Practical Guidelines and Consideration of Using RRHP for 5hmC Detection

The mechanisms regulating myelin repair in the adult central nervous system (CNS) are unclear. Here, we identify DNA hydroxymethylation, catalyzed by the Ten-Eleven-Translocation (TET) enzyme TET1, as necessary for myelin repair in young adults and defective in old mice. Constitutive and inducible oligodendrocyte lineage-specific ablation of Tet1 (but not of Tet2), recapitulate this age-related decline in repair of demyelinated lesions. DNA hydroxymethylation and transcriptomic analyses identify TET1-target in adult oligodendrocytes, as genes regulating neuro-glial communication, including the solute carrier (Slc) gene family. Among them, we show that the expression levels of the Na+/K+/Cl− transporter, SLC12A2, are higher in Tet1 overexpressing cells and lower in old or Tet1 knockout. Both aged mice and Tet1 mutants also present inefficient myelin repair and axo-myelinic swellings. Zebrafish mutants for slc12a2b also display swellings of CNS myelinated axons. Our findings suggest that TET1 is required for adult myelin repair and regulation of the axon-myelin interface.

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“…Data deposited in GEO: accession number GSE122446. 85,86 . The raw sequencing reads were aligned to the mouse reference genome (mm10).…”

Section: Methodsmentioning

confidence: 99%

TET1-mediated DNA hydroxymethylation regulates adult remyelination in mice

et al. 2021

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“…Reduced representation 5hmC profiling (RRHP) analysis (46,47) was performed as previously described. (21) Briefly, 1 μg of DNA was processed using the RRHP 5-hmC Library Prep Kit from Zymo Research (Cat.…”

Section: Reduced Representation 5hmc Profiling Analysis and Callingmentioning

confidence: 99%

“…To assess which specific genes were losing 5hmC, and how this compared with the 5hmC observed in ATDC5 cells, we performed genomewide sequencing. Because of the scarcity of tissue material from cartilage microdissected from E17.5 embryos (see Materials and Methods section), we utilized a RRHP of CCGG sites, (46,47) which allowed us to probe the 5hmC status of a select number of sites in the mouse genome (Supplementary Table S5).…”

Section: Tet1 Loss In Growth Plates Changes the 5hmc Landscapementioning

confidence: 99%

TET1 Directs Chondrogenic Differentiation by Regulating SOX9 Dependent Activation of Col2a1 and Acan In Vitro

et al. 2020

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Skeletal development is a tightly orchestrated process in which cartilage and bone differentiation are intricately intertwined. Recent studies have highlighted the contribution of epigenetic modifications and their writers to skeletal development. Methylated cytosine (5mC) can be oxidized to 5‐hydroxymethylcytosine (5hmC) by the Ten‐eleven‐translocation (TET) enzymes leading to demethylation. We have previously demonstrated that 5hmC is stably accumulated on lineage‐specific genes that are activated during in vitro chondrogenesis in the ATDC5 chondroprogenitors. Knockdown (KD) of Tet1 via short‐hairpin RNAs blocked ATDC5 chondrogenic differentiation. Here, we aimed to provide the mechanistic basis for TET1 function during ATDC5 differentiation. Transcriptomic analysis of Tet1 KD cells demonstrated that 54% of downregulated genes were SOX9 targets, suggesting a role for TET1 in mediating activation of a subset of the SOX9 target genes. Using genome‐wide mapping of 5hmC during ATDC5 differentiation, we found that 5hmC is preferentially accumulated at chondrocyte‐specific class II binding sites for SOX9, as compared with the tissue‐agnostic class I sites. Specifically, we find that SOX9 is unable to bind to Col2a1 and Acan after Tet1 KD, despite no changes in SOX9 levels. Finally, we compared this KD scenario with the genetic loss of TET1 in the growth plate using Tet1−/− embryos, which are approximately 10% smaller than their WT counterparts. In E17.5 Tet1−/− embryos, loss of SOX9 target gene expression is more modest than upon Tet1 KD in vitro. Overall, our data suggest a role for TET1‐mediated 5hmC deposition in partly shaping an epigenome conducive for SOX9 function. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

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“…Furthermore, studies have shown that 5hmC is enriched in active gene bodies (Gross et al, 2017;Mellen et al, 2012), suggesting a potential role of TET1 in transcriptional activation. We generated a TET1 knockdown cell line (A549-shTET1) for further analysis, reduced representation 5-hydroxymethylcytosine profiling (RRHP) identified 2,584 genes with a change of 5hmC in the gene body in A549-shTET1 compared with the wild-type (Petterson, Chung, Tan, Sun, & Jia, 2014;Sun, Chung, Tan, & Kim, 2016). RRHP also revealed additional differentially hydroxymethylated regions in introns rather than upstream sequence (Figure 1c).…”

Section: Mismatch Between the Level Of Promoter Methylation And Gene Expressionmentioning

confidence: 99%

Putative promoters within gene bodies control exon expression via TET1‐mediated H3K36 methylation

Tahir

Wang

et al. 2020

Journal Cellular Physiology

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Hypermethylation of gene promoter has been indicated for the contribution of gene silencing, and DNA demethylating drugs, such as 5-aza-2'-deoxycytidine (DAC), has been used clinically for cancer treatment. However, the reason why a proportion of genes with hypermethylated promoter exhibit high expression levels remains unclear and this drug is not much successful as expected in use. Furthermore, CpG islands (CGIs) are found to be located in not only promotors, but also in gene bodies. By RNA-seq and reduced representation bisulfite sequencing, we found the mismatch

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Practical Guidelines and Consideration of Using RRHP for 5hmC Detection

Cited by 8 publications

References 18 publications

TET1-mediated DNA hydroxymethylation regulates adult remyelination in mice

TET1-mediated DNA hydroxymethylation regulates adult remyelination in mice

TET1 Directs Chondrogenic Differentiation by Regulating SOX9 Dependent Activation of Col2a1 and Acan In Vitro

Putative promoters within gene bodies control exon expression via TET1‐mediated H3K36 methylation

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