At birth, the mammalian brain contains tens of billions of neurons. Although the number does not increase much as the animal grows, there are many dramatic changes to their size and structure. These changes allow the neurons to communicate with one another, develop into networks, and learn the tasks of the adult brain. One way that these changes occur is by the accumulation of chemical marks on each neuron’s DNA that help dictate which genes switch on, and which turn off.

One of the most common ways that DNA can be marked is through the addition of a chemical group called a methyl group to one of the four DNA bases, cytosine. This process is called methylation. When methylation occurs, cytosine becomes 5-methylcytosine, or 5mC for short.

In 2009, researchers found another modification present in the DNA in the brain: 5-hydroxymethylcytosine, or 5hmC. This modification appears when a group of proteins called the Tet hydroxylases turn 5mC into 5hmC. Converting 5mC to 5hmC normally helps cells remove marks on their DNA before they divide and expand. This is important because the newly generated cells need to be able to accumulate their own methylation marks to perform their roles properly. However, neurons in the brain accumulate 5hmC after birth, when the cells are no longer dividing, indicating that 5hmC may be required for the neurons to mature.

Stoyanova et al. set out to determine whether mouse neurons need 5hmC to get their adult characteristics by tracking the chemical changes that occur in DNA from birth to adulthood. Some of the mice they tested produced 5hmC normally, while others lacked the genes necessary to make the Tet proteins in a specific class of neurons, preventing them from converting 5mC to 5hmC as they differentiate. The results reveal that neurons do not mature properly if 5hmC is not produced continuously following the first week of life. This is because neurons need to have the right genes switched on and off to differentiate correctly, and this only happens when 5hmC accumulates in some genes, while 5hmC and 5mC are removed from others. The data highlight the role of the Tet proteins, which convert 5mC into 5hmC, in preparing the marks for removal and demonstrate that active removal of these marks is essential for neuronal differentiation.

Given the role of 5hmC in the development of neurons, it is possible that problems in this system could contribute to brain disorders. Further studies aimed at understanding how cells control 5hmC levels could lead to new ways to improve brain health. Research has also shown that if dividing cells lose the ability to make 5hmC, they can become cancerous. Future work could explain more about how and why this happens.

Development of the mammalian brain requires generation of hundreds of millions of neuronal progenitors that differentiate into distinct cell types with refined functional properties. Although morphological and physiological maturation of most neurons occurs between mid-gestation and a few months or years after birth, the vast majority of CNS neurons must maintain a stable differentiated state for the life of the organism and remain sufficiently plastic to participate in novel behaviors. Studies of signaling molecules and transcriptional programs have identified many mechanisms that orchestrate critical steps in neurogenesis, cell type diversification, neuronal migration, axonal pathfinding, and differentiation. Although it has been established that epigenetic regulatory mechanisms are critical for proper development of all cell types, our knowledge of the precise roles of these mechanisms in neuronal differentiation, function, and vitality remains rudimentary.

5-Hydroxymethylcytosine (5hmC) is produced from 5-methylcytosine (5mC) by the Ten-eleven translocation dioxygenases (Tet1, Tet2, Tet3) (Iyer et al., 2009; Tahiliani et al., 2009). It is present at approximately 10-fold higher levels in neurons than peripheral cell types (Globisch et al., 2010; Kriaucionis and Heintz, 2009), and its distribution across the genome of adult neurons is cell specific and correlated with active gene expression (Mellén et al., 2017; Mellén et al., 2012; Szulwach et al., 2011). The initial discovery that 5hmC accumulates within active gene bodies, coupled with the discovery that MeCP2 can bind probes containing 5hmC with high affinity, led to the proposal that MeCP2 binding within active genes facilitates their expression (Mellén et al., 2012). Further studies demonstrating that MeCP2 binds 5hmC at high affinity in non-CG dinucleotides but does not bind to 5hmCG overturned this model (Ayata, 2013; Gabel et al., 2015; Mellén et al., 2017) by revealing that accumulation of 5hmCG and the depletion of 5hmCH in gene bodies is correlated with less MeCP2 binding and increased expression (Ayata, 2013; Gabel et al., 2015; Mellén et al., 2017). Imaging of the binding and diffusion of single MeCP2 molecules in living neurons lacking Dnmt3a or Tet1, Tet2, and Tet3 demonstrate that its binding is exquisitely sensitive to the levels of both 5mC and 5hmC (Piccolo et al., 2019). Given these data and the sensitivity of neuronal function to MeCP2 gene dosage (Chahrour et al., 2008; Nan and Bird, 2001), relief of the repressive functions of MeCP2 through Tet-mediated conversion of high-affinity 5mCG-binding sites to low-affinity 5hmCG sites, which we have referred to as functional demethylation (Mellén et al., 2017), provides an important mechanism for modulation of chromatin structure and transcription.

In dividing cells, 5hmC serves as an intermediate in DNA demethylation because maintenance DNA methyltransferases do not recognize hemi-hydroxymethylated cytosines in order to reestablish methylation (Wu and Zhang, 2017). Consequently, 5hmC is lost passively and replaced by C due to replicative dilution. Loss of function studies in mouse embryonic stem cells (ESCs) (Dawlaty et al., 2013) and lymphocyte lineages Lio and Rao, 2019 have demonstrated that the role of Tet-mediated replicative DNA demethylation is to provide full accessibility to regulatory regions necessary for expression of genes required for differentiation. For example, at the activation-induced deaminase (AID) locus in B cells, Tet activity is required for demethylation and activation of enhancer regions that modulate AID expression to enable class switch recombination (Lio et al., 2019). Tet-mediated replication-dependent passive demethylation is thought to be common in many dividing cell types, including progenitor cells in the developing nervous system (MacArthur and Dawlaty, 2021).

Most neurons exit the cell cycle during mid-gestation and remain relatively simple and undifferentiated until birth. Following parturition, they initiate an elaborate program of differentiation that includes dramatic increases in size, morphological complexity, and connectivity. Since these neurons are postmitotic and remain so throughout life, removal of the repressive effects of 5mC by passive demethylation cannot occur. However, in addition to functional demethylation and passive demethylation, a third pathway for DNA demethylation, often referred to as active demethylation, has been proposed based on the finding that 5hmC can be further oxidized by Tet proteins to produce 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Removal of 5fC and 5caC by thymine DNA glycosylase (TDG)-dependent base excision repair (BER) provides another mechanism for 5hmC-mediated DNA demethylation (He et al., 2011). Clear evidence that this pathway can operate in cultured cells has been presented, and detailed biochemical studies have delineated the mechanisms operating in TDG-BER DNA demethylation (Weber et al., 2016). Active DNA demethylation is ideally suited for remodeling DNA methylation in postmitotic neurons.

Evidence that continued accumulation of 5hmC is required in differentiating neurons and that it can participate in active demethylation in vivo is beginning to emerge. In studies of cerebellar granule cell development in ESC-derived GC cultures, primary GC cultures, and slice preparations, manipulations of Tet activity and 5hmC levels provide strong evidence that 5hmC is required for expression of axon guidance and ion channel genes, and that proper development of the GC dendritic arbor requires 5hmC (Zhu et al., 2016). Demethylation of 5hmC-GFP transfected DNA fragments retrieved after several days in culture display reduced 5hmC levels at several sites as assessed by bisulfite sequencing (BSSeq) in HEK 293 cells and primary hippocampal neurons (Guo et al., 2011). Changes in methylation levels at GGCC sites in the genome of hippocampal dentate gyrus neurons in response to electroconvulsive shock have been documented using the methylation-sensitive cut counting method that employs the methylation or hydroxymethylation-sensitive restriction enzyme HpaII and its methylation-insensitive isoschizomer Msp1 (Guo et al., 2011). Global measurements of 5mC and 5hmC by mass spectroscopy in the hippocampus have shown that both decrease in response to the induction of seizure (Kaas et al., 2013). And in germline Tet1 knockout mice, analysis of the Npas4 and c-Fos promoters has shown that Tet1 is required for their proper regulation in both the cerebral cortex and hippocampus (Rudenko et al., 2013). Despite these observations, our knowledge of the roles of continued 5hmC accumulation in active DNA demethylation and postmitotic differentiation remains rudimentary.

We report here single nucleotide resolution studies of DNA methylation and hydroxymethylation, transcription, chromatin accessibility, and H3K4me3 and H3K27me3 histone marks in postmitotic, differentiating Purkinje cells (PCs). As PCs transition from relatively small, multipolar immature cells to fully elaborated large neurons with complex dendritic arbors and hundreds of thousands of synapses (McKay and Turner, 2005), 5hmC continues to accumulate, and DNA methylation and hydroxymethylation are reconfigured as epigenetic and transcriptional programs progress. Our data confirm previous studies of the relationships between transcription, DNA methylation, DNA hydroxymethylation, and chromatin organization (Lister et al., 2013; Mellén et al., 2017; Tsagaratou et al., 2017; Tsagaratou et al., 2014), and they reveal several novel modes of transcriptional activation and repression. Notably, we identify a class of developmentally induced PC-specific genes that are highly expressed and lose both 5mC and 5hmC in the final stages of PC differentiation.

We report also studies of newly generated Tet1, Tet2, Tet3 PC-specific triple knockout (Pcp2TetTKO) mouse lines in which recombination is activated in the first postnatal week. These data demonstrate that postmitotic transcriptional and epigenetic maturation in PCs, including transcription of many ion channels and active demethylation of late expressed genes, requires continued oxidation of 5mC to 5hmC. Taken together, our data demonstrate that active demethylation occurs in select genes in postmitotic neurons, and that 5hmC plays an essential role in refining the transcriptional and epigenetic status of PCs during the final stages of differentiation.

In mice, PC progenitors complete their final cell cycle between e10.5 and e13.5 within the ventricular zone of the developing cerebellar anlage (Leto et al., 2016). As they exit cell cycle and commit to a PC fate, they express the transcription factors Lhx1/Lhx5 (Chizhikov et al., 2006; Morales and Hatten, 2006). To characterize 5hmC accumulation, transcription and chromatin organization during their postmitotic development, we chose to analyze PCs at the start of their accelerated differentiation (P0), during their rapid phase of morphological development (P7) and as fully mature, differentiated neurons (adult) (Figure 1A–B). We employed fluorescence-activated nuclear sorting (FANS) to purify Itpr1-positive PC nuclei in two biological replicates (Figure 1C–E, Figure S1A) (Xu et al., 2018). Since PCs are extremely rare and difficult to isolate, we optimized low input protocols for genomic profiling. We used ~20,000 nuclei for transcriptional profiling, ~200,000 nuclei for BSSeq and oxidative bisulfite sequencing (OxBSSeq), 25,000 nuclei for the assay for transposase-accessible chromatin sequencing (ATACSeq), and 25,000 nuclei for H3K27me3 and H3K4me3 chromatin immunoprecipitation sequencing (ChIPSeq) (Figure 1E–F). Analysis of biological replicates for each of the assays revealed that the sorted nuclei expressed markers of Purkinje neurons, and did not express genes marking the two most abundant cerebellar cell types, granule cells and glia (Figure S1B). The Pearson correlation coefficients between each replicate at all timepoints and techniques (RNASeq, OxBSSeq, ATACSeq, and ChIPSeq) were high (over 0.9), establishing that our data are reproducible and of very high quality (Figure 1—figure supplement 1C-F). Bisulfite conversion and oxidation rates for the OxBSSeq datasets were within the expected range (Figure 1—figure supplement 1G).

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It is important to note that we chose to focus on OxBSSeq (Booth et al., 2013) as a definitive technology for analysis of DNA methylation because, in contrast to BSSeq alone, it allows us to assess the contributions of both 5mC and 5hmC to neuronal development. Given the distinct functions of these two modifications, analysis of their separate contributions to epigenetic regulation of the genome is more informative than BSSeq alone. For example, in many genes, 5mCG is depleted and 5hmCG accumulates in the gene body as its expression increases between P0 and adult. This is clearly evident for the gene encoding the cyclic GMP-dependent protein kinase (Prkg1), whose expression in PCs has been shown to be essential for long-term depression (Feil et al., 2003). In this case, the OxBSSeq data reveal a transition from 5mCG to 5hmCG that cannot be detected in the BSSeq data as the gene is activated between P0 and adult (Figure 1—figure supplement 1H). These data illustrate that a precise evaluation of DNA methylation status in relation to other programs unfolding during development for this gene and many others is advanced significantly by inclusion of OxBSSeq data.

Transcriptional programs altered during PC differentiation

PC progenitors complete their final divisions in the cerebellar primordium between e11 and e13 (Butts et al., 2014) and remain as a multilayered, simple migrating cell population until birth (P0). In the first postnatal week, they organize into a monolayer as the cerebellum enlarges and begin the transition from a multipolar primitive neuronal morphology to one of the largest neurons in the brain with a characteristic, highly elaborate planar dendritic arbor. As they begin the second postnatal week (P7), PCs undergo an important developmental transition that includes refinement of their climbing fiber input, formation of many thousands of parallel fiber synapses, and myelination of their axons (Leto et al., 2016; McKay and Turner, 2005). The tremendous increase in synaptogenesis and connectivity continues until PCs attain their mature morphology and functions at 3–4 weeks of age. As these programs unfold, there is a global decrease of 5mCG and global increase of 5hmCG (Figure 2—figure supplement 1A).

To identify genes whose transcription is stable and those that are dynamically regulated during PC differentiation, we conducted differential expression analysis between P0 and adult Purkinje neurons, filtering for significance of p < 0.01 and log2 fold change of >2 in either direction (Figure 2A–B, Supplementary file 1). Using these criteria, there were 922 developmentally repressed genes (down-regulated in adult) enriched in categories involved in cell signaling and axon guidance pathways (e.g. Grid1, Pde1c; Figure 1F). We identified 432 genes with increased expression as PCs differentiate encoding proteins known to be important for the mature functions of PCs including calcium ion buffering and transport, regulation of the inositol triphosphate signaling pathway, and RNA splicing. As expected, similar analysis comparing P0 and P7 or P7 and adult data revealed genes overlapping with those identified in the overall P0 and adult comparative data (Figure 2—figure supplement 1B-D), although additional categories of RNA metabolism, synaptic signaling, and ion transport are enriched in the P7 to adult analysis (Figure 2—figure supplement 1C). As anticipated, the P7 profiles we have included in our analysis have been essential in assessing both the developmental course of epigenetic events studied here and the consequences of loss of 5hmC in PCs (see below).

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Loss of 5mC and 5hmC in active genes during PC differentiation

Our data reveal additional, surprising features that have not been documented in postmitotic cells. Thus, in a small subset of highly expressed genes (e.g. Cep76, Itpr1, Mtss1) there is a profound loss of both 5hmCG and 5mCG during the terminal, postmitotic stage of PC differentiation (Figures 1F and 3). To investigate the apparent demethylation over this class of genes, we computationally identified DNA methylation valleys (DMVs) as previously described (Jeong et al., 2014; Mo et al., 2015; Xie et al., 2013). In brief, DMVs were characterized by filtering undermethylated regions (UMRs) for length (>5 kb) and merging any regions within 1 kb (Burger et al., 2013; Figure 3—figure supplement 1A-B, Supplementary file 2). This revealed multiple regions longer than 15 kb, an unusual finding given that the average length of DMVs is ~5 kb (Figure 3—figure supplement 1D-E; Jeong et al., 2014). As expected from previous studies, a small number of these genes are inactive, fully demethylated, inaccessible, and covered by high levels of H3K27me3 repressive marks (Figure 3A, Foxd1, Figure 3—figure supplement 1C). These genes acquire their characteristics early in development, and their epigenetic features are stable.

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The majority of very large DMVs in PCs, however, arise during differentiation through loss of 5mC and 5hmC over active, highly expressed genes as PCs mature (Figure 3, Itpr1, Mtss1). In these genes, the timing of changes in DNA methylation, chromatin accessibility, and dynamic histone marks varies from gene to gene, but the overall progression of events associated with their expression is similar (Figure 3). At birth, these genes are actively transcribed (Figure 3A), 5hmCG is enriched in the gene body (Figure 3B) and 5mCG is depleted (Figure 3C), their promoters are accessible (Figure 3D), and H3K4me3 is present over their promoters (Figure 3E). These epigenetic properties resemble moderately expressed genes in many cell types. As differentiation proceeds and transcription increases, there is a gradual decrease in both 5mCG, 5hmCG, and H3K27me3 histone marks, and ATAC accessibility and H4K4me3 marks spread from the promoter into the gene body. To further support these data, we have included analysis of independent BSSeq experiments that detect the combined levels of 5mC and 5hmC (Figure 3A, BSSeq). These data also reveal that hypo-DMRs (differentially methylated regions) present at P0 expand and fuse as PCs lose both methylation and hydroxymethylation to form the very large DMRs that are characteristics of these genes (Figure 3A, hypo-DMR track). As shown in Figure 3B–E and Figure 3—figure supplement 1I-K, quantitation of these features demonstrates that that loss of DNA methylation and hydroxymethylation occurs over the entire gene body for these genes as H3K4me3 activating histone marks accumulate and ATAC accessibility increases.

Interestingly, many of the active genes associated with broad DMVs are PC-specific and highly expressed (Figure 3—figure supplement 1F). GO analysis indicates that they are involved in the inositol triphosphate/calcium signaling pathways, and a subset is associated with ataxia and autism (Figure 3—figure supplement 1H). They do not accumulate modified cytosines in CpH context (Figure 3—figure supplement 1G). These data provide strong evidence that DNA demethylation can occur in postmitotic neurons, and that it is enhanced in a specific class of genes that are very highly expressed and functionally important. Although our mass spectroscopic analysis of genomic DNA from differentiating PCs (Figure 3—figure supplement 1L) failed to detect 5fC or 5caC, their involvement as transient intermediates in the loss of 5mC and 5hmC cannot be ruled out because the small fraction of the genome covered by this gene class may preclude their detection (Guo et al., 2011; He et al., 2011; Ito et al., 2011).

PC-specific Tet1, Tet2, Tet3 triple knockout mouse lines

Despite convincing evidence that Tet-mediated active DNA demethylation can occur in mouse ESCs through the TDG-BER pathway (He et al., 2011; Weber et al., 2016), evidence that this can occur in vivo has been difficult to obtain because such a proof requires loss or complete inhibition of all three Tet oxidases in a single cell type after cells have exited the cell cycle permanently. To provide this evidence, we generated PC-specific Tet1/Tet2/Tet3 triple knockout (Pcp2TetTKO) mouse lines (Figure 4A). We chose to drive Cre recombinase expression using an engineered Pcp2 BAC employed previously to generate accurate Cre driver lines (Gong et al., 2007) because the onset of expression of the Pcp2 gene and the corresponding BAC vector occurs approximately 1 week after birth as the cerebellum enters its terminal phase of development and maturation. The Pcp2Cre BAC was introduced by pronuclear injection into ova from females carrying floxed alleles of all three Tet genes (Figure 4—figure supplement 1A-B) yielding several founder lines. PCR analysis of the floxed regions of each Tet gene in purified PC genomic DNA, and RNASeq analysis confirmed the deletion of exons from all three TET proteins (Figure 4—figure supplement 1C-D). The lines chosen for analysis displayed no gross motor phenotype as assessed by rotarod performance, survival was normal, and recombination activity was specific to PCs (Figure 4B–D, Figure 4—figure supplement 2A-D). The deletion of the Tet proteins did not affect the expression of Itpr1 significantly, and we confirmed that it could still be used for purification of PCs (Figure 4—figure supplement 1E-F). Evaluation of the quality control metrics of the datasets showed strong correlation coefficients and enrichment for Purkinje markers (Figure 4—figure supplement 1G-H). Cre recombinase activity, and thus recombination in the Tet genes, began at approximately P7 more than 2 weeks after PCs have completed their last division (Figure 4—figure supplement 2A-D). These lines, therefore, allow assessment of the consequences of loss of Tet activity during the rapid phase of somatic and dendritic growth in postmitotic, differentiating Purkinje neurons.

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Since the discoveries that 5hmC is present at high levels in mammalian neuronal genomes (Kriaucionis and Heintz, 2009) and that it is produced from 5mC by the Tet oxidases (Tahiliani et al., 2009), its possible roles as a stable epigenetic mark and as an intermediate in DNA demethylation have been intensively investigated (Wu and Zhang, 2017). The present study adds to a growing body of work demonstrating that 5hmC plays a critical role in development and function of the nervous system. A central finding of this study is that continued Tet activity and 5hmC accumulation during PC differentiation is necessary for acquisition of their refined transcriptional and epigenetic properties. It is noteworthy that these phenotypes are evident despite loss of Tet function in the Pcp2TetTKO late in PC differentiation. It seems likely based on these findings and the gradual accumulation of 5hmC evident in our data that there is a continual requirement for 5hmC as a driver for epigenetic remodeling in most differentiating, postmitotic neurons. Although studies of neural stem cells (Li et al., 2015; Xu et al., 2012), cerebellar granule cells (Zhu et al., 2016), and embryonic brain development have suggested that 5hmC plays a significant role in neural progenitors and developing granule cells, a continual requirement for 5hmC may be particularly important for PCs, pyramidal cells, and other long range projection neurons whose differentiation program includes postmitotic development of sophisticated morphological features and abundant synapses.

A second principle that has emerged from our studies is that Tet-mediated active DNA demethylation occurs in postmitotic neurons. Our finding that loss of both 5mC and 5hmC in a specific subset of highly expressed gene bodies and ATAC accessible regulatory sites requires Tet oxidase activity provides critical in vivo evidence supporting studies in vitro (Weber et al., 2016) and in cultured cells (He et al., 2011) that have defined this pathway. For some genes (Gpr63, Grid2) the progression toward loss of modified C is easily appreciated. In these cases, conversion of 5mCG to 5hmCG over the gene body is clearly evident between P0 and P7, and continued Tet activity is required to remove both residual 5mCG and 5hmCG to result in DNA demethylation in the adult. Although we have been unable to detect 5fC and 5caC as transient intermediates in this process (Figure 3—figure supplement 1L), this is not surprising given the very small fraction of the PC genome that undergoes active DNA demethylation and the small amounts of genomic DNA that can be obtained from this very rare cell population. Identification of additional components of the of the DNA demethylation pathway that is occurring in PCs will require further genetic studies disrupting candidate genes in the TDG-BER pathway. Since important non-cell autonomous events are required for PC differentiation (Leto et al., 2016), we believe that additional PC-specific knockouts that are restricted to postmitotic cells will be most informative.

An important question arising from these findings is which cells in the nervous system require Tet-dependent active DNA demethylation to complete their developmental programs. Given these data, it seems likely other large neurons with complex and prolonged postmitotic differentiation programs share this requirement. Furthermore, reports that remodeling of DNA methylation may occur in response to a wide variety of stimuli in adult mice (Kaas et al., 2013; Rudenko et al., 2013) suggest that additional studies of Pcp2TetTKO and other precisely constructed mouse models will help elucidate possible functions of 5hmC in neuronal plasticity.

A third advance reported here is the collection of very high-resolution data to understand details of the relationships between DNA methylation and hydroxymethylation, chromatin accessibility, and chromatin organization in a single complex neuronal cell type as it develops from a primitive neuronal precursor to a fully articulated adult neuron. These data have highlighted several features of epigenetic development that remain to be explored including the definition of two distinct relationships between non-CG DNA methylation/hydroxymethylation and transcriptional repression, and the mechanism for selection of specific genes as substrates for active DNA demethylation. Taken together, our data demonstrate that disruption of continued 5hmC formation in the Pcp2TetTKO postmitotic PCs leads to altered transcription of late expressed genes, including a set of ion channels that may be responsible for refining their adult electrophysiological properties.

Although our data reinforce prior studies of the general relationships between DNA methylation, transcription, and chromatin structure, we note that there are many exceptions that require further investigation. One intriguing example is the very different relationships between genes and de novo methylation in CG and non-CG contexts (Lister et al., 2013). In PCs, as in other neurons, substantial enrichment of 5hmCG relative to its general accumulation genome wide occurs in active transcription units, suggesting a mechanism that targets Tet activity to these regions (Figure 2C). While accumulation of 5mCH and 5hmCH can occur within a repressed transcription unit with very nice discrimination between the gene and its surroundings (e.g. Pitpnc1), there are many regions of 5mCH and 5hmCH accumulation that either partially overlap with a gene, or that are present in intergenic regions (e.g. Myo5b/Scarna17/Lipg locus). In all of these regions, however, 5hmC accumulation directly reflects 5mCH levels suggesting 5hmCH localization reflects the pattern of de novo DNA methylation rather than targeted Tet activity. We hope that continued analysis of these datasets will provide a stimulus for additional studies of the mechanisms determining the relative distributions of these important events.

Pcp2TetTKO mice will be made available with a Material Transfer Agreement (MTA). Sequencing data is available on NCBI Gene Expression Omnibus under accession number GSE166423. Code for data analysis is available at https://github.com/estoyanova/EStoyanova_eLife_2021 copy archived at https://archive.softwareheritage.org/swh:1:rev:4c2fa6c102e4f868fc91e0dcb0b8c7155b8f8712.

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[Editor’s note: review comments and author responses relating to electrophysiology data, which were included in an earlier version, have been omitted.]

Congratulations, we are pleased to inform you that your article, "5-hydroxymethylcytosine mediated active demethylation is required for neuronal differentiation and function", has been accepted for publication in eLife.

Thank you for submitting your work entitled "5-hydroxymethylcytosine mediated active demethylation is required for neuronal differentiation and function" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The reviewers have opted to remain anonymous.

Reviewer #1 (Recommendations for the authors):

In this manuscript the authors carry out epigenomic profiling purified Purkinje cells across key stages of cerebellar development to study the regulation and function of 5hmC. The authors have previously published a method for isolation of FACS-based nuclear sorting for these cells and here they use it to isolate from P0, P7, and adult cerebellum with high quality RNA, mC/hmC, ATAC and ChIP-seq data at each time point in biological replicate. The chromatin data are correlated against developmental changes in gene expression and suggest broad mechanisms of chromatin control that are, for the most part, in line with previous studies of other cell types. The authors are particularly interested in regions of the genome that show loss of mCG (and hmCG) over developmental time and are near genes that show enhanced expression in adult PCs. They test the requirement for the Tets in the developmental demethylation of these regions by knocking out all three Tets in a PC specific manner at P7, then running RNA/mCG/hmCG sequencing in adult to compare between cTKO and control.

PCs are a biologically important class of neurons and they are a very different cell type from others that have been developmentally studied. For these reasons the descriptive chromatin data gathered here are of use to the community. However, the analyses performed on the data as presented here are somewhat underdeveloped. I

Reviewer #2 (Recommendations for the authors):

TET dioxygenases (TET1, TET2, TET3) mediate the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which may lead to additional forms of DNA modifications and ultimately DNA demethylation. In dividing cells, the maintenance DNA methyltransferase recognizes poorly of 5hmC, which leads to passive DNA demethylation through replacing the 5hmC sites with unmethylated cytosine after rounds of DNA replication. However, whether 5hmC undergoes demethylation in non-dividing cells, for example through TDG-BER dependent active DNA demethylation, and how it may impact gene transcription and cellular properties remain elusive. To answer these questions will particularly expand our current understanding of TET catalyzed methylcytosine oxidation in the brain, the organ that has not only the highest levels of 5hmC, but also thousands of millions of non-dividing neurons that remain plastic throughout life.

Purkinje neurons in cerebellum subject to orchestrated postnatal migration, arborization, and synaptogenesis after their final cell division at mid-gestation stage. The study focuses on this neuron type and generates single base resolution 5mC and 5hmC mapping at three developmental stages (P0, P7, young adult). In addition, it profiles the Purkinje neuron specific transcriptiome (nuclei RNAseq), epigenome (H3K4me3, H3K27me3), and chromatin accessibility (ATACseq). The study therefore displays novel modes of epigenomic alterations that are associated with transcription regulation, which also confirms DNA methylation turnovers during Purkinje neuron post-mitotic maturation. Furthermore, the study generates a conditional knockout mouse line with all three TET enzymes selectively deleted in Purkinje neurons. The deficiency of TET enzymes not only impedes the DNA methylation changes during Purkinje cell postnatal maturation, but also alters neuronal excitability that increases susceptibility to an excitotoxic drug. Therefore, the study demonstrates a requirement of dynamic 5mC/5hmC changes during cerebellum Purkinje neuron post-mitotic maturation and their implication in neuronal function.

The study has successfully addressed a few technical challenges, such as the low abundancy of Purkinje neurons, to profile 5mC and 5hmC separately, and to generate a cell type specific triple TET knockout mouse line. The data are of good quality (e.g. Figure S1). Overall, the conclusions are supported by the data. Epigenetic modifications have been known to play cell type specific functions. However, the majority of current research is limited by the heterogeneity of tissue samples that lack a cell type specific resolution. To study DNA modifications in a defined neuron type, as shown in this manuscript, provides valuable novel insights (e.g. as mentioned in the Discussion, the changes are specific to Purkinje cells, but not observed in cerebellum granule neurons). In addition to sequencing 5mC and 5hmC side by side, the study also analyzes CG and CH regions separately. The study recognizes unique patterns of 5mC, 5hmC, CG and CH changes, respectively, which suggest the complexity of DNA modification in the brain. The set of genes that are associated with DNA methylation valley dynamics are particularly interesting. The genomic datasets and the mouse line derived from this study will also serve as a good resource for related work.

Through RNAseq, less than 2000 genes are found to be differentially expressed during Purkinje neuron maturation. The study therefore spends major efforts on analyzing the epigenetic changes at these genes. However, it is not sufficiently clear of the epigenetic status of the vast majority of genome beyond the differentially expressed genes, even though some indicative findings are revealed at regions with "gain" or "loss" of chromatin accessibility (Figure 2K-N). To assess this, it will be helpful to further characterize intergenic regions in the future, for example through genomic profiling of enhancer markers H3K4me1 and H3K27ac.

An enhancer marker ChIPseq is preferred, but not required. Below are some suggested revision points.

1. It will be informative to demonstrate the transcription levels of the three TET family members during Purkinje neuron maturation. This can be obtained from the RNAseq data that the authors have generated.

2. The transcriptome and epigenome differential lists should be provided in supplements.

3. It is unclear if both males and females are included in the genomic assays and animal work.

4. Though the number of cells utilized in each genomic assay is stated in methods, it will be helpful to describe the corresponding number of animals.

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for resubmitting your work entitled "5-hydroxymethylcytosine mediated active demethylation is required for neuronal differentiation and function" for further consideration by eLife. Your revised article has been evaluated by Catherine Dulac (Senior Editor) and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

Reviewer #1 (Recommendations for the authors):

In the first round of review I raised two main concerns about the molecular data – the novelty of the findings and the depth of the analysis linking Tet-mediated DNA demethylation with PC maturation. One of the other reviewers raised concerns about the physiology of the Tet cKO mice.

1. Novelty of the findings

The first concern was the degree of novelty in showing Tet-mediated DNA demethylation in post-mitotic neurons. My concern arose from the language used by the authors in their manuscript. In the abstract of the revised manuscript they state, "is it not known whether 5hmC is required for terminal differentiation or if it can serve as an intermediate in active DNA demethylation in postmitotic neurons." This assertion does not jibe with my reading of the literature. My concern was amplified when I read the sentence at line 68 in the introduction that says, "Tet mediated replication dependent passive demethylation is thought to be common in many dividing cell types, including progenitor cells in the developing forebrain (Rudenko et al. 2013), hippocampus (Guo et al. 2011) and cerebellum (Zhu et al. 2016)." This is an inaccurate statement regarding these papers from the literature.

Rudenko et al. studied DNA methylation in adult brains of a Tet1 knockout mouse and found hypermethylation of activity-regulated gene promoters in the knockout compared with wildtype. I appreciate that because this was a germline knockout that this study does not rule out the possibility that the increases in methylation occurred in dividing progenitor cells and were maintained. However, the authors of the current manuscript are over-reaching to conclude that the changes in methylation seen in the Rudenko study were indeed the result of passive demethylation in progenitors, given that this was not tested in the study. The Guo and Zhu studies knocked down Tets in postmitotic neurons. In the Guo 2011 study the authors injected shRNA-bearing AAVs targeting Tet1 in adult dentate gyrus. In the Zhu 2016 study the authors lentivirally infected primary CGN cultures with shRNAs targeting Tets 1 and 3. Both studies showed increases in DNA methylation in knockdown compared with wildtype neurons. In the Guo study this was in the context of seizure-induced demethylation of the Bdnf and Fgf1b genes, and the demethylation was blocked by Tet1 KD. Although there are a few progenitors in the adult dentate gyrus, it is not reasonable to attribute the methylation effects to only these few cells if that is what the authors mean by saying this study was in progenitors. The authors of the Guo paper concluded that Tet1 acted to actively demethylate DNA in postmitotic hippocampal neurons, and their data support that conclusion. The Zhu 2016 used primary CGNs in culture, which have been shown by many groups (using the specific protocol derived from the Hatten lab) to be fully post-mitotic within the first 24 hours after plating. shRNA-mediated knockdown occurs on the order of days (cells were studied at 3DIV). Therefore, the assumption of Guo 2016 in their conclusions that the Tets are acting to mediate these changes in post-mitotic neurons is justified.

Later (line 274) the manuscript references Lio 2016 to cite that prior studies have established that the activation of enhancers is accompanied by increased accessibility and loss of DNA methylation. At line 291 the authors suggest that the DNA demethylation they find in PCs is novel because "in this case…DNA demethylation does not require cell division." I agree it is true the PC data shows postmitotic demethylation, however it was also shown in PMID 32589877. In that study the authors purified nuclei of SST+ and VIP+ neurons from the mouse cortex during development. These experiments were done during comparing interneuron nuclei isolated at P7 with those from P13, both stages at which these neurons are already postmitotic.

These discrepancies in the manuscript between the authors' assertions of what these studies showed and the published data need to be corrected to be accurate.

It was because the authors suggested the novelty of their study as the first to show Tet-mediated active DNA demethylation in postmitotic neurons that I questioned that novelty. As the literature review above shows, this manuscript is not the first to show Tet-mediated active DNA demethylation in postmitotic neurons.

However, this manuscript does do a particularly nice job of it. I think the novelty of the study lies in linking the molecular characterization of PC chromatin regulation during development with the biology of this cell population. With this in mind, I agree with the authors that the body of the data and the analysis do make a significant new addition to the literature that will be of broad interest.

2. Depth of the analysis linking Tet-mediated DNA demethylation with PC maturation

In this revision the authors have significantly expanding and clarified the section on the molecular sequencing from Tet_1/2/3 cKO mice, strengthening the impact of the data. However, even though this is a revised manuscript there are some important omissions and errors in this section that undermine the power of the data to support the model.

– Line 193, 5hmc continues to increase over highly expressed genes – where is that evidence?

– Line 202 "Furthermore, 5hmCG accumulated over the genes bodies early in development is not removed (Figure 2C)," actually what this figure shows is that for the downregulated genes there is an increase in 5hmCG over the gene bodies between P7 and adult, so it is not only not lost, it is gained. It looks no different for the downregulated genes than the upregulated genes.

– Where is the data that goes with line 372 – growth of the DMV preceded by 5hmC and starts at 5' end

– Line 376 -The text that follows this (to line 381) is entirely speculative and does not belong in the methods.

– Figure 4S2 E-G are not cited in the text.

– Line 496 – the authors state they have not seen demethylation in CGNs with no attribution and no data. Also, this contrasts with the Zhu 2016 paper so should be discussed in comparison.

Furthermore, one of the important parts of this section regards the relationship of the chromatin changes in the Tet cKO to the gene expression effects and this part would benefit from more discussion. For example, one of the best example genes for Tet mediated demethylation the authors show it Iptr and yet they specifically say this epigenomic change does not influence Iptr expression (which is key since this is the gene product used for PC isolation). Also, it is surprising that in the Tet_1/2/3 cKO mice, expression of genes encoding synaptic proteins and differentiation markers are the major class elevated, not repressed. Might this suggest that the Tets actually slow differentiation rather than promoting it as the text seems to imply? Either would be interesting, but the data shown needs to be reconciled with the statement (line 106) that "maturation in Purkinje Cells … require continued oxidation of 5mC to 5hmC".

Reviewer #1 (Recommendations for the authors):

In this manuscript the authors carry out epigenomic profiling purified Purkinje cells across key stages of cerebellar development to study the regulation and function of 5hmC. The authors have previously published a method for isolation of FACS-based nuclear sorting for these cells and here they use it to isolate from P0, P7, and adult cerebellum with high quality RNA, mC/hmC, ATAC and ChIP-seq data at each time point in biological replicate. The chromatin data are correlated against developmental changes in gene expression and suggest broad mechanisms of chromatin control that are, for the most part, in line with previous studies of other cell types. The authors are particularly interested in regions of the genome that show loss of mCG (and hmCG) over developmental time and are near genes that show enhanced expression in adult PCs. They test the requirement for the Tets in the developmental demethylation of these regions by knocking out all three Tets in a PC specific manner at P7, then running RNA/mCG/hmCG sequencing in adult to compare between cTKO and control. Finally, the authors assess the functional importance of Tet-dependent gene regulation by recording from cTKO and control PCs and assessing behavioral features of PC function (rotarod and harmaline-induced tremor).

PCs are a biologically important class of neurons and they are a very different cell type from others that have been developmentally studied. For these reasons the descriptive chromatin data gathered here are of use to the community. However, the analyses performed on the data as presented here are somewhat underdeveloped. It is not clear to me that the evidence for developmental demethylation is novel and the data in Figure 4 (which could be very powerful) are not strongly deployed to argue that Tets are key in the demethylation process.

Reviewers 1 and 2 both appreciate that the data we present for transcription, DNA methylation and hydroxymethylation, chromatin accessibility, H4K4me3 and H3K27me3 histone marks are high quality and valuable for the community. We agree with this assessment, and we present them both to support our specific interests and as a resource for further analysis. With respect to our analysis being characterized as “somewhat underdeveloped”, these data are so massive that a complete study of all of their features will require many additional sets of analyses and follow up studies. There is little doubt in our minds that the data will be embraced by the community and additional biological inferences will emerge from them. Although we could populate several more figures with further analysis of these data, we have chosen to focus on only those findings supporting the most novel and important conclusions relative to the direct roles of 5hmC in differentiation.

Novelty. It has been demonstrated that the distribution of 5hmC in neuronal genomes is cell type specific and correlates with active gene expression, and that germline deletions of Tet oxidases disrupt development of the brain. Reviewer 1 is familiar with this “developmental role” of 5hmC covering the course of neuronal development – including proliferative phases of neuronal progenitor expansion where 5hmC is required for replication dependent DNA demethylation and chromatin remodeling. Replication dependent DNA demethylation is a fundamental feature of DNA methylation refinement that occurs in many cell types. However, the term “developmental demethylation” does not distinguish between the role of 5hmC mediated, replication dependent passive demethylation in progenitors and the proposed, but not proven, role of 5hmC mediated active demethylation in the absence of cell division (Ito et al., 2011). Our study was designed to arrest 5hmC formation in postmitotic differentiating PCs to address specifically this issue. The design of the study, the use of new cell type specific triple Tet1, Tet2, Tet3 triple knockout lines that activate well after the final division of PC progenitors, and the results obtained are novel and of high interest.

Figure 4. To address the remaining comments of Reviewer 1, we have edited substantially the section of the manuscript subtitled “Loss of 5mC and 5hmC in active genes during PC differentiation” presenting the data documenting loss of both 5mC and 5hmC in a specific class of highly expressed genes during PC differentiation. We believe that these new edits present the data in a concise yet very clear manner that is accessible to even a general reader. In doing so, they also augment the following section “Loss of 5mC and 5hmC in putative regulatory sites during PC differentiation”. We think this improved presentation of the data in Figure 3 establishes for the reader both that DNA demethylation occurs as PC differentiation proceeds, and that 5hmC accumulation is present in the P7 data at these sites prior to the evident loss of both 5mC and 5hmC in the adult. These new edits provide an improved introduction to Figure 4, and we believe they will help the reader understand more readily the molecular events that are disrupted as a consequence of loss of 5hmC.

Of course, to provide proof that loss of DNA methylation over the PC DMVs is dependent on 5hmC required disruption of continued 5hmC production in the Pcp2TetTKO mice. Figure 4 presents first the basic characteristics of the Pcp2TetTKO lines, establishing that their survival and body weight is normal (panels B,C), and that no gross motor phenotype is evident in rotarod performance measures (panel D). The remaining data in Figure 4 are meant to address directly the molecular events that occur in Pcp2TetTKO PCs and the requirement for 5hmC in the DNA demethylation described in Figure 3.

The description of the molecular events that occur in PCP2TetTKO PCs has been extensively lengthened to clarify each point revealed by the data. We have also changed panel G to box and whisker plots so that the reader can more easily evaluate the alterations in methylation over ATAC peaks (putative regulatory sites) that both increase in adult PCs and are associated with genes whose expression changes in the Pcp2TetTKO PCs. We hope that the additional detailed discussion of the data and our conclusions will address the comment that this section of the manuscript is “somewhat underdeveloped”.

Reviewer #2 (Recommendations for the authors):

[…] Through RNAseq, less than 2000 genes are found to be differentially expressed during Purkinje neuron maturation. The study therefore spends major efforts on analyzing the epigenetic changes at these genes. However, it is not sufficiently clear of the epigenetic status of the vast majority of genome beyond the differentially expressed genes, even though some indicative findings are revealed at regions with "gain" or "loss" of chromatin accessibility (Figure 2K-N). To assess this, it will be helpful to further characterize intergenic regions in the future, for example through genomic profiling of enhancer markers H3K4me1 and H3K27ac.

An enhancer marker ChIPseq is preferred, but not required. Below are some suggested revision points.

1. It will be informative to demonstrate the transcription levels of the three TET family members during Purkinje neuron maturation. This can be obtained from the RNAseq data that the authors have generated.

2. The transcriptome and epigenome differential lists should be provided in supplements.

3. It is unclear if both males and females are included in the genomic assays and animal work.

4. Though the number of cells utilized in each genomic assay is stated in methods, it will be helpful to describe the corresponding number of animals.

We thank Reviewer 2 for a general appreciation of our study, and we believe the new edits to the manuscript will strengthen the already positive description of our work “To study DNA modifications in a defined neuron type, as shown in this manuscript, provides valuable novel insights (e.g. as mentioned in the Discussion, the changes are specific to Purkinje cells, but not observed in cerebellum granule neurons). In addition to sequencing 5mC and 5hmC side by side, the study also analyzes CG and CH regions separately. The study recognizes unique patterns of 5mC, 5hmC, CG and CH changes, respectively, which suggest the complexity of DNA modification in the brain. The set of genes that are associated with DNA methylation valley dynamics are particularly interesting. The genomic datasets and the mouse line derived from this study will also serve as a good resource for related work.” We agree with Reviewer 2 that it “will be helpful to further characterize intergenic regions in the future, for example through genomic profiling of enhancer markers H3K4me1 and H3K27ac.” While these and many other types of experiments are certainly of interest, we view these as future studies that will continue to reveal interesting details that occur during the prolonged and complex differentiation programs characteristic of Purkinje cells and other principal neurons.

We have addressed in the revised manuscript all of the additional, detailed comments of Reviewer 2 and included additional data on the levels of expression of the Tet enzymes as PCs mature into Figure 4 Supplement 1A. Of course, the final transcriptome and epigenome lists will be provided with final manuscript. We believe these changes have improved the manuscript and thank Reviewer 2 for highlighting these issues.

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Reviewer #1 (Recommendations for the authors):

In the first round of review I raised two main concerns about the molecular data – the novelty of the findings and the depth of the analysis linking Tet-mediated DNA demethylation with PC maturation.

1. Novelty of the findings

The first concern was the degree of novelty in showing Tet-mediated DNA demethylation in post-mitotic neurons. My concern arose from the language used by the authors in their manuscript. In the abstract of the revised manuscript they state, "is it not known whether 5hmC is required for terminal differentiation or if it can serve as an intermediate in active DNA demethylation in postmitotic neurons." This assertion does not jibe with my reading of the literature. My concern was amplified when I read the sentence at line 68 in the introduction that says, "Tet mediated replication dependent passive demethylation is thought to be common in many dividing cell types, including progenitor cells in the developing forebrain (Rudenko et al. 2013), hippocampus (Guo et al. 2011) and cerebellum (Zhu et al. 2016)." This is an inaccurate statement regarding these papers from the literature.

We agree with Reviewer 1 that our interpretation of these studies is rather strict. In the revised manuscript we have expanded this section to delineate the findings in each of these studies. We have added a reference n line 94 for a review of studies of 5hmC and passive demethylation in neural progenitors (MacArthur and Dawlaty, 2021). We have also included, in accord with first Reviewer’s comments, a new paragraph that explicitly states the findings of previous studies of demethylation including the mentioned references. We state that these studies provide evidence that is consistent with the hypothesis that there is active DNA demethylation in post-mitotic neurons. However, in our judgement they do not constitute definitive proof of active DNA demethylation in vivo. Consequently, this paragraph concludes with the sentence “Despite these observations, our knowledge of the roles of continued 5hmC accumulation in active DNA demethylation and postmitotic differentiation remain rudimentary. “

Rudenko et al. studied DNA methylation in adult brains of a Tet1 knockout mouse and found hypermethylation of activity-regulated gene promoters in the knockout compared with wildtype. I appreciate that because this was a germline knockout that this study does not rule out the possibility that the increases in methylation occurred in dividing progenitor cells and were maintained. However, the authors of the current manuscript are over-reaching to conclude that the changes in methylation seen in the Rudenko study were indeed the result of passive demethylation in progenitors, given that this was not tested in the study.

We agree with the Reviewer 1 that it is possible that the “hypermethylation” observed in this study results from demethylation in post-mitotic neurons. However, this is a germline KO so these changes could have occurred by passive demethylation in dividing progenitors during development. Furthermore, the assay used to assess these changes is bisulfite sequencing – it does not distinguish between 5mCG and 5hmCG. We agree with Reviewer 1 that no data are presented to directly address this issue, so we have edited the text to discuss this possibility in the revision.

The Guo and Zhu studies knocked down Tets in postmitotic neurons. In the Guo 2011 study the authors injected shRNA-bearing AAVs targeting Tet1 in adult dentate gyrus. In the Zhu 2016 study the authors lentivirally infected primary CGN cultures with shRNAs targeting Tets 1 and 3. Both studies showed increases in DNA methylation in knockdown compared with wildtype neurons. In the Guo study this was in the context of seizure-induced demethylation of the Bdnf and Fgf1b genes, and the demethylation was blocked by Tet1 KD. Although there are a few progenitors in the adult dentate gyrus, it is not reasonable to attribute the methylation effects to only these few cells if that is what the authors mean by saying this study was in progenitors. The authors of the Guo paper concluded that Tet1 acted to actively demethylate DNA in postmitotic hippocampal neurons, and their data support that conclusion.

We agree that it is possible that the increase in “methylation” observed by Guo et al. could arise by the inability to demethylate these sites. However, the assay here was again bisulfite sequencing that cannot distinguish 5mC and 5hmC. Furthermore, the protocol was to use electro convulsive stimulation to induce these changes. Since many studies have documented a strong relationship between 5hmC accumulation and transcription, since ECS causes a large increase in transcription from these promoters, and since both the Tet1 and Apobec1 knock downs leave at least Tet 2 and Tet3 activity intact, it is equally likely that the increased “methylation” seen in the KD is 5hmC rather than 5mC. Again, there are no data presented in the study to distinguish between these two possibilities. We have edited the text to clarify this issue and discuss the possibility that demethylation could occur.

The Zhu 2016 used primary CGNs in culture, which have been shown by many groups (using the specific protocol derived from the Hatten lab) to be fully post-mitotic within the first 24 hours after plating. shRNA-mediated knockdown occurs on the order of days (cells were studied at 3DIV). Therefore, the assumption of Guo 2016 in their conclusions that the Tets are acting to mediate these changes in post-mitotic neurons is justified.

The CGC cultures were prepared at P7, the time of maximal GC division in the EGL (Fujita et al. 1966, Fujita, 1967). As demonstrated by the Hatten lab (Gao et al., 1991), cell division in cultured granule cells depends on the plating density. There are no data in the paper to rule out the possibility that a subpopulation of the plated GCs divide for a day or two after being cultured. Furthermore, this paper is dedicated to showing that 5hmC is required for gene expression and does not mention demethylation.

The discussion of Figure 6 states that “5hmC levels decreased dramatically in transfected cells, while 5mC levels increased slightly. There are no statistics for this slight increase in 5mC. The word demethylation occurs only in the titles of papers cited in the References. In my opinion, this “slight increase” can’t be cited as proof of demethylation. However, we agree that this is a nice demonstration of the role of 5hmC in differentiation of granule cells and have edited the manuscript accordingly.

Later (line 274) the manuscript references Lio 2016 to cite that prior studies have established that the activation of enhancers is accompanied by increased accessibility and loss of DNA methylation. At line 291 the authors suggest that the DNA demethylation they find in PCs is novel because "in this case…DNA demethylation does not require cell division." I agree it is true the PC data shows postmitotic demethylation, however it was also shown in PMID 32589877. In that study the authors purified nuclei of SST+ and VIP+ neurons from the mouse cortex during development. These experiments were done during comparing interneuron nuclei isolated at P7 with those from P13, both stages at which these neurons are already postmitotic.

Although we thank Reviewer 1 for stimulating us to more accurately discuss the three papers cited above, we do not agree with the statement that postmitotic demethylation “was also shown in PMID 32589877”. The paper in this citation “An Activity-Mediated Transition in Transcription in Early Postnatal Neurons” (Stroud et al., Neuron, 2020) is a very nice study of enhancer activation and decommissioning in the developing brain. In the relevant section concerning the role of DNA methylation, the authors use Sst and Vip Cre drivers to knockout Dnmt3a in these interneuron types. The result is that the increase in methylation that occurs normally at these sites does not occur in the KO. The fact that there is less methylation in the KO reflects the failure of DNMT3A to methylate these sites, not DNA demethylation. The word “demethylation” does not appear in the paper. While the Stroud et al. study is very well done, it is not directly relevant and we have not cited it.

These discrepancies in the manuscript between the authors' assertions of what these studies showed and the published data need to be corrected to be accurate.

It was because the authors suggested the novelty of their study as the first to show Tet-mediated active DNA demethylation in postmitotic neurons that I questioned that novelty. As the literature review above shows, this manuscript is not the first to show Tet-mediated active DNA demethylation in postmitotic neurons.

However, this manuscript does do a particularly nice job of it. I think the novelty of the study lies in linking the molecular characterization of PC chromatin regulation during development with the biology of this cell population. With this in mind, I agree with the authors that the body of the data and the analysis do make a significant new addition to the literature that will be of broad interest.

To address these comments, we have edited the manuscript to state accurately what has been established in the literature. My strict technical interpretation of the cited papers is that the caveats discussed above prevent a definitive statement that DNA demethylation occurs in postmitotic neurons. This is the reason we went to great lengths to obtain precise data using the most advanced technology (oxBS) that can distinguish 5mC from 5hmC, and why we constructed PC specific triple knockout lines that are activated at P7 to complete this study. we understand, however, that we have been too strident and we have tried to edit the manuscript according to the comments of Reviewer 1.

2. Depth of the analysis linking Tet-mediated DNA demethylation with PC maturation

In this revision the authors have significantly expanding and clarified the section on the molecular sequencing from Tet_1/2/3 cKO mice, strengthening the impact of the data. However, even though this is a revised manuscript there are some important omissions and errors in this section that undermine the power of the data to support the model.

– Line 193, 5hmc continues to increase over highly expressed genes – where is that evidence?

This is shown in Figure 2S1E. As a result of an editorial error, we did not include the figure citation in the manuscript, and the text has now been edited to correct this error.

– Line 202 "Furthermore, 5hmCG accumulated over the genes bodies early in development is not removed (Figure 2C)," actually what this figure shows is that for the downregulated genes there is an increase in 5hmCG over the gene bodies between P7 and adult, so it is not only not lost, it is gained. It looks no different for the downregulated genes than the upregulated genes.

We agree with the reviewer, and we have edited the manuscript to state that 5hmC continues to increase over these genes.

– Where is the data that goes with line 372 – growth of the DMV preceded by 5hmC and starts at 5' end

These two points are illustrated in (Figure 4H, I). In panel 4H, one can clearly see in the IGV panel that the level of 5hmC at significantly greater at P7 than in the adult over nearly the entire gene body for GPR63, and that this is also the case at the 5’end of Grid2. This is more difficult to appreciate for Grid2 because it is such a large gene and the demethylation occurs only over the first 50-100 kb of the gene. The metagene plots in Figure 4I also indicate that for this whole class of genes, the 5hmC levels are high at P7 and much less in the adult indicating that 5hmC accumulated during development is lost as the cells differentiate.

– Line 376 -The text that follows this (to line 381) is entirely speculative and does not belong in the methods.

Quote in question “These data revealed a large variation in the length of the DMV that does not form in the Pcp2TetTKO PCs. We believe this reflects a complex relationship between the timing at which transcription is initiated, the rate of transcription over the entire gene body, the local activity of Tet oxidase within the gene as differentiation proceeds, and the timing of Tet oxidase loss from each cell following recombination at the Tet1, Tet2, and Tet3 loci. “

We agree with Reviewer 1 that this is speculation. The fact that the sentence starts with “We believe” clearly indicates that this is our conjecture and not a verified result. We see no reason why we can’t let the reader evaluate this idea for themselves, and we would like to leave this in the Results as it is stated.

– Figure 4S2 E-G are not cited in the text.

We made an unfortunate typo while referring to the supplemental figures. Figures 4S2 E-G were incorrectly referred to as 4S1 E-G. This has been corrected in the revision.

Furthermore, one of the important parts of this section regards the relationship of the chromatin changes in the Tet cKO to the gene expression effects and this part would benefit from more discussion. For example, one of the best example genes for Tet mediated demethylation the authors show it Iptr and yet they specifically say this epigenomic change does not influence Iptr expression (which is key since this is the gene product used for PC isolation). Also, it is surprising that in the Tet_1/2/3 cKO mice, expression of genes encoding synaptic proteins and differentiation markers are the major class elevated, not repressed. Might this suggest that the Tets actually slow differentiation rather than promoting it as the text seems to imply? Either would be interesting, but the data shown needs to be reconciled with the statement (line 106) that "maturation in Purkinje Cells…..require continued oxidation of 5mC to 5hmC".

We appreciate this comment and we understand that in the simplest case one might interpret the findings as consistent with slowing differentiation. However, we don’t know whether late expressed genes in PCs may be expressed at higher levels during synaptogenesis than they are once they reach their steady state levels in the adult. We chose the word maturation to reflect this uncertainty. We have also edited the last sentence of this paragraph to state more precisely our conclusion “Taken together, our data demonstrate that active demethylation occurs in select genes in postmitotic neurons, and that 5hmC plays an essential role in refining the transcriptional and epigenetic status of Purkinje cells during the final stages of differentiation.”

Author details

1. Elitsa Stoyanova

   Laboratory of Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, United States

   Contribution

   Conceptualization, Formal analysis, Investigation, Visualization, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6400-6119

2. Michael Riad

   Laboratory of Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, United States

   Contribution

   Formal analysis, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

3. Anjana Rao

   1. Sanford Consortium for Regenerative Medicine, La Jolla, United States
   2. La Jolla Institute for Allergy and Immunology, La Jolla, United States
   3. Department of Pharmacology, University of California San Diego, La Jolla, United States

   Contribution

   Resources, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1870-1775

4. Nathaniel Heintz

   Laboratory of Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, United States

   Contribution

   Conceptualization, Funding acquisition, Resources, Supervision, Writing – original draft, Writing – review and editing

   For correspondence

   heintz@rockefeller.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8874-8704

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