ARTICLE https://doi.org/10.1038/s41467-021-25353-5 OPEN Ten-eleven translocation 1 mediated-DNA hydroxymethylation is required for myelination and remyelination in the mouse brain Ming Zhang 1,8, Jian Wang1,8, Kaixiang Zhang1,8, Guozhen Lu1, Yuming Liu1, Keke Ren1, Wenting Wang 1, Dazhuan Xin2, Lingli Xu3, Honghui Mao1, Junlin Xing1, Xingchun Gao4, Weilin Jin5, Kalen Berry 2, ✉ ✉ ✉ Katsuhiko Mikoshiba 6,7, Shengxi Wu 1 , Q. Richard Lu 2 & Xianghui Zhao 1 1234567890():,; Ten-eleven translocation (TET) proteins, the dioxygenase for DNA hydroxymethylation, are important players in nervous system development and diseases. However, their role in myelination and remyelination after injury remains elusive. Here, we identify a genome- wide and locus-specific DNA hydroxymethylation landscape shift during differentiation of oligodendrocyte-progenitor cells (OPC). Ablation of Tet1 results in stage-dependent defects in oligodendrocyte (OL) development and myelination in the mouse brain. The mice lacking Tet1 in the oligodendrocyte lineage develop behavioral deficiency. We also show that TET1 is required for remyelination in adulthood. Transcriptomic, genomic occupancy, and 5-hydroxymethylcytosine (5hmC) profiling reveal a critical TET1-regulated epigenetic program for oligodendrocyte differentiation that includes genes associated with myelina- tion, cell division, and calcium transport. Tet1-deficient OPCs exhibit reduced calcium activity, increasing calcium activity rescues the differentiation defects in vitro. Deletion of a TET1-5hmC target gene, Itpr2, impairs the onset of OPC differentiation. Together, our results suggest that stage-specific TET1-mediated epigenetic programming and intracellular signaling are important for proper myelination and remyelination in mice. 1 Department of Neurobiology, School of Basic Medicine, Fourth Military Medical University, Xi’an, Shaanxi, China. 2 Division of Experimental Hematology and Cancer Biology, Brain Tumor Center, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA. 3 Center for molecular medicine, Pediatric Research Institute, Children’s Hospital of Fudan University, Shanghai, China. 4 Shaanxi Key Laboratory of Brain Disorders, Xi’an Medical University, Xi’an, Shaanxi, China. 5 Institute of Cancer Neuroscience, Medical Frontier Innovation Research Center, The First Hospital of Lanzhou University, Lanzhou, China. 6 Faculty of Science, Toho University, Funabashi, Japan. 7Present address: Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, ✉ Shanghai, China. 8These authors contributed equally: Ming Zhang, Jian Wang, Kaixiang Zhang. email: [email protected]; [email protected]; [email protected] NATURE COMMUNICATIONS | (2021)12:5091 | https://doi.org/10.1038/s41467-021-25353-5 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-25353-5 yelination by oligodendrocytes (OLs) enables saltatory gene set enrichment analysis (GSEA) for 5hmC peaks in the gene Mconduction of action potentials and provides long-term body regions indicated that genes associated with the bipotent trophic support for axons, maintaining integrity progenitor, OL progenitor, and postmitotic OL were enriched in throughout the central nervous system (CNS)1. The formation of OPCs (Fig. 1c), while pluripotent stem cell-associated genes were mature myelinating OLs is a complex process that is tightly enriched in NPCs (Fig. 1c). Comparison with a neural cell-type coordinated spatially and temporally by genetic and epigenetic transcriptome dataset22 (Supplementary Fig. 1a–c) showed that events2,3. Epigenetic regulation by DNA methylation, histone the 5hmC signals were higher in OPCs than NPCs, in gene loci of modification, and chromatin remodeling is critical for multiple OPC-associated genes, e.g., Cspg4 (chondroitin sulfate pro- aspects of OL development, function, and regeneration4–6. For teoglycan 4) (Fig. 1d), immature OL-associated genes, e.g., Kndc1 instance, proper maintenance of genomic 5-methylcytosine (kinase non-catalytic C-lobe domain containing 1) (Fig. 1e), and (5mC) is essential for normal development, homeostasis, and mature OL-associated genes, e.g., Mag (myelin-associated glyco- function of mammalian cells7,8. Genetic ablation of Dnmt1, protein) (Fig. 1f). In contrast, the genes with 5hmC peaks enri- which encodes the DNA methyltransferase that maintains ched in NPCs were associated with negative regulation of OPC DNA methylation after replication, results in impaired OL pre- differentiation, such as Ngf and Zfp28 (Fig. 1g). We further ver- cursor cell (OPC) expansion and differentiation during early ified the presence of loci-specific hydroxylmethylation in repre- development9. sentative genes with a qPCR assay based on a combination of The modified nucleotide 5-hydroxymethylcytosine (5hmC) has bisulfite and subsequent cytosine deaminase (APOBEC) treat- been shown to be an intermediate product generated during ment (Supplementary Fig. 1d and Supplementary Table 1). These cytosine demethylation10,11. DNA demethylation, like methyla- data suggested a unique distribution pattern of genomic 5hmCs tion, is a highly regulated process. DNA demethylation is medi- in the gene loci associated with OL lineage specification during ated by the ten-eleven translocation (TET) family of dioxygenases. the transition from NPCs to OPCs. The TET enzymes oxidize 5mC into 5hmC to initiate the DNA demethylation process11,12. Dynamic regulation of cytosine methylation or demethylation has been established as a common Deletion of Tet1 in OL lineage causes myelination deficits at epigenetic modification regulating various processes from devel- early postnatal stages. TET1-3 enzymes are present in OL lineage opment to diseases in a cell-type and context-dependent cells16. As TET2 had no detectable effects in OL lineage manner13–15. TET enzymes are present in OL lineage cells16, development23, we assessed the functions of TET1 and TET3 in and here we interrogated how DNA demethylation contributes to OL development. We crossed Tet1flox/flox mice24 and Tet3flox/flox OL lineage development, myelination, and remyelination after mice18 with the Olig1-Cre line25 to knockout the catalytic injury. domains of these TET enzymes early in OL lineage development In this study, we demonstrate that there is a genome-wide shift (Fig. 2a and Supplementary Fig. 2a). The resulting in the 5hmC landscape during OL specification and identify an Tet1flox/flox;Olig1Cre+/− (Tet1 cKO) and Tet3flox/flox;Olig1Cre+/− age-dependent function of TET1 in OL lineage specification and (Tet3 cKO) mice were born at Mendelian ratios and appeared myelination. The mice with Tet1 deletion in OL lineage develop normal at birth. We did not detect significant differences in either behavioral deficiency. In addition, we show that a TET1-regulated the number of CC1+ mature OLs or myelin protein expression epigenetic program is required for efficient remyelination as between heterozygous Tet1-floxed mice (Tet1flox/+;Olig1Cre+/−), depletion of Tet1 in OPCs impairs myelin recovery after Cre control (Tet1+/+;Olig1Cre+/−), or wild-type mice (Supple- demyelinating injury in adult animals. Moreover, Tet1 depletion mentary Fig. 3a, b). Also, we did not detect any OLIG2 expression resulted in genome-wide alterations in 5hmC and transcriptomic changes within the Olig1-Cre (+/−) heterozygous line (Supple- profiles that are associated with OPC differentiation and myeli- mentary Fig. 3c, d). Therefore, heterozygous littermates were used nation, as well as calcium transport. Ablation of Itpr2, one of the as controls. To assess Cre-mediated Tet1 depletion in OL lineage, TET1-5hmC targets that responsible for calcium release from we quantified TET1 expression in OPCs from Tet1 cKO and endoplasmic reticulum (ER) in the OL lineage significantly control mice at P4. Immunostaining revealed that expression of impairs OL differentiation. These data suggest that TET1 and TET1 in SOX10+ OLs was significantly reduced in Tet1 cKO than DNA hydroxymethylation mediated transcriptional and epige- in control mice (Fig. 2b, c). TET1 levels were also decreased in netic programming regulate OL intracellular signaling and are purified OPCs from Tet1 mutant than from control mice assayed required for proper myelination and animal behaviors. by quantitative real-time PCR (Supplementary Fig. 3e). To examine the cell-type or lineage-specificity of Olig1-Cre in the postnatal brain, we have generated Olig1-Cre; R26-tdTomato Results mice by crossing the Olig1-Cre mice with R26-tdTomato reporter Dynamic DNA hydroxymethylation landscape during OL line (Ai14). We found that most Olig1-Cre-TdTomato cells were lineage specification. To investigate the 5hmC landscape during OLIG2-positive OL lineage cells (Supplementary Fig. 3f, g), while OL lineage specification, we carried out antibody-based 5hmC TdTomato+ cells rarely co-label with the markers for astrocytes immunoprecipitation (IP) combined with Illumina sequencing (ALDH1L1+), neurons (NeuN+), or interneurons (PV+, SST+, (hMeDIP-seq)17,18 and analyzed 5hmC distribution across the and VIP+) (Supplementary Fig. 3f, h, i), indicating that the Olig1- genome. We compared the 5hmC distribution within OPCs iso- Cre line is predominantly restricted to the OL lineage. lated via immunopanning from the cortices at postnatal day 6 To investigate the effects of TET1 on OL development in the (P6) to that in neural progenitor cells (NPCs)19 and identified brain, we examined the expression of OL lineage marker SOX10