylation (DNAme) is an important epigenetic mark in diverse species. Our current understanding of DNAme is based on measurements from bulk cell samples, which obscures intercellular differences and prevents analyses of rare cell types. Thus, the ability to measure DNAme in single cells has the potential to make important contributions to the understanding of several key biological processes, such as embryonic development, disease progression and aging. We have recently reported a method for generating genome-wide DNAme maps from single cells, using single-cell bisulfite sequencing (scBS-seq), allowing the quantitative measurement of DNAme at up to 50% of CpG dinucleotides throughout the mouse genome. Here we present a detailed protocol for scBS-seq that includes our most recent developments to optimize recovery of CpGs, mapping efficiency and success rate; reduce hands-on time; and increase sample throughput with the option of using an automated liquid handler. We provide step-by-step instructions for each stage of the method, comprising cell lysis and bisulfite (BS) conversion, preamplification and adaptor tagging, library amplification, sequencing and, lastly, alignment and methylation calling. An individual with relevant molecular biology expertise can complete library preparation within 3 d. Subsequent computational steps require 1-3 d for someone with bioinformatics expertise.

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Nature protocols, 12, 1750-2799, 534-547, 2017

PMID: 28182018

DNA methylation and gene expression changes derived from assisted reproductive technologies can be decreased by reproductive fluids.
Canovas S, Ivanova E, Romar R, García-Martínez S, Soriano-Úbeda C, García-Vázquez FA, Saadeh H, Andrews S, Kelsey G, Coy P

The number of children born since the origin of Assisted Reproductive Technologies (ART) exceeds 5 million. The majority seem healthy, but a higher frequency of defects has been reported among ART-conceived infants, suggesting an epigenetic cost. We report the first whole-genome DNA methylation datasets from single pig blastocysts showing differences between in vivo and in vitro produced embryos. Blastocysts were produced in vitro either without (C-IVF) or in the presence of natural reproductive fluids (Natur-IVF). Natur-IVF embryos were of higher quality than C-IVF in terms of cell number and hatching ability to. RNA-Seq and DNA methylation analyses showed that Natur-IVF embryos have expression and methylation patterns closer to in vivo blastocysts. Genes involved in reprogramming, imprinting and development were affected by culture, with fewer aberrations in Natur-IVF embryos. Methylation analysis detected methylated changes in C-IVF, but not in Natur-IVF, at genes whose methylation could be critical, such as IGF2R and NNAT.

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eLife, 6, 2050-084X, , 2017

PMID: 28134613

Open Access

Establishment and functions of DNA methylation in the germline.
Stewart KR, Veselovska L, Kelsey G

Epigenetic modifications established during gametogenesis regulate transcription and other nuclear processes in gametes, but also have influences in the zygote, embryo and postnatal life. This is best understood for DNA methylation which, established at discrete regions of the oocyte and sperm genomes, governs genomic imprinting. In this review, we describe how imprinting has informed our understanding of de novo DNA methylation mechanisms, highlight how recent genome-wide profiling studies have provided unprecedented insights into establishment of the sperm and oocyte methylomes and consider the fate and function of gametic methylation and other epigenetic modifications after fertilization.

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Epigenomics, , 1750-192X, , 2016

PMID: 27659720

Open Access

Single-cell epigenomics: powerful new methods for understanding gene regulation and cell identity.
Clark SJ, Lee HJ, Smallwood SA, Kelsey G, Reik W

Emerging single-cell epigenomic methods are being developed with the exciting potential to transform our knowledge of gene regulation. Here we review available techniques and future possibilities, arguing that the full potential of single-cell epigenetic studies will be realized through parallel profiling of genomic, transcriptional, and epigenetic information.

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Genome biology, 17, 1474-760X, 72, 2016

PMID: 27091476

Open Access

Pervasive polymorphic imprinted methylation in the human placenta.
Hanna CW, Peñaherrera MS, Saadeh H, Andrews S, McFadden DE, Kelsey G, Robinson WP

The maternal and paternal copies of the genome are both required for mammalian development and this is primarily due to imprinted genes, those that are mono-allelically expressed based on parent-of-origin. Typically, this pattern of expression is regulated by differentially methylated regions (DMRs) that are established in the germline and maintained after fertilisation. There are a large number of germline DMRs that have not yet been associated with imprinting and their function in development is unknown. In this study, we developed a genome-wide approach to identify novel imprinted DMRs in the human placenta, and investigated the dynamics of these imprinted DMRs during development in somatic and extra-embryonic tissues. DNA methylation was evaluated using the Illumina HumanMethylation450 array in 134 human tissue samples, publically available reduced representation bisulfite sequencing in the human embryo and germ cells, and targeted bisulfite sequencing in term placentas. 43 known and 101 novel imprinted DMRs were identified in the human placenta, by comparing methylation between diandric and digynic triploid conceptions in addition to female and male gametes. 72 novel DMRs showed a pattern consistent with placental-specific imprinting and this mono-allelic methylation was entirely maternal in origin. Strikingly, these DMRs exhibited polymorphic imprinted methylation between placental samples. These data suggest that imprinting in human development is far more extensive and dynamic than previously reported and that the placenta preferentially maintains maternal germline-derived DNA methylation.

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Genome research, , 1549-5469, , 2016

PMID: 26769960

Open Access

Parallel single-cell sequencing links transcriptional and epigenetic heterogeneity.
Angermueller C, Clark SJ, Lee HJ, Macaulay IC, Teng MJ, Hu TX, Krueger F, Smallwood SA, Ponting CP, Voet T, Kelsey G, Stegle O, Reik W

We report scM&T-seq, a method for parallel single-cell genome-wide methylome and transcriptome sequencing that allows for the discovery of associations between transcriptional and epigenetic variation. Profiling of 61 mouse embryonic stem cells confirmed known links between DNA methylation and transcription. Notably, the method revealed previously unrecognized associations between heterogeneously methylated distal regulatory elements and transcription of key pluripotency genes.

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Nature methods, , 1548-7105, , 2016

PMID: 26752769

Open Access

Erratum to: Deep sequencing and de novo assembly of the mouse occyte transcriptome define the contribution of transcription to the DNA methylation landscape.
Veselovska L, Smallwood SA, Saadeh H, Stewart KR, Krueger F, Maupetit-Méhouas S, Arnaud P, Tomizawa S, Andrews S, Kelsey G

Genome biology, 16, 1474-760X, 271, 2015

PMID: 26635312

Dynamic changes in histone modifications precede de novo DNA methylation in oocytes.
Stewart KR, Veselovska L, Kim J, Huang J, Saadeh H, Tomizawa SI, Smallwood SA, Chen T, Kelsey G

Erasure and subsequent reinstatement of DNA methylation in the germline, especially at imprinted CpG islands (CGIs), is crucial to embryogenesis in mammals. The mechanisms underlying DNA methylation establishment remain poorly understood, but a number of post-translational modifications of histones are implicated in antagonizing or recruiting the de novo DNA methylation complex. In mouse oogenesis, DNA methylation establishment occurs on a largely unmethylated genome and in nondividing cells, making it a highly informative model for examining how histone modifications can shape the DNA methylome. Using a chromatin immunoprecipitation (ChIP) and genome-wide sequencing (ChIP-seq) protocol optimized for low cell numbers and novel techniques for isolating primary and growing oocytes, profiles were generated for histone modifications implicated in promoting or inhibiting DNA methylation. CGIs destined for DNA methylation show reduced protective H3K4 dimethylation (H3K4me2) and trimethylation (H3K4me3) in both primary and growing oocytes, while permissive H3K36me3 increases specifically at these CGIs in growing oocytes. Methylome profiling of oocytes deficient in H3K4 demethylase KDM1A or KDM1B indicated that removal of H3K4 methylation is necessary for proper methylation establishment at CGIs. This work represents the first systematic study performing ChIP-seq in oocytes and shows that histone remodeling in the mammalian oocyte helps direct de novo DNA methylation events.

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Genes & development, , 1549-5477, , 2015

PMID: 26584620

Open Access

Continuous Histone Replacement by Hira Is Essential for Normal Transcriptional Regulation and De Novo DNA Methylation during Mouse Oogenesis.
Nashun B, Hill PW, Smallwood SA, Dharmalingam G, Amouroux R, Clark SJ, Sharma V, Ndjetehe E, Pelczar P, Festenstein RJ, Kelsey G, Hajkova P

The integrity of chromatin, which provides a dynamic template for all DNA-related processes in eukaryotes, is maintained through replication-dependent and -independent assembly pathways. To address the role of histone deposition in the absence of DNA replication, we deleted the H3.3 chaperone Hira in developing mouse oocytes. We show that chromatin of non-replicative developing oocytes is dynamic and that lack of continuous H3.3/H4 deposition alters chromatin structure, resulting in increased DNase I sensitivity, the accumulation of DNA damage, and a severe fertility phenotype. On the molecular level, abnormal chromatin structure leads to a dramatic decrease in the dynamic range of gene expression, the appearance of spurious transcripts, and inefficient de novo DNA methylation. Our study thus unequivocally shows the importance of continuous histone replacement and chromatin homeostasis for transcriptional regulation and normal developmental progression in a non-replicative system in vivo.

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Molecular cell, , 1097-4164, , 2015

PMID: 26549683

Deep sequencing and de novo assembly of the mouse oocyte transcriptome define the contribution of transcription to the DNA methylation landscape.
Veselovska L, Smallwood SA, Saadeh H, Stewart KR, Krueger F, Maupetit-Méhouas S, Arnaud P, Tomizawa SI, Andrews S, Kelsey G

Previously, a role was demonstrated for transcription in the acquisition of DNA methylation at imprinted control regions in oocytes. Definition of the oocyte DNA methylome by whole genome approaches revealed that the majority of methylated CpG islands are intragenic and gene bodies are hypermethylated. Yet, the mechanisms by which transcription regulates DNA methylation in oocytes remain unclear. Here, we systematically test the link between transcription and the methylome.

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Genome biology, 16, 1474-760X, 209, 0

PMID: 26408185

Open Access

Keeping methylation at bay.
Kelsey G

A hallmark of CpG islands is their unmethylated state, and determining how DNA methylation can invade these elements is therefore important for understanding developmental gene regulation and disease. A new study shows that FBXL10, a protein commonly altered by mutation in leukemia, is part of a mechanism that blocks methylation of CpG islands.

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Nature genetics, 47, 1546-1718, 427-8, 2015

PMID: 25916897

Epigenetics: Cellular memory erased in human embryos.
Reik W,Kelsey G

Nature, 511, 1476-4687, 540-1, 2014

PMID: 25079550

Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity.
Smallwood SA,Lee HJ,Angermueller C,Krueger F,Saadeh H,Peat J,Andrews SR,Stegle O,Reik W,Kelsey G

We report a single-cell bisulfite sequencing (scBS-seq) method that can be used to accurately measure DNA methylation at up to 48.4% of CpG sites. Embryonic stem cells grown in serum or in 2i medium displayed epigenetic heterogeneity, with '2i-like' cells present in serum culture. Integration of 12 individual mouse oocyte datasets largely recapitulated the whole DNA methylome, which makes scBS-seq a versatile tool to explore DNA methylation in rare cells and heterogeneous populations.

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Nature methods, 11, 1548-7105, 817-20, 2014

PMID: 25042786

Open Access

The specification of imprints in mammals.
Hanna CW,Kelsey G

At the heart of genomic imprinting in mammals are imprinting control regions (ICRs), which are the discrete genetic elements that confer imprinted monoallelic expression to several genes in imprinted gene clusters. A characteristic of the known ICRs is that they acquire different epigenetic states, exemplified by differences in DNA methylation, in the sperm and egg, and these imprint marks remain on the sperm- and oocyte-derived alleles into the next generation as a lifelong memory of parental origin. Although there has been much focus on gametic marking of ICRs as the point of imprint specification, recent mechanistic studies and genome-wide DNA methylation profiling do not support the existence of a specific imprinting machinery in germ cells. Rather, ICRs are part of more widespread methylation events that occur during gametogenesis. Instead, a decisive component in the specification of imprints is the choice of which sites of gamete-derived methylation to maintain in the zygote and preimplantation embryo at a time when much of the remainder of the genome is being demethylated. Among the factors involved in this selection, the zinc-finger protein Zfp57 can be regarded as an imprint-specific, sequence-specific DNA binding factor responsible for maintaining methylation at most ICRs. The recent insights into the balance of gametic and zygotic contributions to imprint specification should help understand mechanistic opportunities and constraints on the evolution of imprinting in mammals.

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Heredity, 113, 1365-2540, 176-83, 2014

PMID: 24939713

Open Access

Detailed analysis of the genetic and epigenetic signatures of iPSC-derived mesodiencephalic dopaminergic neurons.
Roessler R, Smallwood SA, Veenvliet JV, Pechlivanoglou P, Peng SP, Chakrabarty K, Groot-Koerkamp MJ, Pasterkamp RJ, Wesseling E, Kelsey G, Boddeke E, Smidt MP, Copray S

Induced pluripotent stem cells (iPSCs) hold great promise for in vitro generation of disease-relevant cell types, such as mesodiencephalic dopaminergic (mdDA) neurons involved in Parkinson's disease. Although iPSC-derived midbrain DA neurons have been generated, detailed genetic and epigenetic characterizations of such neurons are lacking. The goal of this study was to examine the authenticity of iPSC-derived DA neurons obtained by established protocols. We FACS purified mdDA (Pitx3 (Gfp/+) ) neurons derived from mouse iPSCs and primary mdDA (Pitx3 (Gfp/+) ) neurons to analyze and compare their genetic and epigenetic features. Although iPSC-derived DA neurons largely adopted characteristics of their in vivo counterparts, relevant deviations in global gene expression and DNA methylation were found. Hypermethylated genes, mainly involved in neurodevelopment and basic neuronal functions, consequently showed reduced expression levels. Such abnormalities should be addressed because they might affect unambiguous long-term functionality and hamper the potential of iPSC-derived DA neurons for in vitro disease modeling or cell-based therapy.

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Stem cell reports, 2, 2213-6711, 520-33, 2014

PMID: 24749075

Open Access