Canonical and non-canonical PRC1 differentially contribute to the regulation of neural stem cell fate

Neocortex development is characterized by sequential phases of neural progenitor cell (NPC) expansion, neurogenesis and gliogenesis. Polycomb -mediated epigenetic mechanisms are known to play important roles in regulating the lineage potential of NPCs during development. The composition of Polycomb Repressive Complex 1 (PRC1) is highly diverse in mammals and was hypothesized to contribute to context-specific regulation of cell fate. Here, we have performed side- by-side comparison of the role of canonical PRC 1.2/1.4 and non -canonical PRC1.3/1.5, all of which are expressed in the developing neocortex, in NSC proliferation and differentiation. We found that the deletion of Pcgf2/4 in NSCs led to a strong reduction in proliferation and to altered lineage fate, both during the neurogenic and gliogenic phase, whereas Pcgf3/5 played a minor role. Mechanistically, genes encoding stem cell and neurogenic factors were bound by PRC1 and differentially expressed upon Pcgf2/4 deletion. Thus, rather than different PRC1 sub -complexes contributing to different phases of neural development, we found that canonical PRC1 played a more significant role in NSC regulation during proliferative, neurogenic and gliogenic phases compared to non- canonical PRC1.

During the development of the neocortex, stem and progenitor cells initially proliferate, before sequentially giv ing rise to neurons destined to different cortical layers and subsequently generate astrocytes and oligodendrocytes (Lodato & Arlotta, 2015; Qian et al , 2000) . Precise spatial and temporal regulation of NPC proliferation and .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 7, 2024. ; https://doi.org/10.1101/2024.08.07.606990doi: bioRxiv preprint 2 differentiation is key for the proper formation of the intricate structure of the neocortex. Transcription factors and epigenetic mechanisms play important roles in orchestrating dynamic changes in gene expression that underlie coordinated neural differentiation programs in the developing neocortex (Albert & Huttner, 2018; Desai & Pethe, 2020; Tsuboi et al, 2019). Chromatin modifiers of the Trithorax and Polycomb groups maintain active and repressed gene activity states during embryonic development (Piunti & Shilatifard, 2016; Ringrose & Paro, 2004; Schuettengruber et al , 2017) . Polycomb proteins assemble into two major complexes, PRC1 and PRC2, which catalyse mono- ubiquitination of histone 2A lysine 119 (H2AK119ub1) and tri-methylation of histone 3 lysine 27 (H3K27me3), respectively. These complexes are important determinants of the ability of NPCs to either proliferate or to give rise to neurons or glial cells (Hirabayashi et al , 2009; Tyssowski et al , 2014) , and mutations in Polycomb components were reported to cause neurodevelopmental disorders (Bölicke & Albert, 2022; Mastrototaro et al, 2017; Pierce et al, 2018). Specific deletion of the PRC2 histone methyltransferase Ezh2 in the early developing neocortex causes an up-regulation of gene expression and a shift of apical radial glia fate from self-renewal to differentiation (Pereira et al, 2010), reducing the neuronal output and leading to a substantially smaller neocortex. Moreover, deletion of Ring1b, an integral component of PRC1, during the neurogenic phase results in altered neuronal subtype specification (Morimoto-Suzki et al, 2014). In this context , the E3 ubiquitin ligase activity of Ring1b was found to be necessary for the temporary repression of key neuronal genes in neurogenic NPCs (Tsuboi et al , 2018) . These data indicate that Polycomb complexes control important aspects of corticogenesis. Epigenome profiling in specific neural cell populations isolated from the developing mouse neocortex has revealed dynamic changes in H3K4me3 and H3K27me3 during neocortical lineage specification (Albert et al, 2017). An important question is h ow Polycomb target gene specificity is achieved in different neocortical cell types . One way to dynamically control Polycomb function and targeting is by altering the composition of Polycomb complexes, which in mammals is highly diverse, enabling the assembly of various sub-complexes with different functionalities (Kim & Kingston, 2020; Luis et al, 2012; Tsuboi et al., 2019). During neocortex development, chromatin remodelers of the chromodomain helicase DNA -binding (Chd) family, which interact with PRC2 complexes, show differential expression. Whereas Chd5 is expressed in neurons and controls neuronal differentiation (Egan et al, 2013), Chd4 is expressed in neural progenitor cells during early neurogenesis , where it functions in the inhibition of astroglial differentiation (Sparmann et al , 2013) . Such a switch in subunit composition may contribute to the re-targeting of PRC2 during neocortex development. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 7, 2024. ; https://doi.org/10.1101/2024.08.07.606990doi: bioRxiv preprint 3 PRC1 complexes are classified as canonical or non -canonical, depending on which of the complex members are included, with all complexes containing the central Ring1a/b E3 ubiquitin ligase core (Blackledge & Klose, 2021; Piunti & Shilatifard, 2016; Tsuboi et al., 2019). Canonical PRC1 complexes (PRC1.2/1.4) contain Pcgf2/4, one of three polyhomeotic (Phc) proteins and one of five chromobox (Cbx) proteins that recognize H3K27me3 mediated by PRC2. Non -canonical PRC1 is targeted to chromatin independently of H3K27me3 and is characterized by the inclusion of Pcgf1 (PRC1.1), Pcgf3/5 (PRC1.3/1.5) or Pcgf6 (PRC1.6) (Blackledge & Klose, 2021) , even though non-canonical PRC1.2/1.4 lacking Cbx and Phc has also been described (Gao et al, 2012). Non-canonical PRC1 has high ubiquitin ligase activity, whereas canonical PRC1 was reported to promote higher order chromatin structures, but to display low ligase activity and low contribution to target gene repression (Blackledge & Klose, 2021; Fursova et al, 2019). In embryonic stem cells, the interchange of Cbx family protein s in PRC1 (Kim & Kingston, 2020) has been reported to modulate the balance between self-renewal and lineage commitment (Morey et al, 2012; O'Loghlen et al, 2012; Santanach et al, 2017), and different Cbx paralogs are required for different cell lineages (Klauke et al, 2013; Luis et al, 2011). Likewise, Pcgf homologs were suggested to promote context - and stage-specific functions during differentiation and development (Kloet et al , 2016; Morey et al, 2015). While the canonical PRC1 components Pcgf2 and Pcgf4 (Akasaka et al, 2001; Fasano et al, 2007; He et al, 2009; Leung et al, 2004; Molofsky et al, 2005; Molofsky et al, 2003; Zencak et al, 2005) as well as the non -canonical PRC1 components Pcgf3 and Pcgf5 (Gao et al, 2014; Meng et al, 2020; Yao et al, 2018) have been implicated in neural differentiation and brain development, here we set out to perform a systemic comparative analysis of the role of different PRC1 subcomplexes in NSC proliferation and differentiation. Specifically, we delete d canonical ( Pcgf2/4) and non -canonical PRC1 ( Pcgf3/5) in proliferating , neurogenic and gliogenic NSCs to elucidate the function of different PRC1 sub-complexes in key phases of cortical development.

and discussion Pcgf homologs are differentially expressed in the mouse and human developing neocortex To analyse the expression of canonical and non -canonical PRC1 components (Figure 1A) in the human developing neocortex (Figure 1B), we first mined RNA-seq data of microdissected human foetal cortex (Fietz et al, 2012). The core components of PRC1, RING1A and RING1B, showed a fairly uniform distribution across the germinal zones (VZ, ISVZ, OSVZ), which are enriched in NPCs, and the cortical plate, where neurons .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 7, 2024. ; https://doi.org/10.1101/2024.08.07.606990doi: bioRxiv preprint 4 reside (Figure 1C) . This was confirmed by immunohistochemistry of human foetal tissue, in which RING1B and H2AK119ub1 showed a comparable expression across all cortical layers with a minor increase in the ventricular zone (VZ) and cortical plate (CP) (Figure 1D, E). In contrast, PCGF2, PCGF3 and PCGF4 were expressed at higher levels in the CP compared to germinal zones, whereas PCGF5 was specifically enriched in the VZ (Figure 1C –E). These data indicate that PCGF homologs are differentially expressed across neural cell types of the human developing neocortex. Figure 1. PRC1 components are differentially expressed in the human and mouse developing neocortex. (A) Schematic illustration of canonical and non -canonical PRC1 complexes containing the RING1A/B core and either PCGF2/4 or PCGF3/5 subunits, respectively. (B) Schematic illustration of the human developing neocortex, divided into the ventricular zone (VZ), inner subventricular zone (ISVZ), outer subventricular zone (OSVZ), PCGF2 RING1B H2A K119ub1 VZ SVZ IZ CP D Pcgf2 Ring1B H2A K119ub1 H VZSVZIZCP 0.0 0.5 1.5 1.0 H2AK119ub1 2.0 ✱✱ ✱ VZSVZIZCP 0.0 0.5 1.5 1.0 2.0 Pcgf4 Ring1a ✱✱✱ ✱✱✱ 0.00 0.04 0.08 0.12 VZ CP VZ SVZ CP IZ VZ SVZ IZ E14.5 0.00 0.04 0.08 0.12 ✱ ✱✱ Ring1b VZ SVZ CP Expression rel. to ActB 0.00 0.04 0.08 0.12 ✱✱✱ ✱✱ Pcgf2 VZ SVZ CP 0.00 0.04 0.08 0.12 ✱✱✱ ✱✱✱ Pcgf3 VZ SVZ CP Pcgf4 0.00 0.04 0.08 0.12 VZ SVZ CP Pcgf5 ✱✱✱ ✱✱✱ 0.00 0.04 0.08 0.12 VZ SVZ CP CP Figure 1 VZ OSVZ ISVZ IZ CP Canonical Non-canonical PRC1 RING1A/B PCGF1-6 RING1A/B PCGF2/4 RING1A/B PCGF3/5 GW14 ✱✱ ✱ ✱✱ 0.00 0.01 0.02 0.03 0.04 PCGF5 VZISVZOSVZ CP VZSVZ IZ CP 0.0 0.5 1.5 1.0 2.0 2.5 RING1B ✱✱ ✱✱ ✱✱ ✱✱ VZSVZ IZ CP 0.0 0.5 1.5 1.0 2.0 2.5 H2AK119ub1 VZSVZ IZ CP 0.0 0.5 1.5 1.0 2.0 2.5 PCGF2 ✱✱ ✱✱ VZSVZ IZ CP 0.0 0.5 1.5 1.0 2.0 2.5 PCGF4 Intensity rel. to VZIntensity rel. to VZIntensity rel. to VZIntensity rel. to VZ E PCGF4 CTIP2 SOX2 ✱ 0.00 0.01 0.02 0.03 0.04 RING1B VZISVZOSVZ CP ✱ 0.00 0.01 0.02 0.03 0.04 RING1A VZISVZOSVZ CP ✱ ✱ ✱ 0.00 0.01 0.02 0.03 0.04 PCGF2 VZISVZOSVZ CP ✱ ✱ ✱ 0.00 0.01 0.02 0.03 0.04 PCGF4 VZISVZOSVZ CP 0.00 0.01 0.02 0.03 0.04 PCGF3 VZISVZOSVZ CP Expression rel. to ACTB PRC1 expression in the mouse developing neocortex A B C F G VZ IZCP 0.0 0.5 1.5 1.0 2.0 Pcgf2 Intensity rel. to VZSVZ VZ IZCP 0.0 0.5 1.5 1.0 2.0 Intensity rel. to VZ Ring1B SVZ Ctip2 Sox2 Pcgf4 I SVZ PRC1 expression in the human developing neocortex .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 7, 2024. ; https://doi.org/10.1101/2024.08.07.606990doi: bioRxiv preprint 5 intermediate zone (IZ) and cortical plate (CP). ( C) PRC1 mRNA expression levels in the human developing neocortex at gestation week (GW) 14 analysed by RNA -seq (data from Fietz et al. (2012)), relative to the house keeping gene ACTB. (D) Immunofluorescence for the radial glia marker SOX2, neuronal marker CTIP2, and PRC1- related RING1B, H2AK119ub1, PCGF2 and PCGF4 of human foetal tissue (GW12/13). (E) Quantifications of mean intensity per cell in the indicated zones of human foetal tissue, relative to the intensity in the VZ. ( F) Schematic illustration of the mouse developing neocortex. ( G) PRC1 mRNA expression levels in the mouse developing neocortex at E14.5 analysed by RNA-seq (data from Fietz et al. (2012)), relative to the house keeping gene Actb. (H) Immunofluorescence of mouse embryonic tissue (E14.5). (I) Quantifications of mean intensity per cell in the indicated zones of mouse embryonic tissue, relative to the intensity in the VZ. Data information: Scale bars, 100 µm. Bar graphs represent mean values. Error bars represent SD; C, D, of 3 tissue samples from different individuals; G, I, of 3 –5 embryos from at least two different litters. *** p < 0.001, ** p < 0.01, * p < 0.05; Tukey’s multiple comparison test. Next, we analysed expression of PRC1 components in the mouse developing neocortex (Figure 1F). Ring1a/b and H2AK119ub1 were uniformly distributed across all zones, with a slight enrichment in the VZ and CP (Figure 1G –I). In analogy to the human developing neocortex, Pcgf2, Pcgf3 and Pcgf4 showed some enrichment in the CP, whereas Pcgf5 was specifically expressed in the VZ (Figure 1G–I), even though the differences were less pronounced in the mouse compared to the human developing neocortex. The results are in line with previous studies noting the high abundance of Pcgf2 and Pcgf4 in NPCs (Tagawa et al, 1990; Zencak et al., 2005), whereas neuronal expression of Pcgf4 was not seen previously (Zencak et al., 2005). Taken together, the canonical PRC1 components Pcgf2/4 were enriched in neurons compared to NPCs, which is interesting giving their previous implication in the regulation of NSC self-renewal and proliferation (Fasano et al., 2007; He et al., 2009; Molofsky et al., 2005; Molofsky et al., 2003; Zencak et al., 2005). The non-canonical PRC1 components Pcgf3 and Pcgf5 showed differential enrichment in neurons versus NPCs, respectively. Canonical and non -canonical PRC1 contribute to different degrees to the regulation of NSC proliferation To systematically compare the role of canonical and non -canonical PRC1 in NSC proliferation, we isolated NPCs from the developing dorsolateral neocortex of E12.5 mouse embryos from either Pcgf2F/F; Pcgf4F/F; Nes::CreERT2/+ or Pcgf3F/F; Pcgf5F/F; Nes::CreERT2/+ mouse lines (Almeida et al, 2017; Fursova et al., 2019; Imayoshi et al, 2006) and induced the deletion of Pcgf genes in vitro by the addition of 4- hydroxytamoxifen (OHT) (Figure 2A). Deletion of Pcgf genes was highly efficient (Figure 2B, C) and resulted in a complete loss of Pcgf3, Pcgf4 and Pcgf5 proteins after 3 days in vitro (DIV), and a reduction in Pcgf2 levels (Figure 2D, E). Following the validation of conditional knockout (cKO) in NSCs, we then asked how deletion of canonical ( Pcgf2/4) and non -canonical ( Pcgf3/5) PRC1 affects NSC proliferation. Whereas deletion of Pcgf2/4 led to an almost complete inability of NSCs .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 7, 2024. ; https://doi.org/10.1101/2024.08.07.606990doi: bioRxiv preprint 6 to proliferate (Figure 2G, H), the deletion of Pcgf3/5 had a more modest effect (Figure 2I, J). Overall, the cell number s were reduced to less than 10% of control after 6 DIV for Pcgf2/4 cKO compared to roughly 70% of control for Pcgf3/5 cKO (Figure 2K), highlighting the differential contribution of canonical and non-canonical PRC1 to the regulation of NSC proliferation. Figure 2. Deletion of Pcgf2/4 and Pcgf3/5 reduces NSC proliferation to different degrees. (A) Schematic of experimental workflow. NSCs were isolated from E12.5 embryos from either Pcgf2F/F; Pcgf4F/F; Nes::CreERT2/+ or Pcgf3F/F; Pcgf5F/F; Nes::CreERT2/+ mouse lines. After one day in vitro (DIV), the deletion of Pcgf components was induced by the administration of 4-hydroxytamoxifen (OHT) and analysis was performed at 3 DIV. (B, C) Genotyping of Pcgf2/4 (B) and Pcgf3/5 (C) floxed (‘F’) and deletion (‘D’) alleles by PCR analysis following treatment of NSC cultures from control (+/+) or experimental (Cre/+) mice with ethanol (‘EtOH’; control) or OHT. (D, E) Immunoblots of protein lysates from the same NSC cultures shown in (B), using anti-Pcgf2, anti- Pcgf4, anti-Pcgf3/5 and anti-Vinculin antibodies. (F) Schematic of experimental workflow. Deletion of Pcgf genes was induced at 1 DIV, 50,000 cells were seeded at 2 DIV and cells were counted at 6 DIV. (G) Brightfield images of Pcgf2F/F; Pcgf4F/F; Nes::CreERT2/+ NSC cultures treated with EtOH or OHT at 3 DIV and 6 DIV. ( H) Quantification of cell numbers at 6 DIV. (I) Brightfield images of Pcgf3F/F; Pcgf5F/F; Nes::CreERT2/+ NSC cultures treated with EtOH or OHT at 3 DIV and 6 DIV. (J) Quantification of cell numbers at 6 DIV. (K) Data from (H, J) plotted for comparison of cell numbers following deletion of either Pcgf2/4 or Pcgf3/5, shown as percentage relative to the EtOH control. Data information: Scale bars, 300 µm. Bar graphs represent mean values. Error bars represent SD. H, J, dots connected by lines represent 4 embryos from at least two independent litters treated with either EtOH or OHT. * p < 0.05; Tukey’s multiple comparison test. These results are in agreement with previous reports on the role of Pcgf4 in regulating NSC self-renewal and proliferation (Fasano et al., 2007; He et al., 2009; Molofsky et B CPcgf4Pcgf2 Pcgf2/4 Pcgf3/5 E12.5 Isolation 3D I V Analysis 1D I V OHT Pcgf2F/F; Pcgf4F/F; Nes::CreERT2/+ Pcgf3F/F; Pcgf5F/F; Nes::CreERT2/+ A cKO validation D E D F Pcgf3 Pcgf5 D F +/+OHT Cre/+EtOH Cre/+OHT +/+OHT Cre/+EtOH Cre/+OHT α Pcgf2 α Pcgf4 α Vinα Vin F D F α Pcgf3/5 α Vin +/+OHT Cre/+EtOH Cre/+OHT +/+OHT Cre/+EtOH Cre/+OHT EtOH OHT 0 5 10 15 20 ✱ Cell numnber 6 DIV [x105] EtOHOHT 0 5 10 15 20 ✱ Cell number 6 DIV [x105] 3 DIV6 DIV 3 DIV6 DIV EtOH OHT EtOH OHT G H I J K Pcgf2/4Pcgf3/5 0 25 50 75 100 125Cell number rel. to EtOH 6 DIV (%) ✱ Pcgf2/4 Pcgf3/5 E12.5 Isolation 6D I V Analysis 1D I V OHT 2D I V Seed Pcgf2F/F; Pcgf4F/F; Nes::CreERT2/+ Pcgf3F/F; Pcgf5F/F; Nes::CreERT2/+ F Proliferation of NSCs Figure 2 Pcgf3 Pcgf5 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 7, 2024. ; https://doi.org/10.1101/2024.08.07.606990doi: bioRxiv preprint 7 al., 2005; Molofsky et al., 2003; Zencak et al., 2005). Pcgf2 was reported to function antagonistically to Pcgf4 by promoting cell senescence through down -regulation of Pcgf4 (Guo et al, 2007). Yet, double knockout of Pcgf2/4 revealed the synergistic effect of both genes, resulting in strongly exacerbated phenotypes compared to single mutant mice (Akasaka et al., 2001). In line with this, deletion of Pcgf2/4 in NSCs resulted in an almost complete loss of the ability of NSCs to proliferate. Moreover, Pcgf3 has previously been implicated in the regulation of tumour cell proliferation (Hu et al , 2021), whereas Pcgf5 and Pcgf3/5 were dispensable for embryonic stem cell self - renewal (Yao et al., 2018; Zhao et al, 2017). Here, we showed that Pcgf3/5 regulate NSC proliferation. In summary, side-by-side comparison of double knockout of different Pcgf homologs revealed a stronger contribution of canonical PRC1 .2/1.4 compared to non -canonical PRC1.3/1.5 to the regulation of NSC proliferation. Canonical, but not non-canonical, PRC1 regulates the differentiation potential of NSCs Next, we aimed to dissect the contribution of canonical and non -canonical PRC1 to NSC differentiation. For this, we made use of the previously described potential of NSCs to maintain their developmental progression in vitro, initially resulting in the production of neurons (neurogenic phase), followed by the generation of astrocytes (gliogenic phase) (Hirabayashi et al., 2009). Deletion of Pcgf2/4 in freshly isolated NSCs, that were induced to differentiate by the withdrawal of growth factors and the addition of serum to the medium, resulted in the generation of more neurons at the expense of oligodendrocytes, leaving the proportion of astrocytes unchanged (Figure 3A–C). In contrast, deletion of Pcgf3/5 did not result in any significant changes in the proportions of differentiated cell types. This suggests that canonical and non-canonical PRC1 complexes differentially contribute to the regulation of NSC fate during the neurogenic phase. To compare the contribution of different PRC1 subcomplexes during the gliogenic phase, we kept the NSC lines in culture for 20 days and then repeated the differentiation experiment (Figure 3F). While cKO of Pcgf2/4 resulted in the generation of more astrocytes compared to control, cKO of Pcgf3/5 again did not affect the differentiation potential of NSCs . This side -by-side comparison highlights the role of canonical PRC1.2/1.4, but not non-canonical PRC1.3/1.5, in determining the linage potential of NSCs during differentiation. Taken together , deletion of Pcgf2/4 resulted in an increased proportion of neurons during the neurogenic phase and of astrocytes during the gliogenic phase. This is in contrast to the deletion of Ring1b, central to all PRC1 complexes, which was shown to .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 7, 2024. ; https://doi.org/10.1101/2024.08.07.606990doi: bioRxiv preprint 8 not affect neuron numbers during the neurogenic phase, but resulted in more neurons during the gliogenic phase (Hirabayashi et al. , 2009) . These results underscore the differential contributions of PRC1 subcomplexes with different subunit composition during neurogenic and gliogenic phases of neural differentiation. Figure 3. Deletion of Pcgf2/4 but not Pcgf3/5 results in altered lineage potential of neurogenic and gliogenic NSCs. (A) Schematic of experimental workflow. Deletion of Pcgf genes was induced at 2DIV concomitant with seeding of NSCs with neurogenic potential and induction of differentiation. Differentiated cells were analysed at 9 DIV. ( B) DAPI staining and immunofluorescence for the pan -neuronal marker Huc/d, the astrocyte marker Gfap and the oligodendrocyte precursor marker Pdgfrα after 7 days of differentiation of Pcgf2F/F; Pcgf4F/F; Nes::CreERT2/+ NSCs. (C) Quantification of the percentage of marker-positive cells of total DAPI-positive cells following deletion of Pcgf2/4. ( D) DAPI staining and immunofluorescence after 7 days of differentiation of Pcgf3F/F; Pcgf5F/F; Figure 3 E12.5 Isolation 9D I V Analysis 2D I V Seed/OHT/Diff Pcgf2F/F; Pcgf4F/F; Nes::CreERT2/+ Pcgf3F/F; Pcgf5F/F; Nes::CreERT2/+ A Differentiation of neurogenic NSCsEtOHOHT EtOHOHT B C D E DAPI Gfap DAPI Huc/d DAPI Pdgfrα DAPI Gfap DAPI Huc/d DAPI Pdgfrα ✱✱ 0 20 40 60 80 100 EtOH %H u c / d +/t o t a lc e l l s OHT ✱ 0 5 10 15 50 100%P d g f rα+/t o t a lc e l l s EtOHOHT 0 5 10 15 50 100%G f a p +/t o t a lc e l l s EtOHOHT 0 20 40 60 80 100%H u c / d +/t o t a lc e l l s EtOHOHT 0 5 10 15 50 100%P d g f rα+/t o t a lc e l l s EtOHOHT 0 5 10 15 50 100%G f a p +/t o t a lc e l l s EtOHOHT Pcgf2/4 Pcgf3/5 27 DIV Analysis 20 DIV Seed/OHT/Diff Pcgf2F/F; Pcgf4F/F; Nes::CreERT2/+ Pcgf3F/F; Pcgf5F/F; Nes::CreERT2/+ F Differentiation of gliogenic NSCsEtOHOHT EtOHOHT G H I J DAPI Huc/d DAPI Gfap DAPI Pdgfrα DAPI Huc/d DAPI Gfap DAPI Pdgfrα 0 20 40 60 80 100%H u c / d +/t o t a lc e l l s EtOHOHT 0 20 40 60 80 100%G f a p +/t o t a lc e l l s EtOHOHT ✱ %P d g f rα+/t o t a lc e l l s EtOHOHT 0 20 40 60 80 100 %H u c / d +/t o t a lc e l l s EtOHOHT 0 20 40 60 80 100 %P d g f rα+/t o t a lc e l l s EtOHOHT 0 20 40 60 80 100 %G f a p +/t o t a lc e l l s EtOHOHT 0 20 40 60 80 100 Pcgf2/4 Pcgf3/5 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 7, 2024. ; https://doi.org/10.1101/2024.08.07.606990doi: bioRxiv preprint 9 Nes::CreERT2/+ NSCs. (E) Quantification of the percentage of marker-positive cells of total DAPI-positive cells following deletion of Pcgf3/5. (F) Schematic of experimental workflow. Deletion of Pcgf genes was induced at 20 DIV in NSCs with gliogenic potential, concomitant with seeding and induction of differentiation. (G) DAPI staining and immunofluorescence for lineage markers. ( H) Quantification of the percentage of marker -positive cells. ( I) DAPI staining and immunofluorescence for lineage markers. (J) Quantification of the percentage of marker-positive cells. Data information: Scale bars, 100 µm. Bar graphs represent mean values. Error bars represent SD; C, E, H, J, of 4–9 embryos from at least two different litters. ** p < 0.01, * p < 0.05; Tukey’s multiple comparison test. Canonical PRC1 regulates the expression of stem cell and neurogenic factors Lastly, to explore the mechanism by which canonical PRC1 regulates NSC fate during differentiation, we performed gene expression analysis by RNA -seq, 4 days after induction of differentiation, following deletion of Pcgf2/4 in neurogenic NSCs (Figure 4A, B). In line with the repressive function of PRC1, we identified 120 genes that were more than 2-fold up-regulated upon deletion of Pcgf2/4 compared to control, whereas only 24 genes were down -regulated (Figure 4C). The differentially expressed genes (DEG) we re characterized by gene ontology (GO) terms related to the molecular functions ‘DNA-binding‘ and ‘E-box-binding’ (Figure 4D) , which is in accordance with hallmarks of Polycomb -mediated regulation through binding of genes encoding key developmental transcription factors (Bernstein et al, 2006; Schuettengruber et al., 2017). Moreover, GO terms related to biological processes included ‘regionalization’ and ‘pattern specification’ (Figure 4E), extending what has been described for other Polycomb proteins during brain development (Albert et al. , 2017; Eto et al , 2020; Hirabayashi et al., 2009). Of the 120 up-regulated genes, the majority (89 genes) were directly bound by Pcgf2 and/or Ring1b in NPCs (Figure 4F) (Kloet et al. , 2016) . Among the direct PRC1 targets that were up -regulated, we found many transcriptions factors (Figure 4G), including Hox genes (Figure 4H), which represent known targets of PRC1 (Akasaka et al., 2001; Kloet et al., 2016). Next, we aimed to specifically explore the expression of factors that may underlie the shifts in NSC lineage potential that we observed in Pcgf2/4 cKO NSCs. We found that several stem cell factors, including Id2, Pax6 and Hes5, showed reduced expression after Pcgf2/4 deletion, whereas neurogenic factors ( Lhx5, Lhx9, Nr4a2) and neuronal maturation genes ( En1, En2, Pitx3) were increased compared to control (Figure 5A – D). These genes were bound by Pcgf2 and/or Ring1b in NPCs (Kloet et al., 2016), suggesting that they may represent direct targets of canonical PRC1 and may contribute to the enhanced neuronal differentiation of neurogenic NSCs after deletion of Pcgf2/4. In contrast, regulators of astrocyte fate, such as Gfap, Hmgn1 and Hmgn2, did not show altered expression (Figure 5E) and were not bound by Polycomb (Albert et al., 2017; Kloet et al. , 2016), which is in agreeme nt with previous reports suggesting that astrocyte-specific genes are regulated by DNA methylation in NPCs (Fan et al, 2005; Hatada et al, 2008). Several genes encoding oligogenic factors (Olig2, Gli2, Sox9) were .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 7, 2024. ; https://doi.org/10.1101/2024.08.07.606990doi: bioRxiv preprint 10 directly bound by Pcgf2 and/or Ring1b in NPCs (Kloet et al., 2016) and showed a trend for reduced expression, even though not significantly (Figure 5F). Figure 4. Deletion of Pcgf2/4 results in derepression of PRC1 target genes linked to fate specification. (A) Schematic of experimental workflow. Deletion of Pcgf2/4 genes was induced at 2 DIV concomitant with seeding of NSCs with neurogenic potential and induction of differentiation. Gene expression was analysed by RNA-seq after 4 days of differentiation at 6 DIV. (B) Hierarchical clustering analysis with the heatmap of the 100 most differentially expressed genes between control (EtOH) and Pcgf2/4 deletion (OHT) samples, showing the clustering of replicates. (C) Volcano plot of log10 (p value) against log2 fold change representing the differences in gene expression between control samples (EtOH) and Pcgf2/4 cKO (OHT). Grey line repr esents cutoff of p < 0.05, blue line of log2 fold change 1. Numbers in the upper corners indicate number of significantly up- and down-regulated genes, respectively. Genes bound by Ring1b and/or Pcgf2 in NPCs (Kloet et al., 2016) are highlighted (‘PRC1 targets’). (D, E) Gene ontology (GO) term enrichment analysis of upregulated genes (p value < 0.05) was performed for biological processes (D) and molecular function (E). (F) Venn diagram representing the overlap of genes bound by Ring1b and Pcgf2 in NPCs (Kloet et al., 2016) with up-regulated genes, which include several Hox genes. (G) Heat map of the genes up-regulated following Pcgf2/4 deletion (OHT) and bound by Ring1b E12.5 Isolation 6DIV Analysis 2DIV Seed/OHT/Diff Pcgf2F/F; Pcgf4F/F; Nes::CreERT2/+ A RNA expression in differentiating neurogenic NSCs B OHT 3OHT 2OHT 1EtOH 2 EtOH 3EtOH 1 C PRC1 targets n=24 n=120 0 20 40 60 −5 0 5 10 Log2 FoldChange - Log10 (p value) G H appendage development gland development skeletal system mo rphogenesis cell fate commitment embryonic skeletal system morphogenesis embryonic skeletal system development embryonic organ mo rphogenesis anterior/posterior pattern specification patterns p e c i fi c a t i o np r o c e s s regionalization 0 20 40 5e−12 1e−12 p.adjust Number of genes GO - molecular functionEGO - biological processesD OHT 3 OHT 2 OHT 1 EtOH 1 EtOH 3 EtOH 2 Hoxa7 Hoxb13 Hoxc9 Hoxd10 Hoxa5 Hoxd9 Hoxa10 Hoxc4 Hoxa9 Hoxb5 Hoxd8 Hoxc10 Hoxc8 Hoxd11 Hoxd3 Hoxc13 Hoxd4 Hoxd13 Hoxa3 Hoxa4 Hoxb2 Hoxa11 Hoxb1 Hoxc5 Hoxb3 Hoxb9 Hoxc6 Hoxb6 Hoxa2 Hoxa1 Hoxc12 Hoxb4 Hoxa13 Hoxc11 Hoxb8 Hoxa6 Hoxb7 Hoxd1 Hoxd12 ✱ OHT 3 OHT 2 OHT 1 EtOH 1 EtOH 3 EtOH 2 Nr4a2 Lef1 Pitx2 Nkx2−3 Bmp2 Lhx5 Pou4f1 Isl2 Dkk1 Gata3 Lhx9 Fezf1 Nkx2−5 Pax2 Pou4f2 ✱ ✱ ✱ F 3225 1313 Ring1B targets Pcgf2 targets UP genes Hox genes 60 24 31 5 Figure 4 Expression (Log2 counts) Expression (Log2 counts) PRC1 targets Hox genes Expression (Log2 counts) 1272 1272 1062 HMG box domain binding Number of genes transcription co-regulator bind DNA−bind repressor activity,R N A polymerase II−specific neuropeptide hormone activity DNA−bind repressor activity C2H2 zinc finger domain bind RNA polymerase II−specific DNA−bind TF binding E−box binding DNA−bind activator activity DNA−bind activator activity,R N A polymerase II−specific 0.010 0.005 p.adjust 0 10 20 30 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 7, 2024. ; https://doi.org/10.1101/2024.08.07.606990doi: bioRxiv preprint 11 and Pcgf2 in NPCs (Kloet et al., 2016), with a log2 fold change >1. (H) Heat map of up-regulated Hox genes, with a log2 fold change >1. Data information: Replicates represent NSCs from 3 embryos from two different litters. In summary, our side -by-side comparison of Pcgf2/4 and Pcgf3/5 deletion in NSC s revealed a differential contribution of canonical and non-canonical PRC1, respectively, to the regulation of NSC proliferation and lineage potential (Figure 5G). Despite the observation that Pcgf2/4 expression is highest in the neuronal population, we observed that canonical PRC1.2/1.4 contributes to the regulation of NSCs at proliferative, neurogenic and gliogenic phases. Even though Pcgf5 is preferentially expressed in the ventricular zone in vivo , deletion of Pcgf3/5 only ha d a minor effect on NSC proliferation in vitro and d id not impact NSC differentiation, neither during the neurogenic nor the gliog enic phase. It remains possible that additional non -canonical complexes (PRC1.1/1.6) may functionally contribute to the regulation of NSC fate , even though at least Pcgf1 is expressed at low levels in the mouse developing neocortex (Fietz et al., 2012). Figure 5. Deletion of Pcgf2/4 affects expression of stem cell and neurogenic genes. (A) Schematic of NSC differentiation paradigm. (B–F) mRNA expression in transcripts per million (TPM) analysed by RNA-seq for genes encoding (A) stem cell, (B) neurogenic, (C) neuronal maturation, (E) astrogenic and (F) NSC Astrocyte Neuron Oligodendrocyte B 1000 1200 0 200 400 600 800 ✱✱✱ Id2 0 5 10 15 Olig2 En1 ✱ 0.0 0.5 1.0 1.5 2.0 2.5 0 50 100 150 200 Hes5 0 10 20 30 40 Sox9 ✱ 0.0 0.2 0.4 0.6 0.8 Pitx3 Pax6 60 0 20 40 ✱ 0 2 4 6 Gli2 ✱✱✱ 0.0 0.2 0.4 0.6 0.8 En2 Stem cell factorsTPM A Neurogenic factors Neuronal maturation factors Oligogenic factorsAstrogenic factors C 0 1 2 3 4 ✱ Lhx5 0 10 20 40 30 Gfap 0.0 0.2 0.4 0.6 0.8 1.0 ✱✱ Lhx9 0 150 300 600 450 Hmgn1 0 20 40 60 80 ✱ Nr4a2 0 2000 1000 3000 4000 5000 Hmgn2 TPM TPM D F TPM E G TPM Figure 5 EtOH OHT NSC Astrocyte Neuron Oligodendrocyte Neurogenic Gliogenic Pcgf2/4 KO Pcgf3/5 KO = = = = = = = = = Pcgf2/4 KO Pcgf3/5 KO Pcgf2/4 KO Pcgf3/5 KO Pcgf3/5 KO Pcgf2/4 KO Proliferation Differentiation .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 7, 2024. ; https://doi.org/10.1101/2024.08.07.606990doi: bioRxiv preprint 12 oligogenic factors. (G) Summary of the functional role of Pcgf2/4 and Pcgf3/5 in the regulation of NSC proliferation and differentiation during the neurogenic and gliogenic phase. Data information: Bar graphs represent mean values. Error bars represent SD, NSCs from 3 embryos from two independent litters. *** p < 0.001, ** p < 0.01, * p < 0.05; unpaired Student’s t-test. Mechanistically, PRC1 was reported to bind to several stem cell and neurogenic genes, which we found to be differentially expressed upon Pcgf2/4 deletion, suggesting that these genes might be directly regulated by canonical PRC1. Our data suggest that rather than different PRC1 sub -complexes contributing to different phases of neural development, it is canonical PRC1.2/1.4 that regulates NSC proliferation and differentiation, whereas PRC1.3/1.5 plays a minor role in these processes. Overall, this suggests that switches in subunit composition may be more characteristic to the exit from pluripotency and differentiation towards different tissues and organs (Klauke et al., 2013; Luis et al., 2011; Morey et al., 2012; Morey et al., 2015; O'Loghlen et al., 2012; Santanach et al., 2017), but may be less relevant within a given lineage, such as neural development. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 7, 2024. ; https://doi.org/10.1101/2024.08.07.606990doi: bioRxiv preprint 13

Reagents and tools table Reagent/Resource Reference or Source Identifier or Catalog Number Experimental Models GW12/13 human foetal brain tissue This study N/A Mouse: NSC This study N/A Mouse: Pcgf2F/F; Pcgf4F/F (Fursova et al., 2019) N/A Mouse: Pcgf3F/F; Pcgf5F/F (Almeida et al., 2017) N/A Mouse: Nes::CreERT2/+ (Imayoshi et al., 2006) N/A Antibodies Goat anti-Sox2 (IF 1:200) R&D Systems AF2018 RRID: AB_355110 Rat anti-Ctip2 (IF 1:250) Abcam ab18465 RRID: AB_2064130 Mouse anti-Ring1B (IF 1:1,000) Active Motif 39663 RRID: AB_2716831 Rabbit anti-Ubiquityl-Histone H2A Lys119 (IF 1:1,500) Cell Signaling 8240 RRID: AB_10891618 Mouse anti-Mel18 (IF 1:50, WB 1:200) Santa Cruz SC- 515329 RRID: AB_2687587 Mouse anti-Bmi-1 (IF 1:300; WB 1:1,000) EMD Millipore 05-637 RRID: AB_309865 Chicken anti-Nestin (IF 1:500) Abcam ab134017 RRID: AB_2753197 Mouse anti-HuC/HuD (IF 1:300) Invitrogen A21271 RRID: AB_221448 Rat anti-GFAP (IF 1:500) Invitrogen 13-0300 RRID: AB_2532994 Rabbit anti-PDGFR alpha (IF 1:500) Abcam ab203491 RRID: AB_2893014 Mouse anti-APC (CC-1, IF 1:500) Merck OP80 RRID: AB_2057371 Rabbit anti-PCGF3/5 (WB 1:1,000) Abcam ab201510 RRID: AB_2818981 Mouse anti-vinculin (WB 1:1,000) Sigma Aldrich V9131 RRID: AB_477629 Donkey anti-Goat IgG, Alexa Flour 488 conjugated (1:1,000) Invitrogen A-11055 RRID: AB_2534102 Goat anti-Rat IgG, Alexa Flour 633 conjugated (1:1,000) Invitrogen A-21094 RRID: AB_2535731 Donkey anti-Mouse IgG, Alexa Fluor 555 conjugated (1:1,000) Invitrogen A-31570 RRID: AB_2536180 Donkey anti-Rabbit IgG, Alexa Fluor 555 conjugated (1:1,000) Invitrogen A-31572 RRID: AB_2536182 Goat anti-Chicken IgG, Alexa Fluor 488 conjugated (1:1,000) Invitrogen A-11039 RRID: AB_2534096 Donkey anti-Rat IgG, Alexa Flour 555 conjugated (1:1,000) Invitrogen A-48270 RRID: AB_2536114 Donkey anti-Mouse IgG, Alexa Fluor 647 conjugated (1:1,000) Invitrogen A-31571 RRID: AB_162542 Donkey anti-Rabbit IgG, Alexa Fluor 647 conjugated (1:1,000) Invitrogen A-31573 RRID: AB_2536183 Goat anti-rabbit IgG, H&L HRP (1:3,000) Abcam ab6721 RRID: AB_955447 Goat anti-mouse IgG, H&L HRP (1:10,000) Abcam ab205719 RRID: AB_2755049 Oligonucleotides Primers This study Chemicals, Enzymes and other Reagents .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 7, 2024. ; https://doi.org/10.1101/2024.08.07.606990doi: bioRxiv preprint 14 DMEM/F-12, HEPES Gibco Cat. # 31330095 Poly-D-Lysine Invitrogen Cat. # A3890401 Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane Sigma Aldrich Cat. # L2020 Bovine Serum Albumin (BSA) Sigma Aldrich Cat. # A2153 Fetal Bovine Serum (FBS) Sigma Aldrich Cat. # F7524 Heparin sodium salt from porcine intestinal mucosa Sigma Aldrich Cat. # H4784 MEM Non-Essential Amino Acids Solution (100X) Gibco Cat. # 11140050 GlutaMAX™ Supplement Gibco Cat. # 35050038 Human EGF Recombinant Protein Gibco Cat. # PHG0311 Human FGF-basic (FGF-2/bFGF) (aa 10-155) Recombinant Protein Gibco Cat. # PHG0021 B-27™ Supplement (50x), serum-free Gibco Cat. # 17504044 N-2 Supplement (100X) Gibco Cat. # 17502048 OH-Tamoxifen Sigma Aldrich Cat. # H7904 Thermo Scientific™ Halt™ Protease-Inhibitor-Cocktail (100x) Thermo Scientific Cat. # 10516495 DAPI Roche Cat. # 10236276001 Pierce™ Detergent Compatible Bradford Assay Kit Thermo Scientific Cat. # 23246 PVDF Transfer Membranes, 0.45 μm Thermo Scientific Cat. # 88518 SuperSignal™ West Pico PLUS Chemiluminescent Substrate Thermo Scientific Cat. # 34579 Software and Algorithms Fiji/ImageJ Fiji/ImageJ https://imagej.nih.go v/ij/ Prism (8.4.3) GraphPad software N/A Geneious Prime® (2019.2.1) Biomatters Ltd. N/A Affinity Photo + Designer (1.10.5.1342) Serif Ltd. N/A FACSDiva (8.0.2) BD Biosciences N/A FastQC (v0.11.6) Babraham Bioinformatics https://www.bioinfor matics.babraham.ac. uk/projects/fastqc/ Other RNeasy Mini Kit QIAGEN Cat. # 74104 QIAquick Gel Extraction Kit QIAGEN Cat. # 28106 Quick-DNA/RNA Microprep Plus Kit Zymo Research Cat. # D7005 Raw and analyzed RNA-seq data This study RNA-seq (Fietz et al., 2012) NCBI GEO: GSE38805 ChIP-seq Ring1b (Kloet et al., 2016) NCBI GEO: GSM1917303 ChIP-seq Pcgf2 (Kloet et al., 2016) NCBI GEO: GSM1917304 Phylogenetic pictures N/A https://www.phylopic .org/ Phylogenetic pictures Shutterstock N/A .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 7, 2024. ; https://doi.org/10.1101/2024.08.07.606990doi: bioRxiv preprint 15

Mice All experimental procedures were conducted in agreement with the German Animal Welfare Legislation after approval by the Landesdirektion Sachsen (licenses DD24.1- 5131/476/8; 25-5131/521/16). Animals were kept on a 12 -hour/12-hour light/dark cycle with food and water ad libitum. Mice used for PRC1 expression analysis were wildtype mice from the inbred C57BL/6J strain. Embryonic day (E) 0.5 was set as noon on the day on which the vaginal plug was observed. All experiments were performed in the dorsolateral te lencephalon of mouse embryos, at a medial position along the rostro-caudal axis. The developmental time point E14.5 of experimental procedures corresponds to a mid-neurogenic stage, when the production of upper-layer neurons has started. The sex of embryos was not determined, as it is not likely to be of relevance for the results obtained in the present study. For the inducible deletion of Pcgf genes in NSCs, Pcgf2F/F; Pcgf4F/F (Fursova et al., 2019) and Pcgf3F/F; Pcgf5F/F (Almeida et al. , 2017) mice were crossed with Nes::CreERT2/+ mice (Imayoshi et al., 2006). NSCs were isolated from either Pcgf2F/F; Pcgf4F/F; Nes::CreERT2/+ or Pcgf3F/F; Pcgf5F/F; Nes::CreERT2/+ strains. Only male embryos were included for Pcgf3F/F; Pcgf5F/F; Nes::CreERT2/+ to exclude effects attributed to changes in X chromosome inactivation (Almeida et al., 2017). Human foetal brain tissue Human foetal brain tissue was obtained from the Department of Gynaecology and Obstetrics, University Clinic Carl Gustav Carus of the Technische Universität Dresden, following elective pregnancy termination and informed written maternal consents, and with approval of the local University Hospital Ethical Review Committee (IRB00001473; IORG0001076; ethical approval number EK 355092018) , in accordance with the Declaration of Helsinki. The age of foetuses ranged from gestation week (GW) 1 2 to 13 as assessed by ultrasound measurements of crown -rump length and other standard criteria of developmental stage determination. The developmental time point corresponds to an early/mid-neurogenic stage, when the OSVZ expands and the production of upper-layer neurons starts. Due to protection of data privacy, the sex of the human foetuses, from which the neocortex tissue was obtained, cannot be reported. The sex of the human foetuses is not likely to be of relevance for the results obtained in the present study. The foetal neocortex tissue samples used in this study reported no health disorders. Foetal human brain tissue was dissected in Tyrode’s solution and fixed immediately (within 1 hour). Mouse NSC culture and differentiation .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 7, 2024. ; https://doi.org/10.1101/2024.08.07.606990doi: bioRxiv preprint 16 Mouse NSCs were isolated from E12.5 embryos as previously described (Cubillos et al, 2024; Schmitz et al, 2011; Schütze et al, 2022). Briefly, the dorsolateral cortex was isolated from E12.5 mice and treated with trypsin-EDTA (Gibco, #25300054) at 37°C for 20 min. Subsequently, soybean trypsin inhibitor (0.25mg/ml in PBS, Invitrogen, #17075-029) was added, cells were mechanically dissociated by pipetting and pelleted at 300x g for 5 min. NSCs were then plated on poly-D-lysine (at least 2 hours at 37°C; Gibco, #A3890401) and laminin (at least 4 h ours at 37 °C ; Sigma Aldrich, #L2020) coated plates at a density of 40,000 cells/mm2 and cultured under standard conditions (37 °C, 5% CO 2). Culture medium was prepared as a 1:1 mixture of DMEM/F12 and Neurobasal medium (Gibco, #12348 -017) supplemented with 10 ng/mL epidermal growth factor (EGF), 20 ng/mL fibroblast growth factors (FGF), 1X N-2 and 1X B-27 supplements, 1X Penicillin/Streptomycin, 1X Sodium -Pyruvate, 1X GlutaMAX, 1X MEM-NEAA, 4 mg/mL Heparin, 5 mM HEPES, 0.01 mM 2-Mercaptoethanol and 100 mg/L BSA. Differentiation of NSCs was induced with medium deficient of EGF and FGF, supplemented with 2% FBS (Sigma Aldrich, #F7524). NSCs were considered neurogenic in the first 5 DIV and gliogenic for the following passages as described before (Hirabayashi et al. , 2009) . Differentiation assays of neurogenic NSCs were performed at 2 DIV and of gliogenic NSCs after passaging every 3 –4 day at 20 DIV . For immunohistochemistry analysis, NSCs were plated on poly-D-lysin- and laminin- coated coverslips (Marienfeld Superior, #0111520) in a 24 -well plate , with either 10,000 cells per well for the NSC proliferation assay or 50,000 cells per well for the differentiation assay. Medium was changed every second day. Deletion of Pcgf genes was induced by addition of 1 μM 4-hydroxytamoxifen (OHT) to culture medium for 24–48 hours, as previously described (Fursova et al., 2019). 4-hydroxytamoxifen was dissolved in EtOH at a concentration of 1mM and stored for a maximum of 4 months at –20°C. Immunohistochemistry analysis of tissue sections Tissue was fixed in 4% PFA in 120 mM phosphate buffer pH 7.4 for 24 hours at 4°C, washed twice in PBS, transferred to 30% sucrose for 24 hours, embedded in O.C.T. compound (Sakura Finetek, #4583) with 15% sucrose and frozen on dry ice. Tissue was cut into 12 µm cryosections on a Thermo Fisher NX70 cryostat. Immunofluorescence was performed as previously described (Cubillos et al. , 2024; Florio et al , 2015) . Antigen retrieval for 1 hour with 10 mM citrate buffer pH 6.0 at 70°C in a water bath was followed by three washes with PBS, quenching for 30 min in 0.1 M glycine in PBS and blocking for 30 min in blocking solution (10% horse serum and 0.1% Triton in PBS) at room temperature. Primary antibodies were incubated in blocking solution over night at 4°C. Subsequently, sections were washed three times in PBS, incubated with secondary antibodies (1:1,000) and DAPI (1:1,000) in blocking solution for 1 hour at .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 7, 2024. ; https://doi.org/10.1101/2024.08.07.606990doi: bioRxiv preprint 17 room temperature, and washed again three times in PBS before mounting on microscopy slides with Mowiol. Images were acquired with a Zeiss ApoTome2 fluorescence microscope with a 20x

using 1.5-µm thick optical sections. ZEN software was used to generate maximum intensity projections. To quantify the intensity of PRC1 markers, the human foetal and mouse embryonic tissue was divided into the different germinal zones and cortical plate based on SOX2 and CTIP2 staining and alignment of cells in DAPI within a 100 μm-wide image. The Fiji plug-in Stardist 2D (Schmidt et al, 2018) was applied using the versatile model to segment nuclei in DAPI, and if required, segmentation was corrected manually. Mean grey values of segmented nuclei were measured in Fiji and the resulting data processed using Excel and Prism software. Normal distribution of data was tested by Kolmogorov-Smirnov and Shapiro-Wilk tests, followed by Tukey’s multiple comparison test. Immunohistochemistry analysis of NSC differentiation Cells were fixed in 2% PFA in 120 mM phosphate buffer pH 7.4 for 10 min at room temperature, before PFA was washed away twice with PBS. Cells were permeabilized with 0.1% Triton in PBS for 5 min, followed by washing twice with PBS for 5 min and twice with washing solution containing 0.1% Tween in PBS for 5 min. After this, cells were blocked for 30 min in blocking buffer containing 2.5% BSA (Sigma Aldrich, #A2153), 0.1% Tween and 10% horse serum in PBS, before primary antibodies diluted in blocking buffer were added and incubated over night at 4°C. This was followed by three washes with washing solution for 10 min, incubation with secondary antibodies (1:1,000) and DAPI (1:1,000) in blocking buffer for 1 hour at room temperature, three additional washes and embedding in a drop of Mowiol on microscopy slides. Images were acquired with a Zeiss ApoTome2 fluorescence microscope with a 20x

using 1.5-µm thick optical sections. ZEN software was used to generate maximum intensity projections. Samples were blinded after staining, before acquisition of images. For quantification of marker positive cells, nuclei were segmented in DAPI using the Fiji plug -in Stardist 2D (Schmidt et al. , 2018) and counted with the cell counter tool in Fiji. The resulting data was processed using Excel and Prism software. Data was analysed for outliers using the Grubb’s test, followed by Tukey’s multiple comparison test. Protein expression analysis by Western blotting Proteins were isolated from cells in culture using TOPEX Plus buffer containing 300 mM NaCl, 50 mM Tris -HCl pH7.5, 0.5% Triton, 1% SDS, 1 mM DTT (Roche, #10708984001), 1 X protease inhibitor (Roche, #4693116001) and 333.33 U/m L .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 7, 2024. ; https://doi.org/10.1101/2024.08.07.606990doi: bioRxiv preprint 18 Benzonase (Sigma Aldrich, #E1014 -25KU) in water, by incubation at room temperature until viscosity disappeared (5 –15 min). Protein concentration was measured using the Pierce detergent compatible Bradford assay kit (Thermo Scientific, #23246). Subsequently, 40 µg of protein were resolved on a 10% SDS Polyacrylamide gel and transferred to a PDVF transfer membrane (ThermoScientific, #88518). Membranes were blocked for 1 hour at room temperature with 5% skim milk in PBS with 0.05% Tween, and then incubated wit h primary antibodies over night at 4°C. Secondary antibodies were incubated for 1 hour at room temperature. Antibody signal was detected using the SuperSignal West Pico plus kit (ThermoFisher Scientific, #34579). RNA-seq library preparation RNA-seq was performed as previously described (Cubillos et al. , 2024) . RNA of differentiating NSCs was isolated using the Quick-RNA MiniPrep kit (Zymo Research, #R1008). Transcriptome libraries were prepared using an adapted version of the SmartSeq2 protocol (Picelli et al, 2013). Isolated total RNA from an equivalent of one 24-well was denatured for 3 minutes at 72°C in 4 μL hypotonic buffer (0.2% Triton-X 100) in the presence of 2. 4 mM dNTP, 240 nM dT -primer and 4 U RNase Inhibitor (NEB, M0314L). Reverse transcription was performed at 42°C for 90 min after filling up to 10 µ L with RT buffer mix for a final concentration of 1 X Superscript II buffer (Invitrogen), 1 M Betaine, 5 mM DTT, 6 mM MgCl2 , 1 µM TSO-primer, 9 U RNase inhibitor and 90 U Superscript II. The reverse transcriptase was inactivated at 70°C for 15 min. For subsequent PCR amplification of the cDNA, the optimal PCR cycle number was determined with an aliquot of 1 μL unpurified cDNA in a 10 μL qPCR containing 1X Kapa HiFi Hotstart Readymix (Roche), 1X SybrGreen and 0.2 μM UP primer. The residual 9 μL cDNA were subsequently amplified using Kapa HiFi HotStart Readymix (Roche) at a 1X concentration together with 250 nM UP -primer under the follo wing cycling conditions: initial denaturation at 98°C for 3 min, 22 cycles [98°C 20 sec, 67°C 15 sec, 72°C 6 min] and final elongation at 72°C for 5 min. Amplified cDNA was purified using 1X volume of Sera-Mag SpeedBeads (GE Healthcare) resuspended in a buffer consisting of 10 mM Tris, 20 mM EDTA, 18.5% (w/v) PEG 8000 and 2 M sodium chloride solution. The cDNA quality and concentration were determined using a Fragment Analyzer (Agilent). For library preparation, 2 µ L amplified cDNA was tagmented in 1X Tagmentation Buffer using 0.8 µL bead-linked transposome (Illumina DNA Prep, (M) Tagmentation, Illumina) at 55°C for 15 min in a total volume of 4 µL. The reaction was stopped by adding 1 µL of 0.1% SDS (37°C, 15 min). Magnetic beads were bound to a magnet, the supernatant was removed, beads were resuspended in 14 µ L indexing PCR Mix containing 1X KAPA Hifi HotStart Ready Mix (Roche) and 700 nM unique dual indexing primers (i5 and i7), and subjected to a PCR (72°C 3 min, 98°C 30 sec, 12 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 7, 2024. ; https://doi.org/10.1101/2024.08.07.606990doi: bioRxiv preprint 19 cycles [98°C 10 sec, 63°C 20 sec, 72°C 1 min], 72°C 5 min). Libraries were purified with 0.9 X volume Sera -Mag SpeedBeads, followed by a double size selection with 0.6X and 0.9X volume of beads, and a final 0.9X purification to obtain a fragment size distribution of 200–700 bp. Sequencing was performed after quantification using a Fragment Analyzer on an Illumina Novaseq 6000 in 100 bp paired-end XP mode with an average sequencing depth of 40 million fragments per library. RNA-seq data analysis Quality control of the sequencing data was performed with FastQC (version 0.11.9). Kallisto (version 0.64.1) (Bray et al, 2016) was used to align trimmed reads to mouse GRCm39. For further processing, data was imported into R using Tximport (Soneson et al, 2015) and EnsDb.Mmusculus.v79 packages. Raw fragment normalization based on library size and testing for differential expression between genotypes was performed with DESeq2 (version 1.30.1; Wald test) (Love et al, 2014) with a false discovery rate (FDR) of 5% and a Log2 fold change threshold of 1. To quantify gene expression levels within samples, transcripts per million (TPM) values were calculated with Tximport (Soneson et al. , 2015) . Volcano plots and heatmaps were generated with the R - packages Ggplot2 (Wickham et al, 2016) and Complexheatmap (Gu et al, 2016). Venn Diagrams were generated using the online platform DeepVenn.com (Hulsen, 2022). Differentially expressed genes were analysed by KEGG pathway (https://www.genome.jp/) and gene ontology (https://geneontology.org/) (Ashburner et al, 2000; Thomas et al, 2022) analysis. Statistical analysis Sample sizes are reported in each figure legend. Sample sizes were estimated based on previous literature (Albert et al., 2017; Cubillos et al., 2024; Schmitz et al., 2011). All statistical analysis was performed using Prism (GraphPad Software). Normal distribution of datasets was tested by Shapiro-Wilk or Kolmogorov-Smirnov test. Data was analysed for outliers using the Grubb’s test. The tests used included Student’s t- test and Tukey’s multiple comparison test , as indicated in the figure legend for each quantification. Significant changes are indicated by stars for each graph and described in the figure legends. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 7, 2024. ; https://doi.org/10.1101/2024.08.07.606990doi: bioRxiv preprint 20

We are grateful to the facilities of the CRTD and D RESDEN-concept partner institutions for the outstanding support provided, notably R. Hans from the Light Microscopy Facility, the teams for animal husbandry and histology and the DRESDEN- concept Genome Center lab team for technical support. We thank all members of the Albert laboratory for help and discussions. We acknowledge R. Kageyama from Kyoto University for providing the Nes::CreERT2 mouse line. MA acknowledges funding from the Center for Regenerative Therapies TU Dresden, the DFG (Emmy Noether, AL 2231/1-1) and the Schram foundation. Author contribution Janine Hoffmann : Conceptualization; investigation; formal analysis; visualization; writing – review & editing; Theresa M. Schütze: Formal analysis, writing – review & editing; Annika Kolodziejczyk: Investigation; resources; writing – review & editing; Annekathrin Kränkel : Investigation; Susanne Reinhardt : Resources; writing – review & editing; Razvan P. Derihaci: Resources; Cahit Birdir: Resources; Pauline Wimberger: Resources; Haruhiko Koseki : Resources; Mareike Albert: Conceptualization; w riting – original draft; writing – review & editing; f unding acquisition; supervision. Disclosure and competing interests statement The authors declare no competing interests. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 7, 2024. ; https://doi.org/10.1101/2024.08.07.606990doi: bioRxiv preprint 21

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