Dynamic DNA methylation: In the right place at the right time

Tissue-type-specific differentially methylated regions are defined by lineage-specific gain and loss of 5mC during embryogenesis.

“These tissue-type specific DMRs are defined by both lineage specific gain and loss of 5mC during embryogenesis.”

• cites [4] Schultz MD, Human body epigenome maps reveal noncanonical DNA methylation variation. Nature. 523, 212– 216 (2015). 26030523 PMC4499021 sourceNot checked yet

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Numerous transcription factors can directly sense cytosine methylation through changes in their binding affinity and specificity.

“In addition, studies suggest that numerous transcription factors (TFs) can also directly sense the state of cytosine methylation through the modulation of their binding affinity and specificity (26).”

• cites [26] Zhu H, Wang G, Qian J, Transcription factors as readers and effectors of DNA methylation. Nat. Rev. Genet 17, 551– 565 (2016). 27479905 PMC5559737 sourceNot checked yet

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Dynamically methylated CpGs cluster into more than one million tissue-specific differentially methylated regions.

“These dynamically methylated CpGs cluster into over 1 million tissue specific differentially methylated regions (DMRs).”

• cites [4] Schultz MD, Human body epigenome maps reveal noncanonical DNA methylation variation. Nature. 523, 212– 216 (2015). 26030523 PMC4499021 sourceNot checked yet

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• cites [5] Ziller MJ, Charting a dynamic DNA methylation landscape of the human genome. Nature. 500, 477– 481 (2013). 23925113 PMC3821869 sourceNot checked yet

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Deletion of REST increases 5mC at lowly methylated regions normally bound by REST.

“The deletion of REST led to increased 5mC at lowly methylated regions normally bound by REST.”

• cites [6] Stadler MB, DNA-binding factors shape the mouse methylome at distal regulatory regions. Nature. 480, 490– 495 (2011). 22170606 sourceNot checked yet

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This gain of 5mC can be reversed by re-introducing REST.

“Notably, such gain of 5mC can be reversed by re-introducing REST.”

• cites [6] Stadler MB, DNA-binding factors shape the mouse methylome at distal regulatory regions. Nature. 480, 490– 495 (2011). 22170606 sourceNot checked yet

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Many genes are expressed during spermatogenesis despite high methylation at their promoters.

“and numerous genes are expressed during spermatogenesis despite high level methylation at their promoters (18).”

• cites [18] Hammoud SS, Chromatin and transcription transitions of mammalian adult germline stem cells and spermatogenesis. Cell Stem Cell. 15, 239– 253 (2014). 24835570 sourceNot checked yet

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Promoter H3K4me3 inhibits DNMT3a activity, preventing methylation at CpG islands.

“Promoter H3K4me3, in turn, inhibits the activity of the de novo DNA methyltransferase DNMT3a thus preventing methylation at CGIs (20).”

• cites [20] Guo X, Structural insight into autoinhibition and histone H3-induced activation of DNMT3A. Nature. 517, 640– 644 (2015). 25383530 sourceNot checked yet

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These CXXC proteins include components of the H3K4 methyltransferase complex.

“These proteins include components of the H3K4 methyltransferase complex (19).”

• cites [19] Smith ZD, Meissner A, DNA methylation: roles in mammalian development. Nat. Rev. Genet 14, 204– 220 (2013). 23400093 sourceNot checked yet

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Global erasure of DNA methylation in primordial germ cells does not cause widespread transcriptional activation.

“For example, global erasure of DNA methylation in primordial germ cells (PGCs) does not lead to widespread transcriptional activation (16, 17)”

• cites [16] Seisenberger S, The dynamics of genome-wide DNA methylation reprogramming in mouse primordial germ cells. Mol. Cell 48, 849– 862 (2012). 23219530 PMC3533687 sourceNot checked yet

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• cites [17] Hill PWS, Epigenetic reprogramming enables the transition from primordial germ cell to gonocyte. Nature. 555, 392– 396 (2018). 29513657 PMC5856367 sourceNot checked yet

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Depletion of 5mC at CpG islands is partly determined by CpG dinucleotide clusters that are recognized and protected from methylation by CXXC zinc finger proteins.

“The depletion of 5mC at CGIs is, in part, determined by the presence of CpG dinucleotide clusters. that are specifically recognized and protected from methylation by proteins containing a CXXC zinc finger domain (19).”

• cites [19] Smith ZD, Meissner A, DNA methylation: roles in mammalian development. Nat. Rev. Genet 14, 204– 220 (2013). 23400093 sourceNot checked yet

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Intragenic large differentially methylated regions can overlap tissue-defining signature genes and predicted super-enhancers.

“Intragenic DMRs that stretch for kilobases (large-DMRs) can overlap with tissue defining “signature genes” as well as with predicted super-enhancers, which are clusters of enhancers that function cooperatively (4, 10, 11).”

• cites [4] Schultz MD, Human body epigenome maps reveal noncanonical DNA methylation variation. Nature. 523, 212– 216 (2015). 26030523 PMC4499021 sourceNot checked yet

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• cites [10] Luo C, Single Cell Methylomes Identify Neuronal Subtypes and Regulatory Elements in Mammalian Cortex. Science (2017). PMC5570439 28798132 sourceNot checked yet

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• cites [11] Whyte WA, Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell. 153, 307– 319 (2013). 23582322 PMC3653129 sourceNot checked yet

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Existing data suggest that the methylome is overall stable beyond adolescence.

“Although few analyses have been systematically performed across the lifespan of mammals, existing data suggests that, overall the methylome is stable beyond adolescence (13).”

• cites [13] Lister R, Global epigenomic reconfiguration during mammalian brain development. Science. 341, 1237905 (2013). 23828890 PMC3785061 sourceNot checked yet

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Under normal physiological conditions, 15% to 21% of CpGs differ in methylation status between human tissues.

“direct comparison of whole genome methylome profiles derived from numerous cell types has revealed that under normal physiological conditions 15%-21% of CpGs vary in methylation status between human tissues (4, 5).”

• cites [4] Schultz MD, Human body epigenome maps reveal noncanonical DNA methylation variation. Nature. 523, 212– 216 (2015). 26030523 PMC4499021 sourceNot checked yet

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• cites [5] Ziller MJ, Charting a dynamic DNA methylation landscape of the human genome. Nature. 500, 477– 481 (2013). 23925113 PMC3821869 sourceNot checked yet

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5mC readers, including methyl-CpG-binding domain proteins such as MECP2 and MBD proteins, recognize 5mC and recruit co-regulators to methylated DNA.

“5mC readers including proteins containing a methyl-CpG-binding domain such as MECP2 or MBD proteins can recognize 5mC and recruit co-regulators to methylated DNA (25).”

• cites [25] Du Q, Luu P-L, Stirzaker C, Clark SJ, Methyl-CpG-binding domain proteins: readers of the epigenome. Epigenomics. 7, 1051– 1073 (2015). 25927341 sourceNot checked yet

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The model that differentially methylated regions reflect collective local demethylation induced by specific transcription factor binding is supported by mouse embryonic stem cell experiments with CTCF and REST.

“This model has been supported by experiments in mouse embryonic stem cell (mESC) using insulator protein CCCTC-binding factor (CTCF) and a transcriptional repressor of neural genes - RE1-Silencing Transcription factor (REST) (6).”

• cites [6] Stadler MB, DNA-binding factors shape the mouse methylome at distal regulatory regions. Nature. 480, 490– 495 (2011). 22170606 sourceNot checked yet

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Aberrant hypermethylation of CpG islands is commonly seen in cancers.

“In contrast, aberrant CGI hypermethylation is commonly observed in cancers (2).”

• cites [2] Jones PA, Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat. Rev. Genet 13, 484– 492 (2012). 22641018 sourceNot checked yet

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In most somatic tissues, more than 80% of CpGs are methylated, except for CpG islands and other regulatory sequences that have reduced 5mC.

“In most somatic tissues, the majority (>80%) of CpGs in the genome are methylated, with the exception of CGIs, and other gene regulatory sequences that show reduced 5mC levels (3).”

• cites [3] Lister R, Human DNA methylomes at base resolution show widespread epigenomic differences. Nature. 462, 315– 322 (2009). 19829295 PMC2857523 sourceNot checked yet

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Depletion of 5mC at CpG islands also depends on binding by other specific transcription factors.

“Beyond CXXC containing proteins, the depletion of 5mC at CGIs is also dependent on the binding of other specific TFs (19).”

• cites [19] Smith ZD, Meissner A, DNA methylation: roles in mammalian development. Nat. Rev. Genet 14, 204– 220 (2013). 23400093 sourceNot checked yet

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Sequential waves of 5mC gain and loss from late fetal to early postnatal development account for most of the 5mC diversity found in differentiated tissues.

“Sequential waves of gain and loss of 5mC in late-fetal to early postnatal development contribute to the majority of 5mC diversity found in differentiated tissues.”

High-throughput assays for transcription factor-DNA interaction have identified many transcription factors whose binding specificity is altered by 5mC in recognition sequences.

“The development of high-throughput assays for TF-DNA interaction has identified many transcription factors with binding specificity altered by the presence of 5mC in recognition sequences (26).”

• cites [26] Zhu H, Wang G, Qian J, Transcription factors as readers and effectors of DNA methylation. Nat. Rev. Genet 17, 551– 565 (2016). 27479905 PMC5559737 sourceNot checked yet

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It remains uncertain how broadly 5mC modulation of transcription factor binding contributes to in vivo gene regulatory programs.

“Although in vitro studies reveal that the majority of TF-DNA interaction can be modulated by 5mC, it is still uncertain how broadly this mechanism contributes to in vivo gene regulatory programs.”

A protein microarray study interrogating more than 1,500 transcription factors found over 40 that can bind methylated motifs.

“A protein microarray study that interrogated more than 1,500 TFs found over 40 TFs that are able to bind methylated motifs (27).”

• cites [27] Hu S, DNA methylation presents distinct binding sites for human transcription factors. Elife. 2, e00726 (2013). 24015356 PMC3762332 sourceNot checked yet

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MECP2- and 5mCA-dependent repression is particularly critical for long genes longer than 100 kb, many of which have neuronal functions.

“MECP2 and 5mCA dependent repression is particularly critical for long genes (with a length greater than 100kb), many of which have neuronal functions (39).”

• cites [39] Gabel HW, Disruption of DNA-methylation-dependent long gene repression in Rett syndrome. Nature. 522, 89– 93 (2015). 25762136 PMC4480648 sourceNot checked yet

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This selective sensitivity to 5mC can be explained by the biophysical properties of CTCF's 11 zinc fingers, which make it sensitive to methylation of specific cytosines in its binding motif.

“This selective 5mC sensitivity can be explained by the different biophysical property of CTCF’s 11 zinc fingers leading to its sensitivity to specific cytosine within its binding motif being methylated (31).”

• cites [31] Hashimoto H, Structural Basis for the Versatile and Methylation-Dependent Binding of CTCF to DNA. Mol. Cell 66, 711– 720.e3 (2017). 28529057 PMC5542067 sourceNot checked yet

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Interaction between DNMT3B and H3K36me leads to higher levels of intragenic 5mC for active genes in mouse embryonic stem cells.

“The interaction between DNMT3B and H3K36me leads to greater levels of intragenic 5mC for active genes in mESC (32).”

• cites [32] Baubec T, Genomic profiling of DNA methyltransferases reveals a role for DNMT3B in genic methylation. Nature. 520, 243– 247 (2015). 25607372 sourceNot checked yet

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5mC affects CTCF binding only at a specific subset of recognition sequences.

“Intriguingly, 5mC only affects CTCF binding at a specific subset of recognition sequences (30).”

• cites [30] Maurano MT, Role of DNA Methylation in Modulating Transcription Factor Occupancy. Cell Rep. 12, 1184– 1195 (2015). 26257180 sourceNot checked yet

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Independent studies report that MECP2 binds both 5mCA and 5mCG sites with high affinity.

“Independent studies have reported that MECP2 binds to both 5mCA and 5mCG sites with high affinity (37, 39, 40).”

• cites [37] Guo JU, Distribution, recognition and regulation of non-CpG methylation in the adult mammalian brain. Nat. Neurosci 17, 215– 222 (2014). 24362762 PMC3970219 sourceNot checked yet

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• cites [39] Gabel HW, Disruption of DNA-methylation-dependent long gene repression in Rett syndrome. Nature. 522, 89– 93 (2015). 25762136 PMC4480648 sourceNot checked yet

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• cites [40] Mellén M, Ayata P, Heintz N, 5-hydroxymethylcytosine accumulation in postmitotic neurons results in functional demethylation of expressed genes. Proc. Natl. Acad. Sci. U. S. A 114, E7812– E7821 (2017). 28847947 PMC5604027 sourceNot checked yet

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Certain interactions between transcription factors and low-affinity binding sites can be enhanced by 5mC.

“These studies unexpectedly uncovered that certain interactions between TFs and low affinity binding sites can be enhanced by the presence of 5mC.”

• cites [28] Yin Y, Impact of cytosine methylation on DNA binding specificities of human transcription factors. Science. 356 (2017), doi: 10.1126/science.aaj2239. PMC8009048 28473536 sourceNot checked yet

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In neurons, intragenic 5mCH quantitatively modulates gene expression by interacting with MECP2 and recruiting the NCoR corepressor complex to chromatin.

“In neurons, intragenic 5mCH quantitatively modulates gene expression by interacting with MECP2 and recruiting NCoR corepressor complex to chromatin (37–40).”

• cites [37] Guo JU, Distribution, recognition and regulation of non-CpG methylation in the adult mammalian brain. Nat. Neurosci 17, 215– 222 (2014). 24362762 PMC3970219 sourceNot checked yet

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• cites [38] Chen L, MeCP2 binds to non-CG methylated DNA as neurons mature, influencing transcription and the timing of onset for Rett syndrome. Proc. Natl. Acad. Sci. U. S. A 112, 5509– 5514 (2015). 25870282 PMC4418849 sourceNot checked yet

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• cites [39] Gabel HW, Disruption of DNA-methylation-dependent long gene repression in Rett syndrome. Nature. 522, 89– 93 (2015). 25762136 PMC4480648 sourceNot checked yet

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• cites [40] Mellén M, Ayata P, Heintz N, 5-hydroxymethylcytosine accumulation in postmitotic neurons results in functional demethylation of expressed genes. Proc. Natl. Acad. Sci. U. S. A 114, E7812– E7821 (2017). 28847947 PMC5604027 sourceNot checked yet

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Human embryonic stem cells and mammalian brain tissues contain abundant non-CG methylation (5mCH), although the underlying three-base sequence context differs.

“Human embryonic stem cells (hESC) and mammalian brain tissues contain abundant non-CG methylation (5mCH, where H equals A, C or T) although the underlying three-base DNA sequence context (5mCAG vs. 5mCAC, respectively) is distinct (3, 13).”

• cites [3] Lister R, Human DNA methylomes at base resolution show widespread epigenomic differences. Nature. 462, 315– 322 (2009). 19829295 PMC2857523 sourceNot checked yet

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• cites [13] Lister R, Global epigenomic reconfiguration during mammalian brain development. Science. 341, 1237905 (2013). 23828890 PMC3785061 sourceNot checked yet

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CA sites are the most frequently methylated non-CG sites.

“CA sites are the most frequently methylated non-CG sites (13).”

• cites [13] Lister R, Global epigenomic reconfiguration during mammalian brain development. Science. 341, 1237905 (2013). 23828890 PMC3785061 sourceNot checked yet

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In PMD-associated tissues such as cultured fibroblasts, genes located in PMDs are both lowly expressed and methylated.

“In PMD associated tissues such cultured fibroblasts, genes located in PMDs are both lowly expressed and methylated (3).”

• cites [3] Lister R, Human DNA methylomes at base resolution show widespread epigenomic differences. Nature. 462, 315– 322 (2009). 19829295 PMC2857523 sourceNot checked yet

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The correlation between intragenic 5mC and transcription appears to be tissue specific.

“However, this correlation appears to be tissue specific.”

Establishment of intragenic 5mC is a controlled process carried out by DNMT3B and the co-transcriptionally regulated histone modification H3K36me.

“Studies with mESC and a heterologous yeast system found that the establishment of intragenic 5mC is a controlled process carried out by de novo DNA methyltransferase DNMT3B and co-transcriptionally regulated histone modification H3K36me (32, 33).”

• cites [32] Baubec T, Genomic profiling of DNA methyltransferases reveals a role for DNMT3B in genic methylation. Nature. 520, 243– 247 (2015). 25607372 sourceNot checked yet

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• cites [33] Morselli M, In vivo targeting of de novo DNA methylation by histone modifications in yeast and mouse. Elife. 4, e06205 (2015). 25848745 PMC4412109 sourceNot checked yet

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In vitro studies show that the majority of transcription factor-DNA interactions can be modulated by 5mC.

“Although in vitro studies reveal that the majority of TF-DNA interaction can be modulated by 5mC, it is still uncertain how broadly this mechanism contributes to in vivo gene regulatory programs.”

For certain tissue types, active genes are more methylated than repressed genes.

“Indeed, for certain tissue types, active genes are more methylated than repressed genes.”

• cites [2] Jones PA, Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat. Rev. Genet 13, 484– 492 (2012). 22641018 sourceNot checked yet

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Promoter 5mC is negatively correlated with transcription.

“Although promoter 5mC shows a negative correlation with transcription, early studies of intragenic 5mC function posited the opposite finding (2).”

• cites [2] Jones PA, Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat. Rev. Genet 13, 484– 492 (2012). 22641018 sourceNot checked yet

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Analysis of whole-genome methylomes of 39 primary tumors in mouse and human shows pronounced loss of methylation at nuclear lamina-associated partially methylated domains located in late-replicating parts of the genome.

“Analysis of the whole genome methylomes of 39 primary tumors in mouse and human shows a pronounced loss of methylation at nuclear lamina associated partially methylated domains (PMDs) located in late replicating parts of the genome (44).”

• cites [44] Zhou W, DNA methylation loss in late-replicating domains is linked to mitotic cell division. Nat. Genet 50, 591– 602 (2018). 29610480 PMC5893360 sourceNot checked yet

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Functional analyses of 5mC have traditionally relied on pharmacological or genetic perturbations that affect 5mC levels genome-wide.

“Functional analyses of 5mC have traditionally relied on pharmacological or genetic perturbations that affect 5mC levels genome-wide (30, 32).”

• cites [30] Maurano MT, Role of DNA Methylation in Modulating Transcription Factor Occupancy. Cell Rep. 12, 1184– 1195 (2015). 26257180 sourceNot checked yet

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• cites [32] Baubec T, Genomic profiling of DNA methyltransferases reveals a role for DNMT3B in genic methylation. Nature. 520, 243– 247 (2015). 25607372 sourceNot checked yet

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Local amplification of regulator concentration through protein or RNA scaffolding has been incorporated into epigenetic editing to enhance the on-target to off-target ratio.

“Local amplification of regulator concentration through protein (SunTag) or RNA scaffolding (e.g. Casilio) has been incorporated into epigenetic editing to enhance the on/off target ratio (Fig. 3).”

• cites [58] Pflueger C, A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs. Genome Res. (2018), doi: 10.1101/gr.233049.117. PMC6071642 29907613 sourceNot checked yet

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Bulk tissue analyses cannot easily distinguish molecular signatures contributed by distinct cell populations.

“Although computational approaches for tissue deconvolution are improving (48), bulk tissue analyses cannot easily distinguish molecular signatures contributed by distinct cell populations.”

• cites [48] Jaffe AE, Irizarry RA, Accounting for cellular heterogeneity is critical in epigenome-wide association studies. Genome Biol. 15, R31 (2014). 24495553 PMC4053810 sourceNot checked yet

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dCas9-TET and dCas9-DNMT3a fusions have been shown to induce effective remodeling of 5mC.

“dCas9-TET and dCas9-DNMT3a fusions have been shown to induce effective remodeling of 5mC (Fig. 3) (56).”

• cites [56] Liu XS, Editing DNA Methylation in the Mammalian Genome. Cell. 167, 233– 247.e17 (2016). 27662091 PMC5062609 sourceNot checked yet

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Despite global DNA hypomethylation, a limited number of genomic loci gain DNA methylation during aging and in cancer.

“Despite global DNA hypomethylation, a limited number of genomic loci gain DNA methylation during aging and in cancer.”

• cites [47] Schlesinger Y, Polycomb-mediated methylation on Lys27 of histone H3 pre-marks genes for de novo methylation in cancer. Nat. Genet 39, 232– 236 (2007). 17200670 sourceNot checked yet

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Unlike single-cell RNA-seq, single-nuclei methylome data allowed prediction of cell-type-specific regulatory elements using 5mC signatures.

“Unlike single cell RNA-seq which captures transcriptomic information, single nuclei methylome data allowed prediction of cell-type specific regulatory elements with 5mC signatures.”

Modern genome engineering tools have opened the possibility for precise site-specific intervention that would potentially avoid pleiotropic effects of global 5mC disruption.

“Modern genome engineering tools have opened up the possibility for a more precise site specific intervention that would potentially avoid pleiotropic effects of global 5mC disruption (55).”

• cites [55] Pulecio J, Verma N, Mejía-Ramírez E, Huangfu D, Raya A, CRISPR/Cas9-Based Engineering of the Epigenome. Cell Stem Cell. 21, 431– 447 (2017). 28985525 PMC6205890 sourceNot checked yet

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5hmC is abundant in the brain, reaching up to 20% of 5mCG plus 5hmCG.

“This is also supported by the abundance of 5hmC in the brain (up to 20% of 5mCG+5hmCG) (13, 40).”

• cites [13] Lister R, Global epigenomic reconfiguration during mammalian brain development. Science. 341, 1237905 (2013). 23828890 PMC3785061 sourceNot checked yet

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• cites [40] Mellén M, Ayata P, Heintz N, 5-hydroxymethylcytosine accumulation in postmitotic neurons results in functional demethylation of expressed genes. Proc. Natl. Acad. Sci. U. S. A 114, E7812– E7821 (2017). 28847947 PMC5604027 sourceNot checked yet

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Single-cell multi-omics approaches have been developed that can analyze both transcriptomic and epigenomic profiles from a single cell.

“Building upon single-cell methylome methods, single cell multi-omics approaches have been developed that are capable of analyzing both the transcriptomic and epigenomic profiles from a single cell (49).”

• cites [49] Kelsey G, Stegle O, Reik W, Single-cell epigenomics: Recording the past and predicting the future. Science. 358, 69– 75 (2017). 28983045 sourceNot checked yet

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The similarity between tumor and placenta methylomes suggests that tumorigenesis may represent a latent developmental epigenomic program.

“The similarity between tumor and placenta methylomes suggests that tumorigenesis may represent a latent developmental epigenomic program.”

• cites [46] Smith ZD, Epigenetic restriction of extraembryonic lineages mirrors the somatic transition to cancer. Nature. 549, 543– 547 (2017). 28959968 PMC5789792 sourceNot checked yet

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Datasets containing thousands of single-cell methylomes, such as neuron classification in human and mouse frontal cortices, have been generated with snmC-seq or sci-MET.

“Datasets containing thousands of single-cell methylomes, such as neuron classification in the human and mouse frontal cortices, have been generated with a well-based nuclei collection method, snmC-seq, or a combinatorial indexing-based method, sci-MET (10, 50).”

• cites [10] Luo C, Single Cell Methylomes Identify Neuronal Subtypes and Regulatory Elements in Mammalian Cortex. Science (2017). PMC5570439 28798132 sourceNot checked yet

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• cites [50] Mulqueen RM, Highly scalable generation of DNA methylation profiles in single cells. Nat. Biotechnol 36, 428– 431 (2018). 29644997 PMC5938134 sourceNot checked yet

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Sequencing-based approaches have found 5hmC only in CG dinucleotides in mouse embryonic stem cells and brain cortical tissue.

“Although sequencing-based approaches have only found 5hmC in CG dinucleotides in mESC and brain cortical tissue (24), 5hmCH accounted for 38% of 5mCH+5hmCH in cerebellar granule cells, suggesting that 5mCH is also targeted for TET mediated methylation turnover (40).”

• cites [24] Wu H, Zhang Y, Reversing DNA methylation: mechanisms, genomics, and biological functions. Cell. 156, 45– 68 (2014). 24439369 PMC3938284 sourceNot checked yet

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Under pathological conditions, global loss of DNA methylation is often associated with an aberrant transcriptional outcome.

“The situation is, however, different under pathological conditions where the global loss of DNA methylation is often associated with an aberrant transcriptional outcome.”

A fluorescent in vivo reporter of genomic methylation enables microscopic examination of 5mC status at a single genomic locus with cellular resolution.

“In addition to sequencing-based single-cell methylome strategies, a fluorescent in vivo reporter of genomic methylation (RGM) enables microscopic examination of the 5mC status at a single genomic locus with cellular resolution (53).”

• cites [53] Stelzer Y, Shivalila CS, Soldner F, Markoulaki S, Jaenisch R, Tracing dynamic changes of DNA methylation at single-cell resolution. Cell. 163, 218– 229 (2015). 26406378 PMC4583717 sourceNot checked yet

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Studies have already identified 5mC heterogeneity both across and within cell types, providing information not accessible with bulk-level analyses.

“Although single-cell methods are just starting to be applied to analyzing 5mC functions, studies have already identified 5mC heterogeneity both across or within cell types, providing information that cannot be accessed with bulk level analyses.”

Inserting a fluorescent protein gene reporter into a methylation-sensitive locus provides a direct readout of methylation status in living cells or a whole animal.

“In an alternative strategy, the fluorescent protein gene reporter can be directly inserted into the methylation sensitive locus which provides a direct readout of the methylation status in living cells or a whole animal (54).”

• cites [54] Van de Pette M, Visualizing Changes in Cdkn1c Expression Links Early-Life Adversity to Imprint Mis-regulation in Adults. Cell Rep. 18, 1090– 1099 (2017). 28147266 PMC5300902 sourceNot checked yet

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A recent methylation editing study demonstrated that deposition of 5mC at thousands of gene promoters led to only moderate transcriptional alterations.

“For example, a recent methylation editing study demonstrating that deposition of 5mC at thousands of gene promoters led to only moderate transcriptional alterations (65, 66).”

• cites [65] Ford EE, Grimmer MR, Stolzenburg S, Bogdanovic O, Frequent lack of repressive capacity of promoter DNA methylation identified through genome-wide epigenomic manipulation. bioRxiv (2017) (available at http://www.biorxiv.org/content/early/2017/08/17/170506.abstract). sourceNot checked yet

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• cites [66] Korthauer K, Irizarry RA, Genome-wide repressive capacity of promoter DNA methylation is revealed through epigenomic manipulation. bioRxiv (2018) (available at https://www.biorxiv.org/content/early/2018/08/01/381145.abstract). sourceNot checked yet

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Scaffolding allows multiple copies of epigenetic effectors to be tethered to a genomic site, providing more robust on-target modulation and reduced non-specific effects.

“This scaffold allows multiple copies of epigenetic effectors (e.g. DNMT3A catalytic domain) to be tethered to a genomic site, providing more robust on-target modulation and reduced non-specific effects (58).”

• cites [58] Pflueger C, A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs. Genome Res. (2018), doi: 10.1101/gr.233049.117. PMC6071642 29907613 sourceNot checked yet

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The genome-wide distribution of 6mA is species-specific, with no conserved pattern across the eukaryotic species analyzed to date.

“Interestingly, the genome-wide distribution of 6mA is species-specific with lack of a conserved pattern across the eukaryotic species analyzed to date (59).”

• cites [59] Luo G-Z, He C, DNA N6-methyladenine in metazoans: functional epigenetic mark or bystander ? Nat. Struct. Mol. Biol 24, 503– 506 (2017). 28586322 sourceNot checked yet

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Reported differences in 6mA distribution between mouse and human may represent tissue-specific patterns rather than species differences, because the tissues used were unique to each species.

“Reported difference in 6mA distribution between mouse and human may represent tissue specific patterns rather than species differences, since the tissues used for the mouse and human studies were unique to each species (mESC and blood DNA, respectively (60, 61)).”

• cites [60] Xiao C-L, N6-Methyladenine DNA Modification in the Human Genome. Mol. Cell 71, 306– 318.e7 (2018). 30017583 sourceNot checked yet

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• cites [61] Wu TP, DNA methylation on N(6)-adenine in mammalian embryonic stem cells. Nature. 532, 329– 333 (2016). 27027282 PMC4977844 sourceNot checked yet

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Animal models such as mouse fear conditioning suggest a role for 5mC in regulating learning and memory.

“Animal models, such as mouse fear conditioning, suggests a role for 5mC in regulating learning and memory (62).”

• cites [62] Day JJ, Kennedy AJ, Sweatt JD, DNA methylation and its implications and accessibility for neuropsychiatric therapeutics. Annu. Rev. Pharmacol. Toxicol 55, 591– 611 (2015). 25340930 PMC4768530 sourceNot checked yet

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Compelling multimodal evidence has been presented for the existence of 6mA in mouse and human genomes.

“Compelling multimodal evidence has been presented for the existence of 6mA in mouse and human genomes.”

• cites [59] Luo G-Z, He C, DNA N6-methyladenine in metazoans: functional epigenetic mark or bystander ? Nat. Struct. Mol. Biol 24, 503– 506 (2017). 28586322 sourceNot checked yet

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These findings are supported by the identification of both a methyltransferase and a demethylase for deposition and removal of 6mA.

“These findings are supported by the identification of both methyltransferase and demethylase for deposition and removal of 6mA, respectively (60, 61).”

• cites [60] Xiao C-L, N6-Methyladenine DNA Modification in the Human Genome. Mol. Cell 71, 306– 318.e7 (2018). 30017583 sourceNot checked yet

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• cites [61] Wu TP, DNA methylation on N(6)-adenine in mammalian embryonic stem cells. Nature. 532, 329– 333 (2016). 27027282 PMC4977844 sourceNot checked yet

  Not checked in the free run.

In mitotically inactive mammalian tissues, moderate changes in 5mC dynamics likely reflect cellular heterogeneity either across cell types or within a cell type.

“In mitotically inactive mammalian tissues, moderate changes in 5mC dynamics likely reflect cellular heterogeneity either across cell types or within a cell type.”