Global DNA methylation remodeling during direct reprogramming of fibroblasts to neurons

Direct reprogramming of fibroblasts to neurons induces widespread cellular and transcriptional reconfiguration. In this study, we characterized global epigenomic changes during direct reprogramming using whole-genome base-resolution DNA methylome (mC) sequencing. We found that the pioneer transcription factor Ascl1 alone is sufficient for inducing the uniquely neuronal feature of non-CG methylation (mCH), but co-expression of Brn2 and Mytl1 was required to establish a global mCH pattern reminiscent of mature cortical neurons. Ascl1 alone induced strong promoter CG methylation (mCG) of fibroblast specific genes, while BAM overexpression additionally targets a competing myogenic program and directs a more faithful conversion to neuronal cells. Ascl1 induces local demethylation at its binding sites. Surprisingly, co-expression with Brn2 and Mytl1 inhibited the ability of Ascl1 to induce demethylation, suggesting a contextual regulation of transcription factor - epigenome interaction. Finally, we found that de novo methylation by DNMT3A is required for efficient neuronal reprogramming.

[1]  Harrison W. Gabel,et al.  Early-Life Gene Expression in Neurons Modulates Lasting Epigenetic States , 2017, Cell.

[2]  Howard Y. Chang,et al.  Rapid Chromatin Switch in the Direct Reprogramming of Fibroblasts to Neurons. , 2017, Cell reports.

[3]  N. Heintz,et al.  5-hydroxymethylcytosine accumulation in postmitotic neurons results in functional demethylation of expressed genes , 2017, Proceedings of the National Academy of Sciences.

[4]  Thomas Vierbuchen,et al.  Myt1l safeguards neuronal identity by actively repressing many non-neuronal fates , 2017, Nature.

[5]  Joseph R Ecker,et al.  Cerebral Organoids Recapitulate Epigenomic Signatures of the Human Fetal Brain. , 2016, Cell reports.

[6]  N. Neff,et al.  Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq , 2016, Nature.

[7]  Terrence J. Sejnowski,et al.  Epigenomic Signatures of Neuronal Diversity in the Mammalian Brain , 2015, Neuron.

[8]  Matthew D. Schultz,et al.  Human Body Epigenome Maps Reveal Noncanonical DNA Methylation Variation , 2015, Nature.

[9]  Wei Li,et al.  MeCP2 binds to non-CG methylated DNA as neurons mature, influencing transcription and the timing of onset for Rett syndrome , 2015, Proceedings of the National Academy of Sciences.

[10]  Christopher M. Vockley,et al.  Regulation of chromatin accessibility and Zic binding at enhancers in the developing cerebellum , 2015, Nature Neuroscience.

[11]  Paul Flicek,et al.  Ascl1 Coordinately Regulates Gene Expression and the Chromatin Landscape during Neurogenesis , 2015, Cell reports.

[12]  Harrison W. Gabel,et al.  Disruption of DNA methylation-dependent long gene repression in Rett syndrome , 2015, Nature.

[13]  Robert J. Schmitz,et al.  MethylC-seq library preparation for base-resolution whole-genome bisulfite sequencing , 2015, Nature Protocols.

[14]  Wei Li,et al.  Dnmt3a and Dnmt3b have overlapping and distinct functions in hematopoietic stem cells. , 2014, Cell stem cell.

[15]  Paul Theodor Pyl,et al.  HTSeq – A Python framework to work with high-throughput sequencing data , 2014, bioRxiv.

[16]  Seung Woo Jung,et al.  Generation of Induced Neuronal Cells by the Single Reprogramming Factor ASCL1 , 2014, Stem cell reports.

[17]  Matthew D. Schultz,et al.  Abnormalities in human pluripotent cells due to reprogramming mechanisms , 2014, Nature.

[18]  K. Conneely,et al.  A Bayesian hierarchical model to detect differentially methylated loci from single nucleotide resolution sequencing data , 2014, Nucleic acids research.

[19]  Guoping Fan,et al.  Distribution, recognition and regulation of non-CpG methylation in the adult mammalian brain , 2013, Nature Neuroscience.

[20]  Howard Y. Chang,et al.  Hierarchical Mechanisms for Direct Reprogramming of Fibroblasts to Neurons , 2013, Cell.

[21]  G. Hon,et al.  Adult tissue methylomes harbor epigenetic memory at embryonic enhancers , 2013, Nature Genetics.

[22]  Matthew D. Schultz,et al.  Global Epigenomic Reconfiguration During Mammalian Brain Development , 2013, Science.

[23]  Michael B. Stadler,et al.  Identification of active regulatory regions from DNA methylation data , 2013, Nucleic acids research.

[24]  T. Südhof,et al.  Acute reduction in oxygen tension enhances the induction of neurons from human fibroblasts , 2013, Journal of Neuroscience Methods.

[25]  Lee E. Edsall,et al.  A map of the cis-regulatory sequences in the mouse genome , 2012, Nature.

[26]  Kevin W Eliceiri,et al.  NIH Image to ImageJ: 25 years of image analysis , 2012, Nature Methods.

[27]  J. Adjaye,et al.  Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells , 2012, Journal of visualized experiments : JoVE.

[28]  Steven L Salzberg,et al.  Fast gapped-read alignment with Bowtie 2 , 2012, Nature Methods.

[29]  Vijay K. Tiwari,et al.  DNA-binding factors shape the mouse methylome at distal regulatory regions , 2011, Nature.

[30]  F. Guillemot,et al.  A novel function of the proneural factor Ascl1 in progenitor proliferation identified by genome-wide characterization of its targets. , 2011, Genes & development.

[31]  William Stafford Noble,et al.  FIMO: scanning for occurrences of a given motif , 2011, Bioinform..

[32]  Thomas Vierbuchen,et al.  Direct conversion of fibroblasts to functional neurons by defined factors , 2010, Nature.

[33]  Davis J. McCarthy,et al.  edgeR: a Bioconductor package for differential expression analysis of digital gene expression data , 2009, Bioinform..

[34]  Mikael Bodén,et al.  MEME Suite: tools for motif discovery and searching , 2009, Nucleic Acids Res..

[35]  Jonathan E. Dodge,et al.  Inactivation of Dnmt3b in Mouse Embryonic Fibroblasts Results in DNA Hypomethylation, Chromosomal Instability, and Spontaneous Immortalization* , 2005, Journal of Biological Chemistry.

[36]  E. Li,et al.  Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting , 2004, Nature.

[37]  Joan Western,et al.  The Human Body , 1991, Nature.

[38]  Wei Li,et al.  Dnmt 3 a and Dnmt 3 b Have Overlapping and Distinct Functions in Hematopoietic Stem Cells , 2014 .

[39]  Samuele G. Marro,et al.  Transdifferentiation of mouse fibroblasts and hepatocytes to functional neurons. , 2014, Methods in molecular biology.

[40]  Thomas R. Gingeras,et al.  STAR: ultrafast universal RNA-seq aligner , 2013, Bioinform..

[41]  Y. Barde,et al.  Neurotrophins are required for nerve growth during development , 2001, Nature Neuroscience.

[42]  N. Sonenberg,et al.  UHRF1 Plays a Role in Maintaining DNA Methylation in Mammalian Cells , 2007, Science.