Modulation of Schwann cell homeostasis by the BAP1 deubiquitinase

Schwann cell programming during myelination involves transcriptional networks that activate gene expression but also repress genes that are active in neural crest/embryonic differentiation of Schwann cells. We previously found that a Schwann cell‐specific deletion of the EED subunit of the Polycomb Repressive Complex (PRC2) led to inappropriate activation of many such genes. Moreover, some of these genes become re‐activated in the pro‐regenerative response of Schwann cells to nerve injury, and we found premature activation of the nerve injury program in a Schwann cell‐specific knockout of Eed. Polycomb‐associated histone modifications include H3K27 trimethylation formed by PRC2 and H2AK119 monoubiquitination (H2AK119ub1), deposited by Polycomb repressive complex 1 (PRC1). We recently found dynamic regulation of H2AK119ub1 in Schwann cell genes after injury. Therefore, we hypothesized that H2AK119 deubiquitination modulates the dynamic polycomb repression of genes involved in Schwann cell maturation. To determine the role of H2AK119 deubiquitination, we generated a Schwann cell‐specific knockout of the H2AK119 deubiquitinase Bap1 (BRCA1‐associated protein). We found that loss of Bap1 causes tomacula formation, decreased axon diameters and eventual loss of myelinated axons. The gene expression changes are accompanied by redistribution of H2AK119ub1 and H3K27me3 modifications to extragenic sites throughout the genome. BAP1 interacts with OGT in the PR‐DUB complex, and our data suggest that the PR‐DUB complex plays a multifunctional role in repression of the injury program. Overall, our results indicate Bap1 is required to restrict the spread of polycomb‐associated histone modifications in Schwann cells and to promote myelin homeostasis in peripheral nerve.


| INTRODUCTION
Schwann cells play an important role in the stability, integrity, and function of the peripheral nervous system. In their terminally differentiated state, the myelin formed by Schwann cells enables saltatory conduction (Mirsky & Jessen, 1996), and most cases of inherited neuropathy are caused by impaired Schwann cell function (Nelis et al., 1999;Saporta & Shy, 2013;Stassart et al., 2018). Several studies have identified molecular determinants of a homeostatic phase upon myelin maturation, and several mouse mutants of neuropathyassociated genes have demonstrated progressive myelin deformations in adult nerve that eventually impact peripheral nerve function (Beirowski et al., 2017;Bolino et al., 2016;Cotter et al., 2010;Domènech-Estévez et al., 2016;Goebbels et al., 2012;Golan et al., 2013;He et al., 2018;Pantera et al., 2020;Rosenberg et al., 2018). Loss of myelin homeostasis can be prompted by alterations in key signaling pathways involved in neuregulin regulation, and also has been observed when epigenetic regulators are disrupted.
In previous studies, we tested the role of the Polycomb Repressive Complex 2 (PRC2), which forms repressive histone H3K27 trimethylation (H3K27me3), by creating a Schwann cell-specific knockout of the EED subunit of PRC2. We observed disruptions in myelin stability with a progressive hypermyelination of small caliber axons (Ma et al., 2015). Moreover, gene expression analysis revealed that EED had a role in maintaining repression of a significant proportion of the injury program that is normally activated only after peripheral nerve injury (Ma et al., 2015(Ma et al., , 2016(Ma et al., , 2018. The injuryinduced reprogramming of mature Schwann cells allows transformation to pro-regenerative cells that support growth and survival of axons, clear myelin debris by autophagy/phagocytic responses, and form elongated Bungner bands to pave the way for nerve regeneration, before eventually myelinating any regenerated axons (Jessen & Mirsky, 2019). Several transcription factors (e.g. c-JUN) are important activators of injury response genes (Arthur-Farraj et al., 2012;Jessen & Mirsky, 2019;Ramesh et al., 2022;Wagstaff et al., 2021).
In addition, activation of the injury program was associated with reduction of repressive H3K27me3 on these injury genes (Ma et al., 2015(Ma et al., , 2016. Overall, these studies indicated that Polycomb repression was important to suppress the injury program in mature Schwann cells (Ma et al., 2018). In addition, this provides additional evidence that the homeostatic phase of myelination depends upon repression of JUN and the injury program (Fazal et al., 2017;Kim et al., 2018).
Polycomb repression is established by coordinate actions of PRC2 and PRC1, which catalyzes monoubiquitination of histone H2A (H2AK119ub1) through RING1A/B subunits (Blackledge et al., 2015). The canonical forms of PRC1 complexes are epigenetic readers of histone H3K27me3 formed by PRC2, leading to further repression through histone H2A ubiquitination and chromatin compaction (Tamburri et al., 2020). Accordingly, H2AK119ub1 is often associated with H3K27me3, and we found that H2AK119ub1 also decreases upon activation of polycomb-repressed injury genes (Duong et al., 2021). While PRC1-mediated repression was originally thought to be secondary to PRC2-dependent H3K27me3, there can be independent regulation of PRC1 complexes to establish H2AK119ub1 that triggers H3K27 methylation (Blackledge et al., 2015;Cooper et al., 2016;Li et al., 2010). Therefore, regulation of H2AK119ub1 can play a coequal or even primary role in establishing and maintaining polycomb repression.
The activity of the PRC1 and PRC2 complexes is opposed by epigenetic eraser proteins that can remove polycomb-associated histone modifications, such as the JMJD3/KDM6B demethylase for H3K27, and the BAP1 deubiquitinase for H2AK119 . We have previously tested the role of H3K27 demethylases in Schwann cell gene regulation. The Schwann cell-specific knockout of Jmjd3/Kdm6b and Utx/Kdm6a had little effect on Schwann cell development, other than some modest perturbations in PRC2-regulated genes. In addition, nerve injury responses in Schwann cells were temporally delayed but ultimately little affected at later stages of nerve injury (Duong et al., 2021).
We therefore decided to determine the role of the BAP1 deubiquitinase in Schwann cells. BAP1 is part of the PR-DUB (Polycomb Repressive-Deubiquitinase) complex, containing also O-Linked N-Acetylglucosamine (GlcNAc) Transferase (OGT) (Dey et al., 2012;Hauri et al., 2016). Based on the loss of H2AK119ub1 on injuryinduced genes (Duong et al., 2021), we originally hypothesized that BAP1 may regulate polycomb repression of injury genes. Accordingly, we created a Schwann cell-specific deletion of the H2AK119 deubiquitinase, BAP1 (Scheuermann et al., 2010). Loss of BAP1 led to impaired myelin homeostasis and premature activation of the injury program, suggesting that writers and erasers of polycomb repression play mutually reinforcing roles in gene regulation and myelin homeostasis.

| Mouse Colony
Animal experiments were performed according to protocols approved by the University of Wisconsin, Madison. Bap1 floxed mice [Jackson Laboratories 031874] were maintained on the C57BL/6 genetic background and mated to mP0TOTA-Cre (Mpz-cre) (Feltri et al., 1999

| Morphometric quantification of myelination
Freshly dissected sciatic nerves were immersion fixed in a solution of 2.5% glutaraldehyde, 2.0% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4, overnight at 4 . The nerves were then post-fixed in 1% osmium tetroxide in the same buffer for 2 h at room temperature. The nerves were dehydrated in a graded ethanol series, and then further dehydrated in propylene oxide and embedded in Epon resin.
Ultrathin transverse sections were either contrasted with Reynolds lead citrate and 8% uranyl acetate in 50% ethanol or stained with toluidine blue. Images were obtained either with a Philips CM120 electron microscope with an AMT BioSprint side-mounted digital camera at the UW Medical School Electron Microscope Facility or with Nikon Ti2 microscopy. Quantification for g ratios was performed using NIS-Elements 4.0. Three mice per genotype were analyzed, and statistical analyses were evaluated using unpaired t-tests. Axons with myelination abnormalities (tomacula, in/outfolding) were not selected for g ratio quantitation.

| Immunofluorescence
Freshly dissected nerves were embedded in Tissue-Tek OCT compound (Sakura Finetek) and snap frozen with liquid nitrogen.
Longitudinal or transverse cryostat sections (10 μm) were air-dried for 5 min and fixed in 4% paraformaldehyde for 10 min. The sections were then blocked in PBS containing 5% donkey serum/1% BSA/0.5% Triton-X 100 for 1 hr at room temperature. Primary antibody incubation was performed overnight at 4 in PBS containing 5% donkey serum/1% BSA/1% Triton-X 100 and secondary incubation was performed in PBS at room temperature for 1 hr. Hoechst 33342 (1:5000 in PBS, Sigma) was applied to stain nuclei for 1 min.
Three 4 min washes were performed in PBS after fixation and blocking, and in PBS containing 0.1% Tween20 after primary antibody incubation and nuclear staining. After coverslips were mounted using Fluoromount-G™ (SouthernBiotech), sections were examined on a Nikon A1R confocal and quantitated by both Columbus imaging software and manual curation.

| Western blot
Freshly dissected nerves were snap frozen with liquid nitrogen and crushed. The nerves were then homogenized in lysis buffer (50 mM Tris-HCl) at pH 6.8, 10% glycerol, 2% SDS, 10% β-mercaptoethanol, 50 mM NaF, 1 mM Na3VO4 and Protease Inhibitor Cocktail (Sigma, P8340) using a motorized pellet pestle. Cells in culture were homogenized in 3x lysis buffer. After a 15 min incubation in ice, lysates were boiled at 95 for 3 min and centrifuged at 4 for 15 min. Subsequently, supernatants were collected and subjected to SDS-PAGE.
After transfer to polyvinylidene fluoride membrane, proteins were blocked in TBST containing 5% nonfat dry milk for 1 hr at room temperature. Primary and Secondary antibody incubations were performed in TBST containing 5% BSA (Sigma, A7906) at 4 for overnight and at room temperature for 1 hr, respectively. Three 5 min-washes were performed in TBST after the incubations. The membranes were scanned and quantitated with the Odyssey Infrared Imaging System (Li-Cor Biosciences). Antibodies are listed in Table 1.

| Quantitative RT-PCR
RNA was isolated from sciatic nerves using the Trizol/chloroform RNA extraction protocol following purification with RNA Clean and Concentrator kit (Zymo). To prepare cDNA, 0.5-1 μg of total RNA was used from each sample. qRT-PCR and data analysis were performed as described previously . qPCR was performed in duplicate per sample. Primers for Bap1 expression were: CTGGGCTCTCGTTGAACTACTCA and CCTCATCAGGGCCCTTCAC.

| RNA seq
Using control and Bap1 cKO mice at 8 weeks of age (n = 6/group), RNA was purified from sciatic nerves, and 1000 ng total RNA was sent to Genewiz (South Plainfield, NJ) for library preparation after PolyA selection and Illumina sequencing (Illumina HiSeq 2x150bp).
Reads were aligned to GRCm38/mm10 genome using the STAR aligner (Dobin et al., 2013). Data were analyzed using DESeq2 (Anders et al., 2013) to determine differentially regulated genes T A B L E 1 List of antibodies used.

| Chromatin immunoprecipitation
Six freshly dissected mouse sciatic nerves per condition (Bap1 fl/fl/ Mpz-cre and Bap1 fl/fl) were minced in 1% formaldehyde for 10 min then quenched for 10 min with 0.125 M glycine. Samples were incubated in LB1 for 10 min, washed with LB2, then bead-homogenized using the Next Advance Bullet Blender for 5 min with speed 12 . Samples were centrifuged to pull down nuclei-containing

| Bioinformatic analysis
The ChIP-seq data sets from mouse and similar data sets (GSE159265) of H2AK119ub1 in rat peripheral nerve (Duong et al., 2021) were used to create the heat maps for the deregulated genes. The data matrix for each heatmap and read density plots were generated using the EAseq suite (Lerdrup et al., 2016) from filtered bam files based on ChIP-seq data sets and then clustered and visualized. Called peak annotations were created using ChIPSeeker (Yu et al., 2015). The gene ontology enrichment analyses were conducted with PANTHER and GSEA software to identify the enriched biological processes based on respective upregulated and downregulated lists of Bap1 cKO genes. (Mi et al., 2021). For comparative analysis in transcriptomes, the statistical significance for the overlap between potential gene lists was tested at Nemaotes bioinformatic tools which utilizes the Fisher's Exact Test.

| Conditional inactivation of Bap1 in Schwann cells
To test the role of BAP1 in regulating Schwann cell development and maturation, we developed a Schwann cell specific conditional knockout (cKO) of Bap1 using the Mpz-cre driver, which is active in embryonic Schwann cell development at E13.5-14.5 (Feltri et al., 1999). The . The qRT-PCR analysis of RNA extracted from the control and BAP1 cKO sciatic nerves was performed using primers within the excised regions. P < .05 unpaired t-test, n = 3/group. (c). Immunofluorescence analysis of the longitudinal sections from the wildtype and BAP1 cKO sciatic nerves was performed using the indicated antibodies. n = 3 for control and n = 3 for cKO. Error bars indicate standard deviation.
the deleted region, showing loss of Bap1 expression in the conditional knockout (Figure 1 a, b). The decrement in Bap1 expression is consistent with the proportion of Schwann cells found in sciatic nerve. We also further confirmed the loss of BAP1 protein through immunostaining with an antibody targeting an epitope in the C-terminus of BAP1.
Coexpression of BAP1 and SOX10, a marker of Schwann cells (Britsch et al., 2001), was absent in the knockout whereas there are cells positive for such markers in control samples (Figure 1 c, triangles).

| Myelin abnormalities in the Bap1 knockout
Mice with a Schwann cell-specific knockout of Bap1 were overtly normal with no evident motor impairment after weaning. To determine if Myelin thickness was assessed using the g-ratio, which is the axon diameter divided by outer diameter of myelin sheath . The overall g-ratio was not significantly different in the Bap1 cKO (Figure 2 d). A binned analysis also showed no significant change in g ratios in any size range (not shown). However, the distribution of axon diameters was skewed with a notable deficit of the higher caliber axons (>4 micron) and an elevated number of smaller diameter axons (Figure 2 e). The average median axon diameter for control was 2.97 microns (n = 3), compared to 2.5 for the Bap1 cKO (p = .03, one-tailed t-test). We separately tested the overall distribution of axons in control versus Bap1 cKO using the Mann-Whitney test, which yields a highly significant change in distribution (p < 10 À14 ), although the effect size is small.
We also assessed femoral motor nerve at 4 months.

| Polycomb-associated histone modifications in BAP1-regulated genes
To determine if BAP1 regulates polycomb histone modifications in Schwann cells, we also measured the levels of H2AK119ub1 by Western blot of sciatic nerve of the Bap1 conditional knockout and found significantly increased levels of both histone marks (Figure 5a,b).
F I G U R E 4 RNA-seq analysis of Bap1 cKO mice. (a). RNA-seq analysis was performed in 6 week old Bap1 cKO and littermate controls (n = 6/ genotype). Volcano plots indicate increased and decreased genes in the Bap1 cKO relative to control after filtering for >2-fold change and <0.05 p-value. (b). GSEA enrichment plots summarize some of the major biological and cellular processes that are affected in the Bap1 cKO.
Elevated H2AK119ub1 is consistent with the loss of H2A deubiquitinase activity, and increased levels of H3K27me3 likely reflects that PRC1 activity can stimulate H3K27me3 deposition by PRC2 (Cooper et al., 2016;Kalb et al., 2014;Tavares et al., 2012).
Other studies have determined that BAP1 regulates the distribution of polycomb histone modifications in studies of cultured cells Fursova et al., 2021), but there has been limited analysis of BAP1 function in specific cell types in vivo. We then determined the genomic distribution of H2AK119ub1 and H3K27me3 in the control and Bap1 conditional knockout mice, as we had done previously in rat sciatic nerve (Duong et al., 2021;Ma et al., 2015Ma et al., , 2016Ma et al., , 2018. Peak calling for H2AK119ub1 in the control sample identified >35,000 peaks, and the heat maps over these peaks showed a We wanted to determine if deregulated genes in the Bap1 cKO from the RNA-seq analysis were associated with polycomb repression, and more than a third of the upregulated genes are associated with H3K27me3 and/or H2AK119ub1 peaks. Specifically, at least 216 upregulated genes in the Bap1 cKO are associated with both or either of histone marks, which includes many key nerve injury genes (e.g. Shh, Gdnf, and Runx2) which had been found to be regulated by Eed (Duong et al., 2021;Ma et al., 2016Ma et al., , 2018

| BAP1 regulation of nerve injury genes
Within the upregulated dataset, we observed several significant nerve injury genes that were prematurely activated in our analysis of PRC2 function in the Eed cKO (Ma et al., 2018), including Shh, Gdnf, Fgf5, Hmga2, Vgf, and Igfbp2. Upregulated genes include growth factors (Neuregulin 1 and Betacellulin) (Newbern & Birchmeier, 2010), cytokines (Cxcl10), and transcription factors (Tfap2a, Runx2, and Pou3f1) Ma et al., 2015;Quintes et al., 2016). Since our previous studies had indicated that loss of EED led to upregulation of a significant proportion of the nerve injury program in Schwann cells (Ma et al., 2015(Ma et al., , 2016(Ma et al., , 2018, we focused on the overlap between the nerve injury genes and the polycomb histone marks: H3K27me3 and H2AK119ub1 (Figure 7 a) (Boyd & Gordon, 2003;Hashimoto et al., 2008). In contrast, only 10 injuryinduced genes associated with either or both histone marks are downregulated in the Bap1 cKO (Figure 7b). Figure 7C shows the peaks of F I G U R E 6 The H3K27me3 histone modification is redistributed similar to H2AK119ub1 in the Bap1 knockout. (a). ChIPseq analysis of H3K27me3 was performed in control and Bap1 cKO sciatic nerve, and heatmaps of wildtype and knockout H3K27me3 reads centered on wildtype peaks show the loss of H3K27me3 at these sites in Bap1 cKO nerve. (b). Peak calling in the Bap1 cKO ChIP-seq analysis of H3K27me3 identified an increased number of peaks compared to control. Heatmaps of wildtype and knockout H3K27me3 reads centered on Bap1 cKO peaks show increased read density of H3K27me3 in the expanded peak set compared to control.
F I G U R E 7 Association of nerve injury genes with polycombassociated histone modifications. (a). The pie chart indicates injuryinduced genes in sciatic nerve as determined by RNA-seq analysis. The proportion of these genes associated with either or both polycomb-associated histone modifications (H2AK119ub1 and/or H3K27me3) is indicated by shading. (b). The overlap of injuryinduced genes and those genes with altered regulation in the Bap1 cKO is shown. (c). Profiles of H2AK119ub1 from the ChIP-seq analysis of control and Bap1 cKO nerve show localization at the transcription start sites for two injury-induced genes: Gdnf and Nrg1 type 1.
H2AK119ub1 in the Gdnf and Nrg1 type 1 promoters, which are diminished in the Bap1 cKO.

| Comparison of the roles of BAP1 and OGT
BAP1 interacts with OGT as subunits of the PR-DUB complex (Dey et al., 2012;Hauri et al., 2016), and a Schwann cell-specific Ogt KO mouse model exhibited deficient remyelination after injury that was traced to the premature activation of JUN in uninjured nerve (Kim et al., 2016(Kim et al., , 2018. sis of the Bap1 knockout. Therefore, we examined the JUN by western blot but observed no significant difference in JUN expression between genotypes at 6 weeks of age (Figure 8a,b).
It is still possible that JUN activity is stimulated post-transcriptionally, and RNA-seq indicates that Fos, a component of AP-1, is upregulated 2.8-fold. Therefore, we compared the RNA seq profile of the BAP1 cKO to RNA-seq analysis of a mouse model with Schwann cell-specific JUN overexpression (Fazal et al., 2017). Using this data set, there are only 11 common genes that are upregulated in both BAP1 cKO and the JUN overexpression model, such as Shh, S100a4, Btc, and Cxcl10 (Supplementary Table 5). The overlap between upregulated Bap1 cKO and c-JUN cKO genes is significant (p < 2 e-10).
While there is overlapping regulation of injury genes by JUN and polycomb repression, the evidence indicates that they also play independent roles since we do not see induction of the entire JUN target gene network in Schwann cells. Therefore, we conclude that BAP1's active role in regulating nerve injury genes is not mediated by JUN.
There is also an overlap of JUN-regulated genes and those that were found to be deregulated in the EED cKO, but we also did not detect measurable induction of JUN in the EED cKO (Ma et al., 2018).

| DISCUSSION
Since premature activation of the Schwann cell injury program has been shown to adversely impact the formation of stable myelin (Fazal et al., 2017;Kim et al., 2018), several mechanisms have been identified that repress injury genes in mature Schwann cells. One of these mechanisms, Polycomb repression, is regulated by diverse complexes that impinge on this type of regulation. In particular, there are multiple assemblies of PRC2 and PRC1 complexes that can play unique roles in establishing polycomb repression in different genes Tamburri et al., 2021). Our previous study of the Eed knockout in Schwann cells revealed roles in the regulation of myelin homeostasis and also in repression of the injury program (Ma et al., 2015(Ma et al., , 2016(Ma et al., , 2018. PRC2 regulation in Schwann cells has been tied to interactions with CTCF and the ACTL6A-containing BAF complex (Park et al., 2022;Wang et al., 2020) and loss of PRC2 regulation is a critical step in the formation of malignant peripheral nerve sheath tumors in Neurofibromatosis (Ma et al., 2018;Sohier et al., 2017). However, PRC2 regulation is intertwined in several respects with PRC1, and we had found changes in PRC1-mediated H2A ubiquitination in Schwann cells after injury (Duong et al., 2021).
To understand how dynamics of H2AK119 ubiquitination affect polycomb repression in Schwann cells, we focused on the BAP1 deubiquitinase, which is a component of PR-DUB complex that also includes other subunits like OGT and ASXL1/2 (Campagne et al., 2019;Dey et al., 2012). Loss of BAP1 in Schwann cells causes an abnormal myelin morphology that is evident at 6 weeks. Specifically, there are increased levels of tomacula and abnormal myelin, and some large caliber axons have thinner myelin in the mutant mouse, although the overall g ratio is unaffected at this time point. Analysis of femoral motor nerve at 4 months showed a more severe phenotype, with a shift of axon distribution to smaller calibers with thicker myelin, accompanied by a reduction in the total number of myelinated axons.
Overall, these phenotypes are similar to those seen in the Schwann cell-specific knockout of Eed (Ma et al., 2015), although somewhat more severe, indicating an important role of BAP1 in myelin homeostasis.
An RNA-seq analysis of sciatic nerve revealed significant changes in gene regulation. Importantly, we found that several key nerve injury genes are prematurely activated in Bap1 cKO mice including Shh, Gdnf, Runx2, Hmga2, Cdkn2a, and Fgf5, which were also activated in the Eed ckO (Ma et al., 2018), but this analysis revealed a broader range of polycomb-repressed genes, many of which are associated with H2AK119 ubiquitination. The gene expression changes are similar in several respects with previous analysis of a Schwann cell knockout of OGT, which interacts with BAP1 in the PR-DUB complex. The Schwann cell-specific Ogt-cKO leads to elevated JUN activity, resulting in the premature activation of nerve injury genes and a significant degree of tomacula and thinner myelination of larger caliber axons.
Specifically, OGT suppresses the nerve injury response by catalyzing O-glcnacylation of the JUN protein (Kim et al., 2016(Kim et al., , 2018, and one of the striking findings of the OGT analysis was that the failed regeneration after injury could be rescued by deletion of one allele of Jun (Kim et al., 2018).
Since BAP1 and OGT are part of the PR-DUB complex, it was possible that loss of Bap1 could phenocopy the myelination defects seen in Ogt-KO (at 3 months), and the myelin defects showed some similarities as noted above. A comparison of the RNA-seq data sets revealed that 153 out of 708 upregulated genes in Bap1 cKO overlaps with those of Ogt-KO genes suggesting that there is some level of functional overlap, but there are also quite significant differences in the transcriptomic effect of BAP1 loss. Some of these 153 genes are Schwann cell nerve injury genes, which could explain the abnormal myelination in Bap1 cKO mouse model. However, Jun was only slightly elevated (1.7-fold) in the RNA-seq analysis. Moreover, we did not observe any change in JUN protein in the Bap1 cKO. Using Ogt-KO RNA seq data (Kim et al., 2018), there were 11 matches out of 28 upregulated JUN target genes with Bap1 cKO genes. Therefore, we conclude that BAP1 and OGT appear to play overlapping but functionally distinct roles within a multifunctional PR-DUB complex by maintaining polycomb repression of the nerve injury program and inhibiting JUN, respectively. These roles are mutually reinforcing since there is considerable overlap of JUN-regulated and polycombregulated genes in the nerve injury response (Ma et al., 2018). Accordingly, OGT is less tightly associated with BAP1 compared to other PR-DUB subunits and can participate in other complexes (Campagne et al., 2019;Hauri et al., 2016).
Several of the deregulated genes could cause the myelination defects in Bap1 mutant Schwann cells. One gene that was derepressed in both the Eed and Bap1 cKO is IGF-binding protein 2 (Igfbp2), which was shown to increase PI3 kinase signaling (Ma et al., 2015). Another leading candidate is the Neuregulin 1 type I (Nrg1) gene, since previous studies have found that efficient remyelination of injured nerves requires the de novo activation of type I NRG1 in denervated Schwann cells (Stassart et al., 2013). We had found that H3K27me3 mediates repression of Nrg1 and there is increased expression of the type I Nrg1 transcript in the uninjured Eed cKO nerves (Ma et al., 2018). Our data also indicate the presence of H2AK119ub1 on the Nrg1 type 1 promoter ( Figure 7). A recent study showed that overexpression of NRG1 type 1 is responsible for hypermyelination of small caliber axons and the onion bulb pathology that has been observed in CMT1A neuropathy (Fledrich et al., 2019). Another potential mechanism could involve BAP1 regulation of the PTEN lipid phosphatase, since a Schwann cellspecific deletion of Pten caused hypermyelination (Cotter et al., 2010;Goebbels et al., 2010). It has been reported that BAP1 could repress Pten at the transcriptional and post-transcriptional levels (Cao et al., 2020;Chen et al., 2021). However, Pten levels were not elevated in our RNA-seq analysis and Western blot showed no change in PTEN protein levels (data not shown).
Since PRC1 and PRC2 regulation are connected at multiple levels, we compared the deregulated genes with those obtained in our previous RNA-seq profiling of a Schwann cell-specific deletion of Eed (Ma et al., 2018). Although we had speculated that deletion of Bap1 could result in strengthened Polycomb repression, we observed derepression of many polycomb-regulated genes; 105 upregulated Bap1-KO genes are in common with those upregulated in the Eed conditional knockout (Supplementary Table 4). Additionally, there are 52 downregulated genes that are regulated by EED. It was unexpected that deletion of the BAP1 deubiquitinase as a polycomb modification eraser would have a similar phenotype and overlapping gene expression changes with inhibition of the PRC2 writer complex in the Eed cKO (Ma et al., 2015). Several cell culture studies have characterized the role of BAP1 and divergent results have been obtained. In some cases, loss of BAP1 leads to increased polycomb repression which is associated with increased levels of H2AK119ub1 (Campagne et al., 2019;LaFave et al., 2015). In contrast, other studies have shown that loss of BAP1 leads to loss of polycomb repression Fursova et al., 2021). This has been attributed to a function of BAP1 to safeguard transcriptionally silent genes by preventing accumulation of extragenic H2AK119 and H3K27me3 modifications that would titrate out polycomb repression if allowed to spread Fursova et al., 2021). Accordingly, the Drosophila homolog of BAP1, calypso, was originally classified as a polycomb group gene (Scheuermann et al., 2010). These mechanisms are highly relevant to the observed mutation of BAP1 in several types of cancer since both gain-and loss-of-function models of polycomb repression can contribute to malignancy Daou et al., 2018;LaFave et al., 2015;Tamburri et al., 2021). In our case, we found both increased levels of H2AK119ub1 and H3K27me3 in peripheral nerve, and also a larger number of called peaks for H2AK119ub1 and H3K27me3 in our ChIP-seq analysis of Bap1 mutant nerve. Our study is unique in that we have analyzed the effect of Bap1 on polycomb repression in post-mitotic Schwann cells in vivo compared to the previous studies in cell culture models. However, based on the earlier studies Fursova et al., 2021), our data are consistent with a model in which derepression of some polycombassociated genes is due to inappropriate accumulation of H2AK119ub1 and H3K27me3 at new sites across the genome.
Numerous pro-regenerative genes in Schwann cells are repressed by both H2AK119ub1 and H3K27me3 and their basal expression can be virtually undetectable in intact, healthy peripheral nerve. Previous studies had suggested that the removal of polycomb repression is required for induction of nerve injury genes such as Shh and Gdnf (Ma et al., 2015(Ma et al., , 2016(Ma et al., , 2018. PRC2 is commonly known to work cooperatively with PRC1, but recent studies had indicated that PRC1-mediated repression can be mediated independently of PRC2 . Moreover, it has emerged that phenotypes of PRC1-related phenotypes are often more severe than PRC2 mutants (Cohen et al., 2020), and the Bap1 knockout phenotype is more severe than either the Eed or the Jmjd3/Utx knockouts in Schwann cells (Duong et al., 2021). Therefore, future experiments will be used to determine if deubiquitinase(s) play a role in regulation of injury genes in Schwann cells.

AUTHOR CONTRIBUTIONS
Phu Duong planned experiments, bred the mice, performed analysis of RNA-seq and histology data, and co-wrote the manuscript. Raghu

Ramesh performed and analyzed ChIP-seq experiments. Andrew
Schneider maintained the mouse colony and analyzed histology.
Seongsik Won performed Western blot analysis. Aaron Cooper performed histology analysis. John Svaren planned experiments, analyzed data and co-wrote the manuscript.