TET1-DNA Hydroxymethylation Mediated Oligodendrocyte Homeostasis is Required for CNS Myelination and Remyelination

Abstract Ten-eleven translocation (TET) proteins, encoding dioxygenase for DNA hydroxymethylation, are important players in nervous system development and diseases. However, their role in oligodendrocyte homeostasis, myelination and remyelination remains elusive. Here, we detected a genome-wide and locus-specific DNA hydroxymethylation landscape shift during oligodendrocyte-progenitor (OPC) differentiation. Ablation of Tet1, but not Tet3, results in stage-dependent defects in oligodendrocyte development and myelination in the brain. The mice lacking Tet1 in the oligodendrocyte lineage develop schizophrenia-like behaviors. We further show that TET1 is also required for proper remyelination after demyelination injury in the adult mice. Transcriptomic and DNA hydroxymethylation profiling revealed a critical TET1-regulated epigenetic program for oligodendrocyte differentiation and identified a set of TET1-5hmC target genes associated with myelination, cell division, and calcium transport. Tet1-deficient OPCs exhibited reduced calcium activity in response to stimulus in culture. Moreover, deletion of a TET1-5hmC target gene, Itpr2, an oligodendrocyte-enriched intracellular calcium-release channel, significantly impaired the onset of oligodendrocyte differentiation. Together, our results suggest that stage-specific TET1-mediated epigenetic programming and oligodendrocyte homeostasis is required for proper myelination and repair.

The copyright holder for this preprint (which was this version posted July 12, 2020. ; https://doi.org/10.1101/821496 doi: bioRxiv preprint Introduction 1 Myelination by oligodendrocytes (OLs) enables saltatory conduction of action potentials and 2 provides long-term trophic support for axons, maintaining integrity throughout the central nervous 3 system (CNS) 1 . The formation of mature myelinating OLs is a complex process that is tightly 4 coordinated spatially and temporally by genetic and epigenetic events 2, 3 . Epigenetic regulation 5 by DNA methylation, histone modification, and chromatin remodeling is critical for multiple 6 aspects of OL development, function, and regeneration 4-6 . For instance, proper maintenance of 7 genomic 5-methyl cytosine (5mC) is essential for normal development, homeostasis, and function 8 of mammalian cells 7,8 . Genetic ablation of Dnmt1, which encodes the DNA methyltransferase 9 that maintains DNA methylation after replication, results in impaired OL precursor cell (OPC) 10 expansion and differentiation during early development 9 .

11
The modified nucleotide 5-hydroxymethylcytosine (5hmC) has been shown to be an 12 intermediate product generated during cytosine demethylation 10,11 . DNA demethylation, like 13 methylation, is a highly regulated process. DNA demethylation is mediated by the Ten-Eleven 14 Translocation (TET) family of dioxygenases. The TET enzymes oxidize 5mC into 5hmC to initiate 15 the DNA demethylation process 11,12 . Dynamic regulation of cytosine methylation or 16 demethylation has been established as common epigenetic modification regulating various 17 processes from development to diseases in a cell-type and context-dependent manner [13][14][15] . TET 18 enzymes are present in OL lineage cells 16 , and here we interrogated how DNA demethylation 19 contributes to OL lineage development, myelination, and remyelination after injury.

20
In this study, we demonstrate that there is a genome-wide shift in 5hmC landscape during 21 OL specification and identify an age-dependent function of TET1 in OL lineage development and 22 homeostasis. The mice with Tet1 deletion in OL lineage develop schizophrenia-like behaviors. In 23 addition, we show that TET1-regulated epigenetic program is required for efficient remyelination 24 as depletion of Tet1 in OPCs impairs myelin recovery after demyelinating injury in adult animals.

25
Moreover, Tet1 depletion resulted in genome-wide alterations in 5hmC and transcriptomic profiles 26 that are associated with OPC differentiation and myelination, as well as calcium transport.

27
Ablation of Itpr2, one of the TET1-5hmC targets that responsible for calcium release from 28 endoplasmic reticulum in the OL lineage significantly impairs oligodendrocyte differentiation.

29
These data suggest that TET1 and DNA hydroxymethylation mediated transcriptional and 30 epigenetic programming regulate oligodendrocyte homeostasis and are required for proper 31 myelination and animal behaviors.

34
Dynamic DNA hydroxymethylation landscape during OL lineage differentiation

35
To investigate the 5hmC landscape during the OL lineage transition, we carried out antibody-36 not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was this version posted July 12, 2020. ; https://doi.org/10.1101/821496 doi: bioRxiv preprint based 5hmC immunoprecipitation combined with Illumina sequencing (hMeDIP-seq) 17, 18 and 1 analyzed 5hmC distribution across the genome. We compared the 5hmC distribution within OPCs 2 to that in neural progenitor cells (NPCs) 19 and identified 1237 genes that were specifically 3 hydroxymethylated in the promoter or transcription start site (TSS) regions of OPCs but not NPCs 4 ( Fig. 1a). Gene ontology analysis revealed that these genes involved in OPC differentiation are 5 highly associated with terms such as cell projection organization, fatty acid transport, and 6 regulation of cytosolic calcium ion concentration and with signaling pathways that are essential 7 for OL development such as the G-protein coupled receptor pathway 20, 21 (Fig. 1b). Similarly, 8 gene set enrichment analysis (GSEA) for 5hmC peaks in the gene body regions indicated that 9 genes associated with bipotent progenitor, oligodendrocyte progenitor and postmitotic 10 oligodendrocyte were enriched in OPCs (Fig. 1c), while pluripotent stem cell associated genes 11 were enriched in NPCs (Fig. 1c). Comparison with a neural cell-type transcriptome dataset 22 12 (Supplementary Fig. 1) showed that the 5hmC signals were higher in OPCs than NPCs, in gene 13 loci of OPC-associated genes, e.g. Cspg4 (chondroitin sulfate proteoglycan 4) (Fig. 1d), 14 immature OL-associated genes, e.g. Tmem141 (transmembrane protein 141) (Fig. 1e) and 15 mature OL-associated genes, e.g. Mag (myelin-associated glycoprotein) (Fig. 1f). In contrast, the 16 genes with 5hmC peaks enriched in NPCs were associated with negative regulation of OL 17 differentiation, such as Id2 and Zfp28 (Fig. 1g). These data suggested a unique distribution were born at Mendelian ratios and appeared normal at birth. We did not detect significant 28 differences in either the number of CC1 + mature OLs or myelin protein expression between 29 heterozygous Tet1 floxed mice (Tet1 flox/+ ;Olig1Cre +/-), Cre control (Tet1 +/+ ;Olig1Cre +/-), or wild-type 30 mice ( Supplementary Fig. 3a-b). Therefore, heterozygous littermates were used as controls. To 31 assess Cre-mediated Tet1 depletion, we quantified TET1 expression in OPCs from Tet1 cKO and 32 control mice at P4. Immunostaining revealed that expression of TET1 in the corpus callosum was 33 significantly lower in Tet1 cKO than control mice (Fig. 2b-c). TET1 levels were also decreased in 34 purified OPCs from Tet1 cKO mice than from control mice assayed by quantitative real-time PCR

35
( Supplementary Fig. 3c). 36 not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was this version posted July 12, 2020. ; https://doi. org/10.1101/821496 doi: bioRxiv preprint To investigate the effects of TET1 on OL development in the brain, we examined the 1 expression of Sox10 (an OL lineage marker) and mature OL markers CC1 and MBP. The number 2 of CC1 + mature OLs was significantly reduced in juvenile Tet1 cKO mice compared to controls 3 ( Fig. 2d-e), but this difference was not observed in P60 adults (Fig. 2e, Supplementary Fig. 4a).

4
Expression of MBP was also substantially decreased in both cortex (gray matter) and corpus 5 callosum (white matter) in Tet1 cKO mice compared to controls at P16 (Fig. 2f), but the levels 6 were similar in adult animals ( Supplementary Fig. 4a). These observations indicate that Tet1 loss 7 causes a delay in OL maturation. Similar experiments in the Tet3 cKO animals did not show any 8 significant differences between mutants and controls ( Supplementary Fig. 2). Therefore, we 9 focused on examining the processes underlying the observed myelination defects in Tet1 cKO 10 mice.

11
In addition, electron microscopy (EM) revealed that the number of myelinated axons was 12 significantly reduced in Tet1 mutants compared to controls at both P14 optic nerves and P27 13 corpus callosum j). Moreover, those myelinated axons in Tet1 cKO mice were 14 characterized by higher G ratios and thinner myelin sheaths than those of control mice (Fig. 2i,   15 k). However, the myelin ultrastructure defects were not observed in P60 adult Tet1 cKO animals 16 . Together, these results suggest a stage-dependent function of TET1 17 in CNS myelination.

18
To evaluate the neurological significance of hypomyelination in Tet1 cKO mice, we analyzed 19 stimulus-evoked compound action potential (CAP) in optic nerves as previously described 26,27 .

20
Suction electrodes back filled with artificial cerebrospinal fluid were used for stimulation and 21 recording. In Tet1 mutants, both the peak amplitudes and the CAP areas, which are indexes of 22 excited myelinated axon numbers and nerve function 26, 27 , were significantly lower than controls 23 under all stimulating currents tested . This observation indicates that hypomyelination 24 impairs action potential transduction in Tet1 cKO mice.

27
Multiple studies have associate TET-5hmC with psychiatric and cognitive disorders 28-30 , 28 and multivariable logistic regression showed that ErbB4, BDNF and TET1 were independent 29 predictors for schizophrenia 31 . To gain insight into the physiological function of TET1 in animal 30 behaviors, Tet1 cKO mice were subjected to behavior tests relevant to schizophrenia. Tet1 cKO 31 mice did not exhibit differences in weight and whisker number in comparison with control 32 littermates.

33
First, we investigated the performance of juvenile Tet1 mutant in Prepulse Inhibition (PPI) of 34 startle, which is a common test of sensorimotor gating ability for schizophrenia patients 32 .

35
Reduced PPI ability due to an exaggerated acoustic startle reflex (ASR) is thought to contribute 36 not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was this version posted July 12, 2020. ; https://doi.org/10.1101/821496 doi: bioRxiv preprint 6 to schizophrenic conditions. We found that the input/output function and the startle response were 1 comparable between control and Tet1 mutant mice ( Supplementary Fig. 5a), indicating the 2 normal hearing and motor abilities (i/o function) in Tet1 cKO mice. However, when using a 3 combination of auditory-evoked startle (120dB) and three levels of prepulse (70, 76 and 82 dB) 4 to compare ARS, we observed that PPI was significantly attenuated in Tet1 cKO mice in 5 comparison with control animals ( Supplementary Fig. 5b), suggesting the impaired sensorimotor 6 gating ability in Tet1 mutant.

7
Since working memory deficits are characteristic features in schizophrenia, Tet1 cKO mice and 8 control littermates were evaluated for their performance in Morris water maze to assess their 9 working memory. Five-day acquisition trials exhibited similar swim paths, swim velocity and 10 escape latency to the platform between control and Tet1 cKO groups ( Supplementary Fig. 5c-e), 11 which indicates that Tet1 mutants had normal swimming and learning abilities. However, in the 12 sixth-day probe trial, the escape latency was significantly higher in Tet1 cKO mice than in control 13 mice ( Supplementary Fig. 5f), and the number of crossing the position was greatly reduced in 14 mutant mice ( Supplementary Fig. 5g). Additionally, in contrast to controls, the Tet1cKO mice 15 showed no preference for the target quadrant over other three quadrants, (Supplementary Fig.   16 5h-i). These observations suggest that Tet1 cKO mice exhibit impaired PPI and working memory, 17 resulting in schizophrenia-like behaviors.

19
Ablation of Tet1 results in defects in OPC cell-cycle progression 20 Concomitant with the myelin deficiency, we observed a marked reduction of Olig2 + cells from 21 embryonic stage E15.5 and at P1 in Tet1 cKO cortex relative to controls ( Fig. 3a-b). Moreover, 22 the number of PDGFRα + cells in the mutant cortex was significantly reduced at E15.5 and P1 23 (Fig. 3a,c), suggesting a downsized OPC pool.

24
To determine the underlying defects that led to the observed reduction in the OPC and OL 25 population in juvenile Tet1 mutants, we first tested the possibility that OPCs are more likely to 26 undergo apoptosis in the mutant with the TUNEL assay. Brain sections from E14.5, E17.5 and 27 P1 mice revealed no distinguishable changes in the number of apoptotic cells among Olig2 + OL 28 lineage cells between Tet1 cKO animals and control littermates (Supplementary Fig. 6a-b).

29
Next, we performed BrdU incorporation assay to examine the proliferation of OPCs. At P1, mice 30 were dosed with BrdU and sacrificed 2 hours later. Compared to controls, intriguingly, the 31 percentage of BrdU + cells in Olig2 + OL lineage cells showed a significant increase in Tet1 cKO 32 cortex ( Fig. 3d-e). The reduction of OPC numbers in Tet1 cKO mice thus promote us to 33 investigate if there is a cell-cycle defect in Tet1-deficient OPCs. We performed flow cytometry for 34 purified OPCs in which DNA was stained with propidium iodide. Significant increases in the 35 percentages of cells in S phase (23.4 ± 0.85%) and G2/M phase (13.39 ± 1.01%) were observed 36 not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was this version posted July 12, 2020. ; https://doi.org/10.1101/821496 doi: bioRxiv preprint 7 in OPCs from Tet1 mutants compared to the controls (9.02 ± 0.99% for S phase and 8.29 ± 0.62% 1 for G2/M phase) ( Fig. 3f-g). Moreover, there was a concomitant reduction in the number of cells 2 in G1 phase in Tet1 mutants compared to the controls ( Fig. 3f-g). These results suggest that the 3 proliferation of OPCs from Tet1 cKO brains is blocked at the transition from G2/M to G1 phase, 4 which likely leads to the observed reduction in OPC numbers in Tet1 cKO mice.

5
To determine if defects in OL differentiation caused by TET1 deletion are cell-autonomous, we 6 isolated primary OPCs from the neonatal cortices of control and Tet1 cKO pups, and then treated 7 them with T3 to promote differentiation. Immunostaining results showed that the number of CNP + 8 cells and MBP + cells in Tet1 deficient OPCs were significantly decreased when compared with 9 control OPCs at Day 3 and Day 5 ( Fig. 3h-i), suggesting that Tet1-depleted OPCs are intrinsically 10 reduced in their differentiation capacity.

11
In addition, we noted that Tet1 deletion did not substantially alter the number of other neural 12 cell types in the brain. Western blot and immunostaining with antibodies against DCX, a marker 13 for newly generated neurons; NeuN, a mature neuron marker; and ALDHL1, an astrocyte marker,  in Tet1 OPC-iKO mice ( Fig. 4d-g). Moreover, a reduction in CC1 + OLs and MBP intensity was 28 observed in corpus callosum from P14 Tet1 OPC-iKO mice ( Fig. 4h-i). These results indicate that 29 TET1 is required for the transition from OPCs to OLs.

32
Given the critical role of TET1 in early oligodendrocyte development, we reasoned that TET1 33 should also be required in the adult brain for remyelination after injury that results in 34 demyelination. We induced demyelinated lesions in the corpus callosum via stereotaxic guided 35 lysolecithin (LPC) injections (Fig. 5a). LPC induces rapid myelin breakdown followed by myelin 36 not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was this version posted July 12, 2020. ; https://doi.org/10.1101/821496 doi: bioRxiv preprint regeneration through an OPC recruitment phase at 7 days post-lesion (7 dpl) induction and a 1 remyelination phase at 14 dpl. TET1 + cell numbers were increased substantially in the lesion site 2 at 7 dpl relative to controls injected with vehicle ( Fig. 5b-c). In particular, the expression levels of 3 TET1 in Olig2 + cells were higher after LPC treatment (Fig. 5b-c).

4
To evaluate the role of TET1 in remyelination, we used NG2-CreER T :Tet1 flox/flox (Tet1 OPC-5 iKO) animals mentioned above. To induce recombination in adult mice, 8-week-old Tet1 OPC-6 iKO mice were injected daily with tamoxifen for 8 days, starting 3 days prior to LPC injection in 7 the corpus callosum (Fig. 5d). Brains were harvested at 7, 14 and 21 dpl from Tet1 OPC-iKO 8 mice and heterozygous controls. To determine the extent of remyelination, we examined the 9 expression of OPC markers and myelin-related genes. Loss of Tet1 did not appear to impair the 10 recruitment of PDGFRα + OPCs, and the numbers of OPCs in the lesions were comparable 11 between control and Tet1 OPC-iKO mice during the recruitment phase at dpl 7 (Fig. 5e, f). In 12 contrast, there were significantly fewer GST-pi + differentiating OLs in the lesion site during the 13 remyelination phase at dpl 14 and dpl 21 in Tet1 OPC-iKO mice relative to controls (Fig. 5e, g).
14 Consistent with a reduction in the number of differentiating OLs, MBP was also reduced in Tet1-

17
Furthermore, the thicknesses of newly generated myelin sheaths around axons were significantly 18 reduced in Tet1-iKO mutants (Fig. 5k). These observations indicate that TET1 is required for the    Tet1 deficient OPCs (Fig. 6g) as were genes involved in cell-cycle regulation (Fig. 6h). The 36 not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was this version posted July 12, 2020. ; https://doi.org/10.1101/821496 doi: bioRxiv preprint transcriptome landscape alterations in Tet1 cKO OPCs were in line with the observations that 1 Tet1 depletion led to cell-cycle progression defects and hypomyelination phenotypes.

2
Since TET1 mediates DNA hydroxymethylation/demethylation, we next tested the level of 3 5hmC in oligodendrocytes from Tet1 mutants. In P27 brain sections, immunostaining of 5hmC 4 simultaneously with the OL marker CC1 revealed a striking reduction in 5hmC intensity in 5 oligodendrocytes ( Fig. 7a-b), which strongly suggested that 5hmC is involved in TET1-mediated 6 regulation of OL differentiation. To further compare the genome-wide 5hmC distributions, we 7 performed hMeDIP-seq in OPC cultures from controls and Tet1 mutants. Tet1 cKO OPCs showed 8 a dramatic reduction in 5hmC peak signals compared to controls ( Supplementary Fig. 7a). In 9 both groups, most 5hmC peaks resided in intergenic regions; less than 40% of peaks were within 10 gene bodies of annotated RefSeq genes ( Supplementary Fig. 7b). This is different from the 11 distribution pattern in mouse embryonic stem cells 18 and neurons 34 . After plotting the distribution 12 of 5hmC peaks over RefSeq genes, we found that 5hmC was reduced near the transcription start

20
We next examined the effects of hydroxymethylation on gene expression. By integrating 21 RNA-seq data with hMeDIP-seq data, we observed that among the genes that had lower   Fig. 7f). Together, these results are highly in consistent with the hypomyelination 36 not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was this version posted July 12, 2020. ; https://doi.org/10.1101/821496 doi: bioRxiv preprint phenotypes in Tet1 cKO mice and indicate the significance of TET1-5hmC mechanisms in OL 1 homeostasis.

3
Impaired calcium transport leads to differentiation defects in OPC cultures from Tet1 cKO 4 mice 5 When searching for TET1-5hmC regulated factors that may involve in OL development and 6 homeostasis, we noticed that there was a cluster of calcium transporter genes among the 7 downregulated and hypohydroxymethylated genes in Tet1 cKO group ( Fig.6a- The decrease in expression of these genes in Tet1 cKO mice was confirmed by qRT-PCR 9 assays (Fig. 7j). CACNA1a, CACNA1c, CACNA2d1, CACNB4, and CACNG5 are plasma 10 membrane voltage-operated Ca 2+ channels (VOCCs) that are expressed in OPCs and contribute 11 to calcium dynamics in these cells 22,37 . In particular, calcium influx mediated by CACNA1c, also 12 known as Cav1.2, is required for oligodendrocyte differentiation 38, 39 . Another target gene Itpr2, 13 which encodes a type 2 IP3 receptor, localized to the endoplasmic reticulum (ER) and expressed 14 exclusively in postmitotic OLs 40, 41 , also had decreased mRNA expression in OPCs from Tet1 15 cKO mice compared to controls (Fig. 7j). These data indicate that TET1-5hmC targets calcium 16 transport genes and may regulate calcium dynamics in oligodendrocytes.

17
To

31
To investigate the consequences of impaired calcium rise in Tet1-deficient OPCs, we examined 32 cell differentiation after activating calcium signaling. Consistent with the results of high K + 33 application 39 , three consecutive pulses (5 min/each) daily with 10 μM calcium channel agonist 34 Bay K 8644 significantly promoted differentiation of OPCs and restored the differentiation defects 35 in Tet1 deficient OPCs as determined by qRT-PCR analysis of myelin genes and MBP + OL 36 not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

3
Ablation Itpr2, modulator for calcium release from endoplasmic reticulum, inhibits OL 4 differentiation 5 To further distinguish calcium signaling as TET1-5hmC target during OL differentiation, or the 6 results of impaired differentiation in Tet1 cKO mice, we then tested the function of Itpr2, one of 7 TET1-5hmC target calcium transport genes, in myelination. As modulator for calcium release   The copyright holder for this preprint (which was this version posted July 12, 2020. ; https://doi.org/10.1101/821496 doi: bioRxiv preprint differentiation from neural progenitor cells, suggesting a role of TET-mediated DNA demethylation 1 in regulation of OL lineage progression.

2
We found that TET1, but not TET3, is critical for OPC proliferation, differentiation, and 3 myelination during early animal development, suggesting a unique function of TET1 in 4 oligodendrogenesis and subsequent myelinogenesis. Despite early developmental defects, we 5 noticed that the developmental myelin deficiencies recovered in adult Tet1 cKO mice, which might 6 be due to the expansion of OL numbers that escaped from Cre-mediated Tet1 depletion. The 7 remyelination capacity after injury was compromised in adult Tet1 OPC-iKO brains, suggesting 8 that TET1 is also critical for the myelin regeneration process.

9
Although OL differentiation defects were not due to increased apoptosis in the Tet1 cKO 10 mutant brain, we found that OPC cell cycle progression was impaired in the developing brain of The copyright holder for this preprint (which was this version posted July 12, 2020. ; https://doi.org/10.1101/821496 doi: bioRxiv preprint 1 promoters or enhancers that are decorated with both activating (H3K4me3) and repressing 2 (H3K27me3) histone marks 59,60 . How 5hmCs cooperate with other epigenetic regulators for OPC 3 differentiation remains to be determined.

4
Intriguingly, ablation of Tet1 led to upregulation of a set of genes in OPCs, indicating that TET1-5 mediated 5hmC deposition may also function as a transcriptional repressor. Consistent with our 6 data, inhibition of Tet1 expression increased expression of a set of genes in ESCs 61, 62 . TET1-7 mediated repression might involve recruitment of the MBD3/NuRD repressor complex, which was 8 shown to co-localize with TET1 in ESCs 63 . TET1 may also coordinate with Sin3A co-repressor 9 complex, which has a similar binding profile to TET1 and is required for a subset of TET1-

24
We find that Itpr2, an intracellular calcium channel that exclusively expressed in postmitotic 25 OLs 40, 41 , is one of TET1-5hmC targets. Expression of Itpr2 is upregulated during a motor learning 26 task 41 , indicating the participation of Itpr2 + OLs in myelin plasticity. We find that deletion of Itpr2 27 in the OL lineage greatly reduces OPC differentiation, suggesting that Itpr2 is critical for an 28 initiation for myelination. Thus, TET1 mediated 5hmC modification, or DNA hydroxymethylation,

29
can modulate the process of oligogenesis and myelinogenesis through at least two critical 30 processes, by fine-tuning cell cycle progression for OPC proliferation and by regulating 31 oligodendrocyte hemostasis e.g., Itpr2-mediated calcium transport, for OL myelination. 4 not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was this version posted July 12, 2020.                                                 54 not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was this version posted July 12, 2020.                         54 not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

10 11
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was this version posted July 12, 2020.     53 not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was this version posted July 12, 2020.

24
Lysolecithin-induced demyelination was carried out in the corpus callosum of 8-week-old mice.

25
Anesthesia was induced and maintained by peritoneal injection of a mixture of ketamine (90 26 mg/kg) and xylazine (10 mg/kg). The skull was exposed, and a hole was cut into the cranium.

28
(L-a-lysophosphatidylcholine, Sigma L4129) into the corpus callosum at coordinates: 0.8 mm 29 lateral, 0.8 mm rostral to bregma, 1.2mm deep to brain surface) using a glass-capillary connected 30 to a 10 μl Hamilton syringe. Animals were left to recover in a warm chamber before being returned 31 into their housing cages. LPC-induced injuries were conducted in a genotype-blinded manner.

34
Analyzing the compound action potential was performed according to previous protocols 27, 74 .

35
Tet1 cKO and control littermates were killed by cervical dislocation and then decapitated. Optic and performed blind to genotype. Image drawing and statistical analysis were performed in 52 not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

2
Behavior test 3 Startle response/prepulse inhibition tests 4 A startle reflex measurement system was used to measure startle response and prepulse 5 inhibition. Throughout the session, the startle system delivered a constant background white 6 noise of 68 dB. The startle response was recorded for 300 ms (measuring the response every 1 7 ms) with the onset of stimulus and a startle response was defined as the peak response during 8 the 300 ms period.

9
Acoustic startle began by placing a mouse in the undisturbed chamber for 5 min. The test consists 10 ten 20-ms bursts of white noise varied in level from 65-125 dB sound stimuli in steps of 5dB, plus 11 ten no-stimulus trials. The order of these stimuli was randomized, and the duration of inter-trial 12 intervals was 15 s. The prepulse inhibition test session began with a 5 min acclimation period 13 followed by three consecutive blocks of test trials. Block 1 and 3 consisted of six startle stimulus-14 alone trials. Block 2 contained 10 startle stimulus-alone trials, 10 prepulse + startle trials per 15 prepulse intensity, and 10 no-stimulus trials. Three combinations of prepulse and startle stimuli 16 (70-120, 76-120 and 82-120 dB) were employed. Trials were presented in a pseudo-random 17 order, ensuring that each trial was presented 10 times and that no two consecutive trials were 18 identical. Inter-trial intervals ranged from 30 to 45 s. Basal startle amplitude was determined as 19 the mean amplitude of the 10 startle stimulus-alone trials. PPI was calculated according to the 20 formula: 100 × [1−(PPx/P120)] %, in which PPx means the 10 PPI trials (PP70, PP76 or PP82 21 and P120 was the basal startle amplitude).

24
The Morris water maze was conducted as described 75 with minor modifications. A white plastic 25 tank 120 cm in diameter was kept in a fixed position and filled with 22°C water, which was made 26 opaque with milk. A 10 cm platform was submerged 1cm below the surface of opaque water and 27 located in the center of one of the four virtually divided quadrants. All animal activities were 28 automatically recorded and measured by a video-based Morris water maze tracking system. The 29 swim training consisted of 5 days of trials, during each day mice were released from four random 30 locations around the edge of the tank with an inter-trial interval of about 30 min and they were 31 allowed to freely swim for a maximum of 60 sec or guided to the platform. Afterwards, mice were 32 allowed to stay on the platform for 15 sec. A probe trial was performed 24 h after the last day of 33 training. During the probe trial, mice were allowed to swim in the pool without the escape platform 34 for 60 s. The performance was expressed as the percentage of time spent in each quadrant of 35 the MWM and swim distance in the target quadrant, which were automatically recorded.

36
Moreover, the latency to reach the platform position (using 10 cm diameter) and the number of 37 crossings through the position were manually recorded.

51
Sequencing library preparation was performed according to a previous study 76 with minor 52 modifications. Genomic DNA was sonicated to ~200-800bp with a Bioruptor sonicator 53 (Diagenode). 800 ng of sonicated DNA was end-repaired, A-tailed, and ligated to single-end 54 not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was this version posted July 12, 2020. ; https://doi.org/10.1101/821496 doi: bioRxiv preprint adapters following the standard Illumina genomic DNA protocol (FC-102-1002, Illumina). After 1 agarose size-selection to remove unligated adapters, the adaptor-ligated DNA was used for 2 immunoprecipitation (IP) with a mouse monoclonal anti-5-hydroxymethylcytosine antibody 3 (Diagenode, C15200200). For this, DNA was heat-denatured at 94°C for 10 min, rapidly cooled 4 on ice, and immunoprecipitated with 1 μL primary antibody overnight at 4°C with rocking agitation 5 in 400 μL IP buffer (0.5% BSA in PBS). To recover the immunoprecipitated DNA fragments, 20 6 μL of magnetic beads were added and incubated for an additional 2 hours at 4°C with agitation.

7
After IP, a total of five washes were performed with ice-cold IP buffer. Washed beads were 8 resuspended in TE buffer with 0.25% SDS and 0.25 mg/mL proteinase K for 2 hours at 65°C and 9 then allowed to cool down to room temperature. DNA was then purified using Qiagen MinElute 10 columns and eluted in 16 μL EB (Qiagen). 14 cycles of PCR were performed on 5 μL of the 11 immunoprecipitated DNA using the single-end Illumina PCR primers. The resulting products were 12 purified with Qiagen MinElute columns, after which a final size selection (300-1,000 bp) was 13 performed by electrophoresis in 2% agarose. Libraries were quality controlled by Agilent 2100 14 Bioanalyzer.

3
Two or four days later, cultures were harvested for qRT-PCR assay or immunocytochemistry as 4 indicated.

Statistical analysis
7 Numerical values were analyzed using Mean ± SEM and are presented as bar graphs. Group 8 meet normal distribution and homogeneity of variance were compared using Two-tailed unpaired 9 t-test. Group with normal distribution but do not meet homogeneity of variance were compared 10 using Two-tailed unpaired separate variance estimation t-test. Group do not meet normal 11 distribution were compared using Mann-Whitney U test. Two factors do not meet normal

20 21
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

10
(e) Quantification of BrdU + cells within Olig2 + OPC population in control and Tet1 cKO brains.

53
(f) Quantification of PDGFRα + OPCs in LPC lesion sites at dpl 7. Data are Means ± SEM (n=3 54 not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

7
(j) The number of myelinated axons in lesion regions from control and Tet1-iKO mutants at 28 8 dpl. Data are Means ± SEM (n=4 slides from 3 animals per group). ***, p<0.001 compared to 9 control, Student's t test.

10
(k) G ratios versus axonal perimeters in lesion regions from control and Tet1-iKO mutants at 28 11 dpl. p<0.001 compared to control, Student's t test (> 130 myelinating axon counts/animal from 3 12 animals/genotype).        54 not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

14
The copyright holder for this preprint (which was this version posted July 12, 2020.

45
(q) G ratios versus axonal perimeters for control and Itpr2 cKO mice reveal significant difference.

49 50
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.