Distinct and separable roles for EZH2 in neurogenic astroglia
It is now appreciated that , like neurons , astrocytes serve multiple functions in the adult brain and are heterogeneous in their identity ( Zhang and Barres , 2010; Molofsky et al . , 2012 ) . While astrocytes are distributed widely throughout the brain , only those located in the subventricular zone ( SVZ ) of the lateral ventricles and subgranular zone ( SGZ ) of the hippocampus serve as neurogenic NSCs for all of adult life ( Kriegstein and Alvarez-Buylla , 2009 ) . Though several transcriptional regulators have been identified that distinguish neurogenic astroglia ( Hsieh , 2012 ) , what makes adult NSCs epigenetically distinct from non-neurogenic astrocytes remains an outstanding question . Understanding these differences may yield novel insights into how adult stem cells retain the competence to self-renew and generate multiple specialized cell types . In particular , Polycomb group ( PcG ) proteins , which comprise a chromatin-remodeling system that mediates transcriptional repression , have been demonstrated to play a crucial role in the development of various cell populations ( Aloia et al . , 2013 ) . PcG proteins assemble into two functionally distinct Polycomb repressive complexes , PRC1 and PRC2 . PRC2 typically contains the core components SUZ12 , EED , RbAp46/48 , and EZH2 , a histone methyltransferase responsible for catalyzing histone H3 lysine 27 trimethylation ( H3K27me3 ) . In the prevailing model of PcG-mediated repression , PRC2 is targeted to specific loci , and the deposited H3K27me3 marks serve as a platform to recruit PRC1 , leading to transcriptional silencing ( Margueron and Reinberg , 2011 ) . Most of what is known about PRC2 developmental function derives from studies of the embryo . In the early developing forebrain , conditional deletion of Ezh2 results in a shortened period of neuronal production related to lack of precursor cell proliferation and premature NSC differentiation ( Pereira et al . , 2010 ) ; in contrast , deletion of Ezh2 a few days later during corticogenesis causes an increase in the duration of neurogenesis and a delay in astrocyte differentiation ( Hirabayashi et al . , 2009 ) . Thus , EZH2 , in concert with other PcG members , appears to orchestrate the temporal alterations in embryonic NSC behavior . In contrast to the dynamic and transient nature of embryonic NSCs , adult NSCs are relatively stable in their differentiation potential and are maintained for all of adult life . Postnatal NSCs lacking PRC1 component BMI1 are defective for self-renewal , in part due to the derepression of cell cycle inhibitors encoded by the Ink4a/Arf locus ( also known as Cdkn2a ) ( Molofsky et al . , 2003 ) . Whether PcG factors are required for other aspects of adult neurogenesis such as lineage specification is not known . In this study , we found EZH2 expression to be a distinguishing feature of neurogenic astrocytes in the adult mouse brain . Ezh2 in SVZ NSCs was required for distinct functions , regulating both cell proliferation and neuronal lineage specification . To enable SVZ NSC self-renewal , EZH2 directly repressed the Ink4a/Arf locus . Ink4a/Arf-deletion reversed the proliferation defect of Ezh2-deleted NSCs , but neurogenesis was still greatly impaired . In SVZ cells , EZH2 directly targeted Olig2 , and repression of this bHLH transcription factor was required for neuronal differentiation . Furthermore , genome-wide transcript and chromatin analyses revealed a role for EZH2 in preventing the aberrant activation of transcriptional regulators that specify alternative neuronal lineages . In the human brain , EZH2 was also expressed in astroglia and neuronal precursors of the early infant SVZ , suggesting that this PRC2 factor plays key roles in postnatal human neurogenesis . Taken together , these data indicate that EZH2 is a critical epigenetic determinant for the maintenance of multipotent astroglia , enabling both proliferation and cell fate specification of an adult stem cell population .
EZH2 is expressed widely in the embryonic brain , including in radial glial cells ( Pereira et al . , 2010 ) , which are the primary precursor of all major neural cells in the adult brain ( Kriegstein and Alvarez-Buylla , 2009 ) . While the PRC1 component BMI1 is widely expressed in mature neurons and astrocytes ( Chatoo et al . , 2009 ) , EZH2 expression in the postnatal brain has not been examined . Using immunohistochemistry ( IHC ) , we detected EZH2 in most cells of the cortex and striatum at postnatal day 7 ( P7 ) ( Figure 1—figure supplement 1B ) . However , by P15 and in adulthood ( ∼P60 ) , EZH2 was no longer detected by IHC in non-neurogenic brain regions ( Figure 1—figure supplement 1B ) . In contrast , EZH2 staining remained robust in SVZ and SGZ cells from P0 to adulthood ( Figure 1D , Figure 1—figure supplement 1C ) . 10 . 7554/eLife . 02439 . 003Figure 1 . EZH2 is specifically expressed in the neurogenic SVZ lineage . ( A ) Schematic of sagittal brain section depicting the RMS ( red ) that connects the SVZ to the OB . Lines indicate locations of Figure 1B , C . ( B and C ) Schematic of coronal sections through the SVZ ( B ) and OB ( C ) depicting the location of cells in the SVZ neurogenic lineage . Boxes indicate the location of subsequent images . ( D ) IHC analysis of the SVZ ( ventricle–left; striatum–right ) staining for EZH2 ( red ) , S100β ( green ) , and 4 , 6- diamidino-2-phenylindole ( DAPI; blue ) to label nuclei from birth ( P0 ) through adulthood ( P62 ) . ( E ) Co-localization of EZH2 ( red ) in the SVZ ( ventricle–left; striatum–right ) by IHC with various markers of the SVZ neurogenic lineage ( green ) in P21 coronal brain sections of control animals ( DAPI; blue ) . Arrowheads highlight a GFAP+ EZH2+ cell . ( F and G ) IHC co-localization of EZH2 with DCX+ neuroblasts ( F ) and NeuN+ neurons ( G ) in the OB . Dotted line indicates the boundary between the OB core and granular cell layer ( GCL ) . Scale bars , 20 µM . DOI: http://dx . doi . org/10 . 7554/eLife . 02439 . 00310 . 7554/eLife . 02439 . 004Figure 1—figure supplement 1 . EZH2 Expression is maintained in the SVZ and SGZ but not the cortex and striatum postnatally . ( A ) Schematic of coronal brain section depicting the location of images in ( B ) . Ctx = Cortex , Str = Striatum . ( B ) IHC analysis of the SVZ , cortex , and striatum staining for DAPI , EZH2 and Vimentin ( VIM ) , a marker for radial glial cells in early postnatal periods and ependymal cells from at P7 , P15 , and P62 . ( C ) Co-localization of EZH2 ( red ) with the proliferation marker Ki67 ( green ) merged with DAPI ( blue ) in the hippocampus of P21 coronal brain sections of control animals . Scale bars , 20 µM . DOI: http://dx . doi . org/10 . 7554/eLife . 02439 . 004 To investigate the function of EZH2 in NSC astroglia , we focused on the SVZ-olfactory bulb neurogenic system ( Ihrie and Alvarez-Buylla , 2011 ) . Co-IHC of EZH2 with markers of SVZ cell types demonstrated EZH2 expression in the entire neurogenic cell lineage , from the GFAP+ type B1 astroglial cells that serve as self-renewing SVZ NSC , to DLX2+ transit amplifying type C cells ( Figure 1E ) , and finally to DCX+ type A neuroblasts that migrate from the SVZ through the rostral migratory stream ( RMS ) to the olfactory bulb ( OB ) ( Figure 1F ) . Of note , EZH2 also co-localized with OLIG2 ( Figure 1E ) , which is expressed in a small subset of both type B1 and C cells in the SVZ ( Menn et al . , 2006 ) . Upon arriving into the core of the OB , DCX+ neuroblasts migrate tangentially into the granule cell layer ( GCL ) where they differentiate into NeuN+ neurons . Interestingly , DCX+ cells away from the OB core had decreased levels of EZH2 , and NeuN+ neurons that populate the GCL were mostly EZH2-negative ( Figure 1G ) . These data indicate that EZH2 is expressed in SVZ neurogenic astroglia , their immature daughter cells , and is gradually downregulated upon terminal differentiation . Both SVZ type B1 cells and local non-neurogenic , postmitotic ependymal cells arise from local radial glia shortly after birth ( Merkle et al . , 2004; Spassky et al . , 2005 ) . Notably , S100β+ ependymal cells were EZH2-negative ( Figure 1D ) . In addition , while almost all EZH2+ cells ( >99% ) in the postnatal SVZ express the cell cycle marker Ki67+ , only a small percentage ( ∼7% ) of Ki67+ cells located away from the SVZ were EZH2+ ( Figure 1E , data not shown ) , suggesting that EZH2 expression is not a strict consequence of cell proliferation . To study Ezh2-deficency in SVZ NSCs , we used mice with conditional alleles of Ezh2 ( Ezh2F/F ) and the hGFAP-cre transgene , which drives Cre-mediated recombination in the precursors of the cerebellar granule cell layer , hippocampal dentate gyrus , and SVZ NSCs ( Han et al . , 2008 ) . hGFAP-cre;Ezh2F/F animals were born at expected Mendelian ratios and did not exhibit gross morphological or behavioral defects as compared to their hGFAP-cre;Ezh2F/+ or non-hGFAP-cre littermates ( hereafter referred to as controls ) . While the cerebellar granule cell layer did not appear abnormal , both the hippocampal dentate gyrus and OB had reduced cellularity ( Figure 2—figure supplement 2 ) . In the P21 OB of hGFAP-cre;Ezh2F/F mice , the density of DCX+ migratory neuroblasts was markedly decreased as compared to controls ( Figure 2A ) , with no evidence of increased cell death as measured by cleaved Caspase 3 ( Casp3 ) IHC ( data not shown ) . To investigate whether this decrease in neuroblasts relates to defective postnatal neuron production , we injected P11 mice with the thymidine analog EdU to label a cohort of cells born in the postnatal SVZ and analyzed the OB 10 days ( 10 d ) later . hGFAP-cre;Ezh2F/F mice had twofold fewer EdU+ NeuN+ OB neurons as compared to controls ( Figure 2B , C ) . This decrease was not due to a developmental defect in the SVZ , as we did not find any significant differences in the type C cell ( DLX2+ , DCX-negative ) population nor a deficit in the type B cell ( GFAP+ , Nestin+ ) population in hGFAP-cre;Ezh2F/F mice ( Figure 2—figure supplement 3 ) . However , hGFAP-cre;Ezh2F/F mice had fourfold fewer DCX+ cells in the dorsal SVZ , which is the initiation of the RMS ( Figure 2D , E ) , indicating that the decrease in OB neurogenesis relates to a deficit of neuroblast production from SVZ NSCs . 10 . 7554/eLife . 02439 . 005Figure 2 . Conditional deletion of Ezh2 in SVZ NSCs both in vivo and in vitro inhibits neurogenesis . ( A ) IHC for the neuroblast marker DCX ( green ) in P21 OB coronal sections comparing Control to hGFAP-Cre;Ezh2F/F slices . ( DAPI; blue ) . ( B and C ) IHC for NeuN+ EdU+ cells in the granule cell layer of the OB ( B ) and quantification ( C ) comparing slices from P21 control to hGFAP-Cre;Ezh2F/F animals injected with Edu 10 days prior to sacrifice ( *p=0 . 0153 , n = 3 ) . ( D and E ) IHC for DCX + cells in the SVZ ( D ) and quantification ( E ) comparing slices from P21 control to hGFAP-Cre;Ezh2F/F animals ( **p=0 . 0079 , n = 3 ) . ( F ) ICC for the neuronal marker Tuj1 and the astrocyte marker GFAP of SVZ NSC control and Ezh2Δ/Δ cultures after 7 days of differentiation . ( G ) Quantification of Tuj1+ and GFAP+ cells in Ezh2-deleted ( orange ) and Control ( blue ) SVZ cultures ( **p=0 . 0063 , n = 3 ) . ( H ) Quantification of ICC experiment co-staining for GFP and either GFAP ( orange ) , Tuj1 ( white ) , or GFP-only ( blue ) colonies that are established from GFP+ Control or Ezh2Δ/Δ SVZ NSCs seeded at a low density amongst wildtype , GFP- SVZ NSCs . Data are represented as ± SEM . Scale bars , 20 µM . DOI: http://dx . doi . org/10 . 7554/eLife . 02439 . 00510 . 7554/eLife . 02439 . 006Figure 2—figure supplement 1 . Loss of EZH2 and H3K27me3 upon conditional deletion of Ezh2 in vivo and in vitro . ( A ) IHC co-localization of EZH2 ( green ) and H3K27me3 ( red ) merged with DAPI ( blue ) in P21 control and hGFAP-cre;Ezh2F/F animals at the SVZ , RMS , and OB . Scale bars , 50µM . ( B ) ICC co-localization of EZH2 ( green ) and H3K27me3 ( red ) merged with DAPI ( blue ) in Control and Ezh2Δ/Δ SVZ NSC cultures . Scale bars , 20 µM . DOI: http://dx . doi . org/10 . 7554/eLife . 02439 . 00610 . 7554/eLife . 02439 . 007Figure 2—figure supplement 2 . Morphology of neurogenic brain regions in Control and hGFAP-cre;Ezh2F/F animals . Hematoxylin and eosin ( H&E ) or DAPI staining of brain slices from the cerebellum , hippocampus , and olfactory bulb in P21 control and hGFAP-cre;Ezh2F/F animals . DOI: http://dx . doi . org/10 . 7554/eLife . 02439 . 00710 . 7554/eLife . 02439 . 008Figure 2—figure supplement 3 . Conditional deletion of Ezh2 in SVZ NSCs does not cause defects in type C or B cells in the SVZ . ( A ) IHC for the type C/A marker DLX2 ( green ) or type A neuroblast marker DCX ( red ) in P21 OB coronal sections comparing Control to hGFAP-Cre;Ezh2F/F slices . Type C cells are DLX2+ DCX− . ( B ) Quantification of DLX2 DCX- type C cells per hpf comparing slices from P21 control to hGFAP-Cre;Ezh2F/F animals ( n = 3 ) . ( C ) IHC for the type B cell markers Nestin ( green ) and GFAP ( red ) along with DAPI in P21 OB coronal sections comparing Control to hGFAP-Cre;Ezh2F/F slices . Data are represented as ± SEM . Scale bars , 20 µM . DOI: http://dx . doi . org/10 . 7554/eLife . 02439 . 008 To further investigate the role of EZH2 in SVZ NSCs , we used a monolayer culture system that recapitulates SVZ neurogenesis ( Scheffler et al . , 2005 ) . Ezh2 was acutely deleted by the addition of 4-hydroxytamoxifen ( 4OHT ) to cultures generated from UBC-CreERT2;Ezh2F/F mice ( henceforth Ezh2 Δ/Δ ) , resulting in a marked decrease in EZH2 and H3K27me3 staining as compared to control ( Figure 2—figure supplement 1B ) . Ezh2 Δ/Δ cultures resulted in > sixfold fewer Tuj1+ neurons after 7 d of differentiation ( Figure 2F , G ) , while cell death as measured by immunocytochemistry ( ICC ) for Casp3 was not increased ( data not shown ) . Furthermore , the number of cells expressing the astrocyte marker GFAP was not significantly changed in Ezh2 Δ/Δ cells , indicating that Ezh2 deletion affects neuronal but not astrocyte differentiation in vitro ( Figure 2F , G ) . To investigate whether Ezh2 functions in a cell-autonomous manner to promote neurogenesis , we followed the fate of Ezh2 Δ/Δ;GFP and non-deleted GFP control SVZ NSCs cells plated at low density among a large number wild-type SVZ cells ( GFP:WT cell ratio , 1:350 ) . After 7 d of differentiation , both control GFP and Ezh2Δ/Δ; GFP cells yielded similar percentages of GFAP+ colonies , consistent with our previous observation that Ezh2 deletion does not affect astrocyte differentiation . However , control GFP cells generated discrete colonies of GFP+ , Tuj1+ neurons , while Ezh2Δ/Δ;GFP cells failed to generate any GFP+ , Tuj1+ colonies in the presence of wild-type SVZ NSCs , indicating that the role of EZH2 in neurogenesis is likely cell-autonomous ( Figure 2H ) . In multiple adult stem cell populations , proliferation is regulated by EZH2 and other PcG members via repression of Ink4a/Arf ( also known as Cdkn2a ) , which encodes the p16 and p19 cell cycle inhibitors ( Ezhkova et al . , 2009; Aoki et al . , 2010; Juan et al . , 2011 ) . Indeed , chromatin immunoprecipitation ( ChIP ) analysis of acutely isolated postnatal SVZ tissue revealed significant enrichment of EZH2 and H3K27me3 at the Ink4a/Arf locus ( Figure 3A , B ) . PRC1 component BMI1—a downstream effector of H3K27me3 repression—is required for SVZ NSC self-renewal , and NSCs with Bmi1-deletion have increased expression of Ink4a ( Molofsky et al . , 2003; Bruggeman et al . , 2005 ) . Consistent with these results , Ink4a expression was increased greater than twofold in Ezh2 Δ/Δ NSCs as compared to controls ( Figure 3C ) . 10 . 7554/eLife . 02439 . 009Figure 3 . Deletion of the Ink4a/Arf locus rescues proliferation but not neurogenesis in Ezh2Δ/Δ SVZ NSCs . ( A and B ) ChIP-qPCR analysis of HoxA2b ( positive control ) , Gapdh ( negative control ) , Arf , Ink4a , and Olig2 genomic loci pulled down by either IgG ( non-specific control ) or antibodies to EZH2 ( A ) and H3K27me3 ( B ) relative to input . ( C ) qPCR analysis of the expression levels of Ink4a , Arf , and Ezh2 relative to the expression of the RPLP control transcript in Control vs Ezh2Δ/Δ SVZ NSC cultures . ( D and E ) Quantification of either primary ( D ) or secondary ( E ) NS sizes grown in soft agar generated from control or Ezh2Δ/Δ SVZ NSCs in either an Ink4a/Arf+/+ or Ink4a/Arf−/− background with each dot representing a measured NS . ( F ) Quantification of BrdU+ and Tuj1+ cells in control and Ezh2Δ/Δ SVZ NSCs in either an Ink4a/Arf+/− or null background . Data is represented as the number of cells with a positive stain in Ezh2Δ/Δ divided by the Control with the dotted line at 1 . 0 representing no change between Ezh2Δ/Δ and Control SVZ NSCs ( n = 3 ) . ( G ) Quantification of Tuj1+ GFP+ cells as a fraction of all GFP+ cells in Ezh2Δ/Δ;Ink4a/Arf−/− SVZ NSCs infected with a lentivirus ( LV ) containing GFP and a control gene encoding for alkaline phosphatase compared to a LV expressing GFP and Ezh2 ( ****p<0 . 0001 , n = 3 ) . ( H ) IHC staining for DCX ( green ) and DAPI ( blue ) of P14 coronal OB slices from Control;Ink4a/Arf−/− animals compared to hGFAP-cre;Ezh2F/F;Ink4a/Arf−/− demonstrating a decrease in DCX+ cells in the OB core . Data are represented as ± SD . DOI: http://dx . doi . org/10 . 7554/eLife . 02439 . 00910 . 7554/eLife . 02439 . 010Figure 3—figure supplement 1 . Schematic of the location of ChIP primers to Ink4a/Arf and Olig2 used in Figure 3A , B . DOI: http://dx . doi . org/10 . 7554/eLife . 02439 . 010 To investigate whether Ezh2 is required for SVZ NSC proliferation , we compared the growth of Ezh2 Δ/Δ to control SVZ NSCs in a soft-agar neurosphere ( NS ) assay , in which SVZ NSCs are cultured at clonal density and grown as immobilized , spheroid colonies . Ezh2-deletion resulted in primary NSs that were significantly smaller than those controls , suggestive of a defect in cell proliferation ( Figure 3D ) . When primary NSs were dissociated and recultured at clonal density , these secondary Ezh2 Δ/Δ NSs were also much smaller than controls , further indicating that the proliferation of self-renewing SVZ NSCs requires Ezh2 ( Figure 3E ) . We next asked whether Ink4a/Arf-deficiency can rescue the proliferation defect of Ezh2-deleted SVZ NSCs . Ink4a/Arf-null SVZ NSCs with Ezh2-deletion ( henceforth Ezh2Δ/Δ;Ink4a/Arf−/− ) generated primary and secondary NSs that were similar in size than those from Control;Ink4a/Arf−/− NSCs ( Figure 3D , E ) . Thus , Ink4a/Arf-deficiency reverses the self-renewing proliferation defect of Ezh2-deleted NSCs . Taken together , these data indicate that EZH2-mediated repression of the Ink4a/Arf locus is critical for the self-renewing proliferation of SVZ astroglia . Given the well-characterized role of EZH2 in the regulation of cell proliferation in other stem cell niches ( Aldiri and Vetter , 2012 ) , we investigated whether the neurogenesis defect of Ezh2 Δ/Δ SVZ NSCs was primarily a result of decreased cell proliferation . Ezh2 Δ/Δ cultures exhibited an approximately 40% decrease in 5-bromo-2-deoxyuridine ( BrdU ) incorporation as compared to control cultures; this decrease in proliferation was accompanied by a >6x decrease in the number of Tuj1+ cells after 7 d of differentiation ( Figure 3F , left blue column ) . Consistent with our NS culture results , Ink4a/Arf-deficiency rescued the proliferation defect associated with Ezh2-deletion ( Figure 3F , right blue column ) . Unexpectedly , Ink4a/Arf-deficiency did not rescue neurogenesis , as Ezh2Δ/Δ;Ink4a/Arf−/− cultures still exhibited greater than sixfold fewer Tuj1+ cells compared to Control;Ink4a/Arf-/- cultures ( Figure 3F , orange columns ) . Furthermore , Ink4a/Arf-deficiency did not rescue OB neurogenesis in vivo , as the density of DCX+ neuroblasts in the OB of hGFAP-cre;Ezh2F/F;Ink4a/Arf−/− mice was greatly reduced as compared to Control;Ink4a/Arf−/− mice ( Figure 3H ) . Taken together , these results indicate that EZH2 has a role in SVZ neurogenesis that is distinct from its function in Ink4a/Arf repression . Furthermore , by rescuing only cell proliferation and not neuronal differentiation , Ink4a/Arf-deficiency enabled us to investigate additional roles for EZH2 in the neurogenic capacity of this astroglial population . We next tested whether reexpression of Ezh2 could rescue neurogenesis in Ezh2-deleted , Ink4a/Arf-null SVZ NSCs . We infected Ezh2Δ/Δ;Ink4a/Arf−/− SVZ NSCs with a lentivirus ( LV ) that expresses Ezh2 and GFP ( Ezh2 LV ) or a control GFP vector ( Control LV ) one day before the initiation of differentiation . Ezh2 LV infected cells produced >10-fold more Tuj1+ cells as compared to Control LV ( Figure 3G ) . This rescue of neurogenesis by Ezh2 re-expression suggests that neuronal differentiation requires Ezh2-dependent gene repression independent of Ink4a/Arf regulation . Given the results of the Ezh2 rescue experiments , we reasoned that EZH2 might promote neurogenesis by catalyzing H3K27me3 at specific loci during differentiation . To identify such genes , we first performed ChIP-seq analysis of SVZ NSC monolayer cultures with antibodies to H3K27me3 to detect loci that did not have this chromatin modification before differentiation . As expected , the housekeeping gene Gapdh was not repressed with H3K27me3 ( Figure 4A ) . In contrast , Hoxa2b , a homeobox gene that is not normally expressed in neural cells , was highly enriched with this chromatin modification ( Figure 4A ) . Olig2 encodes a bHLH transcription factor that is expressed in cultured SVZ NSCs , and this locus was not enriched with H3K27me3 ( Figure 4A ) . Furthermore , ChIP-seq analysis for histone 3 lysine 4 trimethylation ( H3K4me3 ) , a chromatin modification associated with active transcription , revealed peaks of enrichment at both Gapdh and Olig2 , but not Hoxa2b ( Figure 4A ) , correlating with the transcriptional state of these loci . 10 . 7554/eLife . 02439 . 011Figure 4 . Aberrant expression of OLIG2 in Ezh2Δ/Δ;Ink4a/Arf−/− SVZ NSCs inhibits neuronal differentiation . ( A ) ChIP-seq analysis of proliferating SVZ NSCs in vitro with an H3K4me3 or H3K27me3 antibody at the Hoxa2 , Gapdh , and Olig2 genomic loci . ( B ) ChIP-qPCR analysis of FACS-sorted Tuj1+ and GFAP/Nestin + cells after 4 days in differentiation media with an H3K27me3 antibody using primer sets spanning the promoter regions of Hoxa2 , Gapdh , and Olig2 ( black boxes in ( A ) ) . ( C ) Representative images of ICC analysis comparing Control;Ink4a/Arf−/− and Ezh2Δ/Δ;Ink4a/Arf−/− SVZ NSCs after four days of differentiation with DCX and OLIG2 antibodies ( Merge includes DAPI ) . ( D ) Quantification of ICC experiment described in ( C ) after 1 , 2 , and 4 days of differentiation . ( E ) Representative images of IHC analysis of the dorsal SVZ with BrdU and OLIG2 antibodies comparing control and hGFAP-cre;Ezh2F/F P17 slices . ( F ) Quantification of IHC experiment described in ( E ) demonstrating a significant increase of OLIG2+ BrdU+ cells in hGFAP-cre;Ezh2F/F vs Control slices ( *p=0 . 0289 , n = 3 ) . ( G ) Quantification of ICC images generated from a differentiation timecourse analysis ( Figure 4—figure supplement 2B ) demonstrating significant decrease of Tuj1+ cells in Ezh2Δ/Δ;Ink4a/Arf−/− SVZ NSC 7 d post-differentiation cultures infected with GFP-expressing lentiviruses that can conditionally express OLIG2 with exposure to Dox ( **p=0 . 0014 , n = 3 ) . ( H ) Quantification of ICC images generated from a differentiation timecourse analysis ( Figure 4—figure supplement 2E ) demonstrating significant increase of Tuj1+ cells in Ezh2Δ/Δ;Ink4a/Arf−/− SVZ NSC Day 3 post-differentiation cultures infected with GFP-expressing lentiviruses expressing shRNAs specific to Olig2 compared to those expressing Luciferase as a control ( ****p<0 . 0001 , n = 3 ) . Data are represented as ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 02439 . 01110 . 7554/eLife . 02439 . 012Figure 4—figure supplement 1 . FACS analysis of SVZ NSCs after 0 or 4 days of differentiation . ( A ) Scatter plot of FACS performed on undifferentiated SVZ NSCs using GFAP and Nestin antibodies . ( B ) Scatter plot of FACS performed on SVZ NSCs after 4 days in differentiation conditions using a Tuj1 antibody . DOI: http://dx . doi . org/10 . 7554/eLife . 02439 . 01210 . 7554/eLife . 02439 . 013Figure 4—figure supplement 2 . Aberrant expression of OLIG2 in Ezh2Δ/Δ;Ink4a/Arf−/− SVZ NSCs inhibits neuronal differentiation . ( A ) Images of differentiating rtTA-expressing SVZ NSCs infected with a LV expressing GFP and encoding tetO-Olig2 . Arrows point to GFP+ cells expressing OLIG2 upon addition of Dox in top panel . ( B ) Schematic of experimental setup with infection of rtTA-expressing SVZ NSC with LV two days prior to differentiation . OLIG2 is induced by treatment of cultures with Dox from Day 0 to 3 in differentiation conditions by addition of Dox to the media . Images show representative fields from quantification in Figure 4G . ( C ) Representative images and schematic showing Tuj1+ GFP+ cells when OLIG2 was induced by Dox treatment from Day 3 to 7 after differentiation induction . ( D ) Images of proliferating SVZ NSCs infected with a LV expressing GFP and either an Olig2 knockdown ( KD ) or Luciferase KD construct . Arrows point to GFP+ cells expressing OLIG2 in Luciferase KD but not Olig2 KD LV infected cells . ( E ) Schematic of experimental setup with infection of KD LV a day prior to differentiation . Images show representative fields from quantification in Figure 4H . ( F ) Quantification of the proportion of GFAP+ cells infected with Luciferase-KD virus compared to those infected with Olig2-KD virus in Control;Ink4a/Arf−/− or Ezh2Δ/Δ;Ink4a/Arf−/− SVZ NSCs ( n = 3 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 02439 . 01310 . 7554/eLife . 02439 . 014Figure 4—figure supplement 3 . Identification of transcriptional modules that require EZH2 for proper downregulation during differentiation . To identify other potential targets of Ezh2-dependent gene repression besides Olig2 that are related to SVZ NSC neurogenesis , we analyzed by expression microarray the transcriptional profiles of Ezh2Δ/Δ;Ink4a/Arf−/− and Control;Ink4a/Arf−/− SVZ NSCs across a differentiation timecourse ( 0 , 1 , 2 , 4 , and 7 d after differentiation ) . We used gene co-expression analysis ( Zhang and Horvath , 2005; Oldham et al . , 2008; Ramos et al . , 2013 ) , which organizes genes into modules that exhibit similar expression patterns , to analyze the expression data . ( A ) Top panels contain heatmaps representing the expression levels for three modules ( saddlebrown , white , and darkgreen ) of 46 co-expressed transcripts that , like Olig2 , require Ezh2 for repression during neuronal lineage specification and would be expected to exhibit increased expression in Ezh2Δ/Δ;Ink4a/Arf−/− cells early in the differentiation time course , as compared to control cultures . Red = increased expression , Green = decreased expression , Black = no change . Bottom panels contain plots of the eigengene values in each module representing the first principal component of each module ( ‘Materials and methods’ for details of analysis ) . ( B ) Expression patterns for the top 15 genes ranked by their Pearson correlation to the first principal component of each coexpression module ( Oldham et al . , 2008 ) are shown . Samples on the x-axis correspond to those depicted in ( A ) . DOI: http://dx . doi . org/10 . 7554/eLife . 02439 . 01410 . 7554/eLife . 02439 . 015Figure 4—figure supplement 4 . Ezh2-dependent genes during early differentiation are enriched for homeobox-containing neuronal transcriptional regulators and H3K27me3 . ( A ) Selected functional categories of the combined genes identified from the saddlebrown , white , and darkgreen modules that are significantly enriched after DAVID analysis ( Huang da et al . , 2009 ) . ( B ) ChIP-seq analysis of proliferating SVZ NSCs with antibodies to H3K4me3 ( green ) and H3K27me3 ( red ) demonstrating bivalency at three genomic loci identified from the module analysis ( Six1 , En2 , and Isl1 ) . Note that the majority of genes ( 74% , 34/46 ) in these modules had H3K27me3 enrichment within 1 kb of their transcriptional start site , versus only 7% ( 381/5566 ) of genes across all modules , indicating that most of these dysregulated genes are likely direct targets of EZH2 . ( C ) Comparison of our ChIP-seq profiles ( Mouse SVZ ) for selected loci to published ChIP-seq profiles from other mouse and human tissues ( Rosenbloom et al . , 2013 ) . Note the predominance of the repressive H3K27me3 mark on Olig2 in human astrocytes , consistent with our observation of direct EZH2 repression of Olig2 upon differentiation in the mouse SVZ . In addition , genes encoding homedomain-containing transcription factors are marked with chromatin states that correlate with their activity in various tissues ( e . g . , En2 active but Six1 and Isl1 repressed in cerebellum ) , suggesting that epigenetic regulation by factors like EZH2 may be necessary to prevent their inappropriate expression . DOI: http://dx . doi . org/10 . 7554/eLife . 02439 . 01510 . 7554/eLife . 02439 . 016Figure 4—figure supplement 5 . List of genes identified in the saddlebrown , white , and darkgreen modules . Interestingly , many of the genes that exhibited Ezh2-dependent transcriptional repression encode for homeodomain transcription factors involved in the production of neuronal subtypes that are temporally and spatially distinct from OB interneurons . Examples include Six1 , En2 , and Isl1 , which are involved in the embryonic development of olfactory receptor , midbrain dopaminergic , and sensory neurons , respectively ( Simon et al . , 2001; Ikeda et al . , 2007; Sun et al . , 2008 ) ; these loci were ‘bivalent’ for both H3K27me3 and H3K4me3 , consistent with a ‘poised’ transcriptional state ( Figure 4—figure supplement 4B ) , which is typical of key developmental genes in stem cell populations ( Bernstein et al . , 2006 ) . Taken together , these data support a model in which Ezh2-dependent H3K27me3 is required to repress specific sets of homeodomain-containing transcription factors in SVZ NSCs , preventing their inappropriate expression to potentially enable proper neuronal lineage specification . DOI: http://dx . doi . org/10 . 7554/eLife . 02439 . 016 Similar to cultured SVZ NSCs , OLIG2 is coexpressed with EZH2 in a subset of type B and C cells in vivo ( Figure 1E ) . Migratory neuroblasts , however , do not express OLIG2 ( Hack et al . , 2005; Marshall et al . , 2005 ) , which suggested to us that this developmental regulator becomes repressed during neuronal differentiation . To compare the chromatin state of NSC and neuroblast populations , we used fluorescence activated cell sorting ( FACS ) to isolate cultured GFAP+ , Nestin+ SVZ NSCs before differentiation and Tuj1+ neuroblasts 4 d after differentiation ( Figure 4—figure supplement 1A , B ) , As expected , high levels of H3K27me3 at Hoxa2b were similar in both undifferentiated GFAP+ , Nestin+ NSCs and the Tuj1+ neuroblast population ( Figure 4B ) . Also , H3K27me3 levels remained low at Gapdh in neuroblasts ( Figure 4B ) . In contrast , H3K27me3 was strikingly increased at Olig2 in neuroblasts as compared to the GFAP+ , Nestin+ cell population ( Figure 4B ) . Furthermore , ChIP analysis of acutely isolated SVZ cells identified EZH2 and H3K27me3 enrichment at Olig2 ( Figure 3A , B ) , indicating that Olig2 is likely a direct target of EZH2 in the SVZ in vivo . This chromatin analysis suggested that Ezh2 is required for Olig2 repression during neuronal differentiation . To investigate whether Olig2 is expressed aberrantly in Ezh2-deleted SVZ cells , we first performed ICC on Ezh2Δ/Δ;Ink4a/Arf−/− and Control;Ink4a/Arf−/− SVZ NSC cultures . After 1 , 2 , and 4 d of differentiation , Ezh2-deleted cultures had a greater proportion of OLIG2+ cells ( Figure 4C , D ) . To ask whether Ezh2-deleted SVZ cells also generate greater numbers of OLIG2+ cells in vivo , we injected BrdU into mice 1 hr before sacrifice and analyzed the SVZ for BrdU+ , OLIG2+ cells . Compared to controls , there were greater than twofold more BrdU+ , OLIG2+ cells in the SVZ of hGFAP-cre;Ezh2F/F mice ( Figure 4E , F ) . Thus , Ezh2-deletion in SVZ NSCs results in aberrant expression of OLIG2 both in vitro and in vivo . To test whether increased OLIG2 levels in SVZ cells inhibits neurogenesis , we expressed Olig2 with a doxycycline ( Dox ) -inducible Olig2 lentiviral vector ( LV-GFP-tetO-Olig2 ) in SVZ NSC cultures derived from rtTA-mice ( Figure 4—figure supplement 2A ) . When we added Dox during early differentiation ( Day 0–3 ) , the number of Tuj1+ neuroblasts after 7 d of differentiation was greatly reduced as compared to no-Dox control ( Figure 4G , Figure 4—figure supplement 2B ) . Interestingly , when Dox was added shortly after the onset of differentiation ( Day 3–7 ) , neurogenesis was not affected ( Figure 4—figure supplement 2C ) . Thus , aberrant OLIG2 expression during early stages of differentiation inhibits neurogenesis from SVZ NSCs . We next asked whether reduction of Olig2 in Ezh2-deleted SVZ NSCs could rescue neuronal differentiation . Cells infected with a lentiviral short hairpin RNA Olig2 knockdown vector ( LV-GFP-Olig2-shRNA ) exhibited significantly reduced OLIG2 expression as compared to cells infected with the LV-GFP-Luciferase-shRNA control ( Figure 4—figure supplement 2D ) . Ezh2Δ/Δ;Ink4a/Arf−/− SVZ NSCs with Olig2-knockdown produced nearly threefold more Tuj1+ cells after 3 d of differentiation as compared to cells infected with the control vector with no significant increase of GFAP-positive cells ( Figure 4H , Figure 4—figure supplement 2E , F ) . Olig2-knockdown in Control;Ink4a/Arf−/− SVZ NSCs did not increase neurogenesis , suggesting that only aberrant Olig2 expression in Ezh2-deleted cells inhibits neuronal differentiation ( Figure 4H ) . Of note , Ezh2Δ/Δ;Ink4a/Arf−/− NSCs with Olig2-knockdown did not generate Tuj1+ cells with the same efficiency as Control;Ink4a/Arf−/− SVZ NSCs , indicating that EZH2 likely regulates other genes for efficient neurogenesis . Thus , we performed microarray and H3K27me3 ChIP-seq analysis to identify specific transcriptional modules that required EZH2 for proper transcriptional repression ( Figure 4—figure supplements 3 and 4 ) . Interestingly , many of the genes that exhibited Ezh2-dependent transcriptional repression encode for homeodomain transcription factors involved in the production of neuronal subtypes that are temporally and spatially distinct from OB interneurons ( Figure 4—figure supplement 5 ) . Taken together , these data indicate that SVZ NSCs require EZH2 for the repression of specific sets of genes including Olig2 for proper neuronal lineage specification . Astrocytes isolated from the adult human SVZ can serve as NSCs in vitro ( Sanai et al . , 2004 ) , and adult human SVZ neurogenesis has been described in vivo ( Sanai et al . , 2004; Quinones-Hinojosa et al . , 2006; Curtis et al . , 2007 ) . While the infant human SVZ has large numbers of DCX+ neuroblasts , this neurogenesis declines sharply during the first 18 months of life ( Sanai et al . , 2011 ) . To investigate whether EZH2 expression correlates with this early postnatal human neurogenesis , we analyzed human brain sections containing the lateral ventricle SVZ ( Figure 5A , B ) at increasing ages of development . 10 . 7554/eLife . 02439 . 017Figure 5 . EZH2 expression in the infant human subventricular zone decreases post-birth . ( A ) Schematic illustrating the location of the 30 µm thick sections of the anterior subventricular zone stained in ( C ) and ( D ) . RMS = rostral migratory stream; LV = lateral ventricle . ( B ) Magnification of the lateral ventricle region and sectioned region . ( C ) IHC co-staining with DAPI ( blue ) of sections from varying ages of early human development ( gestational week 22 , 1 week , and 18 month timepoints ) with antibodies to EZH2 ( yellow ) , DCX ( red ) , and GFAP ( green ) . Note the presence of a blood vessel ( BV ) surrounded by DCX + cells in the 1 week timepoint ( ** indicates non-specific signal from EZH2 staining ) . Boxes in the 1 week timepoint indicate the region examined in ( D ) . ( D ) Magnified view of a region of the 1 week section in ( C ) co-stained by IHC with antibodies to EZH2 ( grey ) , DCX ( red ) and GFAP ( green ) demonstrating EZH2+ cells that are either DCX+ ( closed arrowhead ) , GFAP+ ( arrow ) , or negative for both DCX and GFAP ( open arrowhead ) . Scale bars , 20 µM . DOI: http://dx . doi . org/10 . 7554/eLife . 02439 . 017 At gestational week 22 , EZH2 was detected in many SVZ cells ( Figure 5C ) as well as cells in the developing neocortex ( data not shown ) . In the 1-week-old infant , EZH2 was prominent in the SVZ ( Figure 5C ) , but sporadic in non-neurogenic regions more distant from the ventricle wall ( data not shown ) . In the 1-week old SVZ , EZH2 was detected in a subset of GFAP+ cells as well as clusters of DCX + cells ( Figure 5D ) , indicating that EZH2 is expressed in cell types that correspond to cells of the neurogenic lineage . However , by 18 months of age , when SVZ neurogenesis is nearly extinct ( Sanai et al . , 2011 ) , EZH2+ cells were not observed in the SVZ ( Figure 5C ) . Thus , EZH2 is expressed in SVZ astrocytes and neuroblasts during the period of postnatal human neurogenesis .
Despite many immunohistological , ultrastructural , and electrophysiological similarities between SVZ astrocytes and those in non-neurogenic brain regions , it is clear that astrocytes in the SVZ are distinct in their ability to produce neurons for all of adult life . Our data indicate that EZH2 is a distinguishing feature of SVZ astrocytes and in part underlies the epigenetic basis of this unique form of astrocyte heterogeneity ( Figure 6 ) . Below , we discuss our results in the broader contexts of neural development , brain tumor malignancy , and postnatal neurogenesis of the human brain . 10 . 7554/eLife . 02439 . 018Figure 6 . Model of EZH2 function in maintaining the neurogenic potential of SVZ astroglia . EZH2 represses multiple targets to promote distinct and separable aspects of adult SVZ neurogenesis . DOI: http://dx . doi . org/10 . 7554/eLife . 02439 . 018 Astrocytes throughout the brain arise from radial glia , the primary multipotent neural precursor cells population of the developing brain ( Kriegstein and Alvarez-Buylla , 2009 ) . While most astrocytes do not generate neurons in the early postnatal brain , SVZ astrocytes retain the ability to produce neurons and glia throughout life . Thus , conceptually , SVZ astrocytes appear to have ‘preserved’ the multi-lineage developmental potential of their radial glial cell origin , whereas non-neurogenic astrocytes apparently ‘lose’ this stem cell characteristic ( Gonzales-Roybal and Lim , 2013 ) . The expression of EZH2 strongly correlated with the neurogenic potential of glial cells along this developmental continuum . EZH2 is expressed in embryonic radial glia and is required for proper cortical neuron differentiation ( Hirabayashi et al . , 2009; Pereira et al . , 2010 ) . We detected EZH2 expression in early postnatal radial glia , including those that give rise to the SVZ astrocytes . While EZH2 expression decreased over time in non-neurogenic brain regions , this PRC2 factor remained highly expressed in SVZ astrocytes into adulthood . Interestingly , PRC1 component BMI1—which is required for SVZ NSC self-renewal ( Molofsky et al . , 2003 ) —does not become similarly restricted to neurogenic astrocytes and is instead also expressed in mature neurons and astrocytes of the adult brain ( Chatoo et al . , 2009 ) . This suggests that BMI1 , while clearly required for adult neurogenesis , is not a distinguishing feature of SVZ astrocytes; instead , BMI1 may function more broadly in a variety of mature and immature neural cell types . During the first 2 weeks of life , astrocytes isolated from the cortex can give rise to multipotent NS cultures ( Laywell et al . , 2000 ) . After these first 2 postnatal weeks , cortical astrocytes lose their ability to generate such neurogenic cell cultures . Intriguingly , this reported loss of neurogenic competence during postnatal development parallels our observation that EZH2 was no longer detected in the cortex by P15 . Furthermore , the ectopic expression of EZH2 in cultured postnatal astrocytes induces the expression of Nestin and other markers of radial glia ( Sher et al . , 2011 ) . Future studies may address whether re-expression of EZH2 in non-SVZ astrocytes can enable neurogenesis or their directed transdifferentiation into neurons in vivo for potential therapeutic purposes . Acute deletion of Ezh2 in SVZ NSCs resulted in decreased neurogenesis , and this deficiency in part related to reduced cell proliferation . As has been found in other stem cell systems ( Aloia et al . , 2013 ) , in SVZ cells , Ink4a was enriched with both H3K27me3 and the EZH2 methyltransferase . Deletion of Ezh2 increased the expression of the p16 cell cycle inhibitor encoded by Ink4a . These findings are coherent with the finding that Ink4a repression by BMI1 is required for SVZ NSC self-renewal ( Molofsky et al . , 2003 ) . Given our current understanding of Polycomb-mediated gene repression ( Margueron and Reinberg , 2011 ) , these data suggest that Ink4a repression in SVZ astrocytes requires EZH2 for the local H3K27me3 enrichment , and that BMI1 is required for the subsequent transcriptional repression mediated by PRC1 . While these data clearly indicated that EZH2 is required for proper SVZ NSC proliferation , the expression of EZH2 in type C and A cells suggested that EZH2 also plays a role in neuronal lineage specification . By deleting Ezh2 in Ink4a/Arf−/− SVZ NSCs , we were able to evaluate the Ink4a/Arf-independent roles of Ezh2 in neurogenesis . This genetic strategy enabled the identification of Olig2 as a key direct target of EZH2-mediated repression . OLIG2 is expressed in a subset of both type B1 and type C cells , but not in migratory neuroblasts ( Hack et al . , 2005; Menn et al . , 2006 ) , suggesting that OLIG2 is expressed only at early stages of the SVZ neurogenic lineage . However , the epigenetic mechanisms required to repress Olig2 during neurogenesis were not known . Our results demonstrate that EZH2 is critical for Olig2 repression , which we found to be a key aspect of SVZ neurogenesis . While EZH2 is well recognized for its role in maintaining stem cells in an undifferentiated state , the EZH2-dependent downregulation of Olig2 illustrates a critical function for EZH2 in cell lineage specification . How EZH2-mediated repression is targeted to specific loci is still not well understood . In a subpopulation of SVZ cells , EZH2 was co-expressed with OLIG2 ( Figure 1E ) , suggesting that while EZH2 is present in these cells , its repressive activities have not been localized to the Olig2 locus . Future work may reveal how other factors , possibly including long non-coding RNAs ( Simon and Kingston , 2013 ) , serve to dynamically target EZH2 to specific loci at different states of the neurogenic lineage . In addition to repressing Olig2 , EZH2 was also required to prevent the aberrant expression of many homeodomain-containing transcription factors ( Figure 4—figure supplement 3 , 4 , and 5 ) , including those involved in the embryonic development of non-OB neurons ( Simon et al . , 2001; Ikeda et al . , 2007; Sun et al . , 2008 ) , suggesting that EZH2 plays a role in restricting SVZ NSCs to neuron subtypes appropriate for the OB . It remains to be determined whether the misexpression of these transcription factors contributes to the neurogenic defect of Ezh2-deleted SVZ NSCs . While EZH2 was not observed in mature , non-neurogenic astrocytes , the expression of this H3K27 methyltransferase is common to high-grade gliomas , including glioblastoma multiforme ( GBM ) ( Crea et al . , 2010; Orzan et al . , 2011 ) . Given that EZH2 directly targeted Ink4a/Arf , a tumor suppressor locus that is frequently inactivated in human GBM ( Brennan et al . , 2013 ) , it is possible that EZH2 in SVZ astrocytes increases their oncogenic potential . Indeed , mouse SVZ precursors give rise to tumors more frequently than non-SVZ cells when oncogenic alterations are induced ( Alcantara Llaguno et al . , 2009 ) . Thus , EZH2 has been investigated as a potential chemotherapeutic target for gliomas . Inhibition of EZH2 in human GBM cell lines results in decreased cell proliferation ( Suva et al . , 2009 ) , and in other GBMs , EZH2 knockdown results in derepression of BMPRIB , which may reduce tumorigenicity ( Lee et al . , 2008 ) . However , recent discoveries raise questions about this therapeutic approach . Expression of H3 . 3 with a K27M mutation results in a dominant-negative inhibition of PRC2 function , leading to a global decrease in H3K27me3 , and this K27M H3 . 3 mutation is a major driver of pediatric high-grade glioma ( Bender et al . , 2013; Lewis et al . , 2013 ) . Our finding that EZH2 can have distinct and separable developmental roles provides a conceptual resolution to these seemingly paradoxical findings . In some gliomas , inhibition of EZH2 may reduce cell proliferation , possibly by de-repressing Ink4a/Arf . However , in other gliomas , loss of EZH2 function could lead to the aberrant expression of factors such as OLIG2 , which can drive tumorogenesis ( Ligon et al . , 2007 ) . Intriguingly , OLIG2 is one of most highly expressed genes in the K27M glioma subtype ( Chan et al . , 2013 ) . Taken together , these findings suggest that the development of EZH2 inhibitors for cancer chemotherapy will benefit from a continued understanding of the multiple , distinct roles that EZH2 plays in development and cell differentiation . While most neurogenesis in the human brain is complete by the time of birth , the infant brain SVZ contains many young migrating neurons ( Sanai et al . , 2011 ) . In the one-week old human brain , EZH2 expression was primarily observed in DCX+ neuroblasts and a subset of GFAP+ astroglia . Thus , EZH2 is expressed in GFAP+ astroglia in the human SVZ when neurogenesis is observed . In the 18-month-old brain , when SVZ neurogenesis is sparse , we did not find EZH2+ cells in the SVZ . In particular , we did not observe GFAP+ , EZH2+ cells at this time point , though we cannot rule out the existence of EZH2+ cells in other parts of the SVZ . Nevertheless , for this region of the lateral ventricle SVZ , the near absence of postnatal neurogenesis correlated with the loss of astroglia expressing EZH2 . This suggests that this specialized , neurogenic astrocyte population is transient in the human brain . Future studies will be required to determine whether the loss of EZH2 expression is a cause or a consequence of this decrease in SVZ neurogenic activity . Taken together , our studies establish EZH2 as a distinguishing feature of astroglial cells in the postnatal neurogenic SVZ of both the mouse and human brain . Furthermore , we identify distinct and separable roles for EZH2 in the production of new neurons from a multipotent stem cell population . These results provide novel insights into the complex role that this Polycomb repressive factor plays in normal development and gliomagenesis .
Ezh2 F/F mice featuring LoxP sites flanking the four exons that encode for the essential SET domain were maintained and genotyped as previously described ( Su et al . , 2003 ) . hGFAP-Cre ( Zhuo et al . , 2001 ) and UBC-Cre/ERT2 mice were purchased from from Jackson Laboratories and described previously . To label proliferating cells , BrdU ( 10 mg/ml solution , 50 μg/gram body weight , Sigma , St . Louis , MO ) or EdU ( 10−2 μmol/gram body weight , Invitrogen , Carlsbad , CA ) were injected intraperitoneally with regimens as described in the figure legends . Animal experiments were performed in accordance to protocols approved by Institutional Animal Care and Use Committee at UCSF . SVZ NSC monolayer cultures were established and maintained as previously described ( Scheffler et al . , 2005 ) . Briefly , SVZ microdissections from the brains of P7 to P9 mice were dissociated with 0 . 25% trypsin and trituration in proliferation medium ( DMEM/F12/N2–Invitrogen , 5% FCS–Hyclone , Logan , UT , 20 ng ml−1 EGF , 20 ng ml−1 bFGF , 35 μg ml−1 bovine pituitary extract–Peprotech , Rocky Hill , NJ ) . SVZ tissue from one mouse was plated into one well of a 12-well plate , grown to confluence and subsequently passaged with 1:2 with 0 . 25% trypsin . For analysis of neuronal or astrocyte differentiation , passage 5 cells were switched to differentiation media ( same as proliferation media except with 2% FCS and no EGF or bFGF ) and let proceed for the specified time . For cell proliferation analysis , BrdU was used at 10 μM . To conditionally delete EZH2 function from UBC-CreERT2;Ezh2F/F ( Ezh2Δ/Δ ) SVZ NSCs with or without Ink4a/Arf deletion , 4OHT dissolved in ethanol was added to proliferation media ( final concentration 50 nM ) of cultures during passages 3 and 4 post-dissection and establishment . Monolayer cells were trypsonized to single cell suspension and then resuspended in NS media ( In neurobasal media minus Glutamine , N2 , B27–Invitrogen , L-Glutamine , Penicillin/Streptomycin–UCSF Cell Culture Facility , San Francisco , CA , supplemented with 10 nM EGF or bFGF every 4 days ) . NS can be passaged by treatment with Accutase ( Millipore , Billerica , MA ) to dissociate into single cell suspension . For soft agar assay , 1 mL of 0 . 8% of low melting point ( LMP ) agarose ( Life Technologies , Carlsbad , CA ) with 1x B27 and Glutamine is solidified in a six-well plate as a base . 5 ml of this 0 . 8% agarose medium is added to 2 . 5 ml of NS media + EGF ( final 40 ng/ml ) and bFGF ( final 20 ng/ml ) to make 0 . 6% media . Using 1 ml of 0 . 6% media , 30 , 0000 cells are resuspended and plated on top of the solidified 0 . 8% agarose base . 0 . 5 ml of NS media + EGF/FGF is added every 4 days until NS are large enough to count and measure . For ectopic expression of EZH2 , Ezh2 was cloned into a pCS-CG vector and high-titer lentiviruses ( LV ) were generated ( Han et al . , 2009 ) from this construct in HEK 293T cells and infected into SVZ cells as described in Figure 3G . For inducible OLIG2 expression , Olig2 was cloned into a TetO-FUW-T2A-EGFP doxycyline inducible vector and packaged into LV as above . Doxycyline at 2 µg/ml was added to media for induction of Olig2 . For Olig2 knockdown experiments , a shRNA specific to Olig2 and a control shRNA sequence for Luciferase were cloned into the lentiviral pSicoR vector ( Ventura et al . , 2004 ) and packaged into LV as above . All specimens were collected with informed consent and in accordance with the University of California San Francisco Committee on Human Research ( IRB no . H11170-19113 ) . Tissues were obtained and processed as in Sanai et al . ( 2011 ) . For ICC , cells were fixed by treatment with 4% paraformaldehyde ( PFA ) for 20 min and were blocked for 30 min with blocking buffer ( PBS with 10% natural goat serum , 1% bovine serum albumin , 0 . 3M glycine , 0 . 3% Tx100 ) . Cells were incubated for 1 hr at room temperature with primary antibody diluted in blocking buffer as described in Supplementary file 1 . Cells are subsequently incubated with secondary antibodies ( Invitrogen ) and DAPI at room temperature for 45 min ( washes in between each step with 0 . 1% Tx100 ) . Slides are mounted with Aqua Poly/Mount ( Polysciences Inc . , Warrington , PA ) . For IHC , brains were fixed by intracardiac perfusion with 4% PFA and sectioned on either a Leica cryostat or vibratome ( 12 μm or 40 μm thickness , respectively ) . For EZH2 staining of cryostat sections , antigen retrieval by steaming was performed as previously described ( Tang et al . , 2007 ) for 20 min in 10 mM sodium citrate . For EZH2 and BrdU staining of vibratome sections , antigen retrieval was performed by acid treatment with 1N HCl for 10 min on ice , 2N HCl for 10 min at room temperature ( RT ) and then 20 min at 37°C , 0 . 1M Borate buffer for 12 min , and subsequent washes with Phosphate Buffered Saline ( PBS ) + 0 . 1% TritonX100 ( Tx100 ) . After blocking as described for ICC , sections were incubated at 4°C overnight with primary antibodies diluted in blocking buffer and then subjected to secondary incubation and mounting as described for ICC . For human sections , additional treatments were required to reduce background autofluorescence and enhance EZH2 staining via TSA Plus Cyanine 5 kit ( PerkinElmer , Waltham , MA ) . After 10 min of steaming in 10 mM Sodium Citrate ( reduced from 20 min for mouse to minimize damage to tissue ) , sections were treated for 30 min in 1% H2O2 to eliminate background peroxidase activity and washed 3x with PBS + 0 . 05% Tx100 ( all subsequent steps at room temperature unless noted ) . Sections were then incubated 20 min in NaBH4 and washed 3x with PBS + 0 . 05% Tx100 before proceeding with blocking and staining . Sections were incubated for 2 days overnight at 4°C with EZH2 primary , washed and then incubated 45 min with anti-mouse conjugated to HRP along with other secondaries and DAPI for costaining . After washes , slides were treated for 5 min in Cy5 Amplification Reagent ( 1:50 ) in 1X Amplification Diluent and rinsed 3x with PBS + 0 . 05% Tx100 . Sections are treated with 4% PFA for 15 min to fix the fluorescence followed by 3x PBS + 0 . 05% Tx100 wash . Slides are incubated with 1 mM CuSO4 in 50 mM ammonium acetate for 1 hr , washed 3x with PBS + 0 . 05% Tx100 , and finally mounted with Aqua Poly/Mount and coverslipped for visualization . For quantification of cell cultures ( triplicate wells per condition ) , images were captured using either the Leica DMI4000B epifluorescent microscope ( 20X objective ) and analyzed using ImageJ ( NIH ) software or the GE IN Cell Analyzer 2000 ( 20X or 10X objective ) and analyzed with the GE IN Cell Investigator software suite . For in vivo SVZ cell quantification , we collected confocal z-section images from tissue sections using a Leica TCS SP5X with 20X objective; from three animals per condition , at least two high powered fields ( HPF ) were z-projected and analyzed using the Fiji distribution of ImageJ ( Schindelin et al . , 2012 ) . Statistical tests of significance were analyzed using t Test in GraphPad Prism 6 . RNA was isolated by using the RNeasy Plus Mini Kit ( Qiagen , Valencia , CA ) and quantified using the NanoDrop spectrophotometer . cDNA was synthesized using Transcriptor First Strand cDNA Synthesis ( Roche , Switzerland ) , and qPCR was performed using SYBR Green I master mix ( Roche ) on a Roche LightCycler 480 ( Roche ) . Relative expression for was normalized to RPLP . ChIP was performed as previously described ( Lim et al . , 2009 ) with minor modifications detailed below . Sonication with using the Biorupter ( Diagenode , Denville , NJ ) was performed with the following parameters: high setting , 20 cycles of 30 s on followed by 30 s off . The lysate was diluted and incubated in 2 µg of either EZH2 , H3K27me3 , or IgG antibody and pulldown was performed using protein G dynabeads ( Invitrogen ) . Primer sequences qPCR analysis can be found in Supplementary file 2 . SVZ NSC cultures ( 0 and 4 d of differentiation ) were fixed as above for ChIP and then rinsed with PBS three times . 0 d cells were incubated with Alexa Fluor 488-conjugated GFAP and Alexa Fluor 647-conjugated Nestin antibodies ( 1:40; BD Pharmingen , San Jose , CA ) in blocking buffer for 30 min on ice . 4 d cells were treated identically except incubated with Alex Fluor 488-conjugated Tuj1 antibody ( 1:40; BD Pharmingen ) . Using a FACSAria ( BD Pharmingen ) cell sorter with a 100 μm nozzle and relatively low pressure , we first set FSC and SSC gates to eliminate debris and potential cell doublets . Unstained SVZ cells , Alexa Fluor 488 or Alexa Fluor 647 stained cells were individually used to set the sort gates . The fixed , FACS-isolated cells were then pelleted by centrifugation at 4°C and washed three times with ice-cold PBS containing protease inhibitor cocktail ( Roche ) . For ChIP-seq experiments , ChIP was performed as described above using cultured SVZ monolayers . Libraries were prepared using Illumina PE primers via standard protocols using reagents found in the Illumina Library Preparation Kit ( NEB , Ipswich , MA ) . DNA was sequenced using the Illumina HiSeq 2000 DNA Sequencer at the UCSF Genomics Core Facilities . Peak-calling was performed using MACS with adjustments for the no model , no lambda method for H3K27me3 as previously described ( Feng et al . , 2011 ) . Data was visualized using the UCSC Genome Browser ( http://genome . ucsc . edu/ ) ( Kent et al . , 2002 ) and can be accessed through the Gene Expression Omnibus ( GEO ) at http://www . ncbi . nlm . nih . gov/geo/query/acc . cgi ? acc=GSE56394 . Total RNA was isolated by Trizol ( Life Technologies ) , purified using the RNeasy Plus Mini Kit ( Qiagen ) and quantified using the NanoDrop spectrophotometer . Samples were checked for quality using an Agilent TapeStation and hybridized to Illumina expression microarrays ( Mouse Ref 8 v 2 . 0 ) by the UCLA Neuroscience Genomics Core ( http://www . semel . ucla . edu/ungc ) . Gene expression data were quantile normalized in R using the Bioconductor suite of software packages . Quality control was performed using the SampleNetwork R function ( Oldham et al . , 2012 ) . This analysis revealed two sample outliers ( 1 control and 1 Ezh2Δ/Δ;Ink4a/ARF−/− sample from day 1 ) , which were hybridized on a different day than the remaining samples and were removed from the analysis . The data can be accessed through the Gene Expression Omnibus ( GEO ) at https://www . ncbi . nlm . nih . gov/geo/query/acc . cgi ? acc=GSE56988 . Gene coexpression modules were identified as described ( Ramos et al . , 2013 ) . Briefly , analysis consisted of four steps: ( 1 ) Pearson correlation coefficients ( cor ) were calculated for all microarray probes across all 24 samples; ( 2 ) probes were clustered using the flashClust ( Langfelder and Horvath , 2008 ) implementation of a hierarchical clustering procedure with complete linkage and 1-cor as a distance measure; ( 3 ) the resulting dendrogram was cut at a static height of ∼0 . 641 ( corresponding to the top 2% of pairwise correlations ) and resulting clusters of at least 10 members were summarized by their first principal component; ( 4 ) highly similar modules were merged if the Pearson correlation coefficients of their first principal components exceeded an arbitrary threshold ( 0 . 85 ) in order of the highest correlation . This procedure yielded 34 coexpression modules , for which the strength of module membership for each transcript was calculated by correlating its expression pattern across all samples with each module's first principal component ( Oldham et al . , 2008 ) . A given transcript was assigned to a module if ( i ) it showed the strongest positive correlation to that module vs all other identified modules , and ( ii ) its correlation was significant ( P value <0 . 05 ) after applying a Bonferroni correction for multiple comparisons ( number of probes X number of modules ) .
The epigenetic mechanisms that enable specialized astrocytes to retain neurogenic competence throughout adult life are still poorly understood . Here we show that astrocytes that serve as neural stem cells ( NSCs ) in the adult mouse subventricular zone ( SVZ ) express the histone methyltransferase EZH2 . This Polycomb repressive factor is required for neurogenesis independent of its role in SVZ NSC proliferation , as Ink4a/Arf-deficiency in Ezh2-deleted SVZ NSCs rescues cell proliferation , but neurogenesis remains defective . Olig2 is a direct target of EZH2 , and repression of this bHLH transcription factor is critical for neuronal differentiation . Furthermore , Ezh2 prevents the inappropriate activation of genes associated with non-SVZ neuronal subtypes . In the human brain , SVZ cells including local astroglia also express EZH2 , correlating with postnatal neurogenesis . Thus , EZH2 is an epigenetic regulator that distinguishes neurogenic SVZ astrocytes , orchestrating distinct and separable aspects of adult stem cell biology , which has important implications for regenerative medicine and oncogenesis .
In addition to the billions of nerve cells called neurons , the brain and spinal cord also contain star-shaped cells called astrocytes . At first it was thought that all astrocytes were the same , but it later became clear that there are several different types of astrocytes that perform different functions . Most neurons are formed in the embryo , but some astrocytes that are found deep within the brain can act as ‘neurogenic stem cells’ and continue to produce new neurons during adult life . However , it was not clear how these neurogenic astrocytes were different from other astrocytes . Now Hwang et al . have found that neurogenic astrocytes contain a protein called EZH2 that is not found in other types of astrocyte in the adult brain . Researchers already knew that this protein , which acts to help keep DNA tightly packed inside the nucleus and to keep genes switched off , was important for brain development . EZH2 was also known to prevent stem cells from prematurely turning into specialized cell types . But , surprisingly , Hwang et al . found that EZH2 has two distinct roles in neurogenic astrocytes: it allows them to multiply to make more astrocytes , and it also helps guide astrocytes into becoming neurons . Hwang et al . showed that different sets of genes were involved in these two roles .