HDAC3 controls gap 2/mitosis progression in adultneural stem/progenitor cells by regulating CDK1 levelsYindi Jiang and Jenny Hsieh1

Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390

Edited by Fred H. Gage, The Salk Institute for Biological Studies, San Diego, CA, and approved July 29, 2014 (received for review June 25, 2014)

The maintenance of the resident adult neural stem/progenitorcell (NSPC) pool depends on the precise balance of proliferation,differentiation, and maintenance of the undifferentiated state.Identifying the mechanisms that regulate this balance in adulthippocampal NSPCs can provide insight into basic stem cell self-renewal principles important for tissue homeostasis and preventingtumor formation. Pharmacological inhibition of histone deacetylases(HDACs), a class of histone-modifying enzymes, have promisingeffects in cancer cells, yet the specific roles of individual HDACs instem cell proliferation is unclear. Here using conditional KO (cKO)mice and in vitro cell culture, we show that histone deacetylase 3(HDAC3) is required for the proliferation of adult NSPCs. Detailedcell cycle analysis of NSPCs from Hdac3 cKO mice reveals a defectin cell cycle progression through the gap 2/mitosis (G2/M) but notthe S phase. Moreover, HDAC3 controls G2/M phase progressionmainly through posttranslational stabilization of the G2/M cyclin-dependent kinase 1 (CDK1). These results demonstrate that HDAC3plays a critical role in NSPC proliferation and suggest that strate-gies aimed at pharmacological modulation of HDAC3 may be ben-eficial for tissue regeneration and controlling tumor cell growth.

adult hippocampal neurogenesis | epigenetic | acetylation |ubiquitination | malignancy

Adult hippocampal neural stem cells, also called radial glial-like (RGL) cells or type 1 cells, self-renew and give rise to

transit-amplifying progenitors (TAPs) before differentiating intogranule neurons and astrocytes in the dentate gyrus subgranularzone (SGZ) (1, 2). Self-renewal of RGLs is tightly regulated topromote proliferation as well as maintain the undifferentiatedstate, which is important for homeostasis and lifelong neuro-genesis. Cell cycle regulators have been extensively studied incell cycle progression in the context of cancer cells (3). However,whether these genes merely control cell proliferation in adultneural stem/progenitor cells (NSPCs) or even control cell fatedecisions, such as remaining quiescent or undergoing differen-tiation, is not entirely clear. Recent studies reveal the roles of G1phase regulators E2f3 and cyclin D not only in cell proliferation,but also in cell fate commitment (4, 5), suggesting a possible roleof cell cycle regulators in mediating self-renewal and differenti-ation of adult NSPCs.Our previous work demonstrated HDAC inhibitors decreased

adult NSPC proliferation and promoted neuronal differentiation(6). This raises the question of the role of specific HDACs in-volved in adult NSPC maintenance and neurogenesis. HDAC1and HDAC2 appear to function redundantly to control theprogression of neural precursors to neurons during brain de-velopment (7). In adult brain, HDAC2 is involved in neuronaldifferentiation and survival (8). Although HDAC3 is highlyexpressed in the brain (9) and has documented roles in learningand memory associated with cocaine-seeking behavior (10), itsrole in adult NSPCs is largely unknown.HDAC3 is best known for its actions as an epigenetic regulator

of gene expression through deacetylation of histone tails (11). Inrecent years, a role of HDAC3 in cell cycle progression has alsobeen uncovered. In human colon cancer cells, HDAC3 levels areelevated, which have been suggested to control cells in bothS and gap 2/mitosis (G2/M) phase (12). Loss of HDAC3 in

hematopoietic progenitor cells results in only S-phase pro-gression defects (13), whereas in HeLa cells, a G1/S transitiondefect was observed after knockdown of HDAC3 (14). However,the underlying mechanisms are still unclear; in particular, whichstage(s) of cell cycle HDAC3 control(s) in NSPCs.Here, we investigate the function and mechanism of HDAC3

in adult NSPCs. From HDAC3 loss of function experimentsin vitro and in vivo, we identified that HDAC3 is required forthe proliferation of adult NSPCs. We further determined thatHDAC3 controls G2/M phase progression in adult NSPCs byblocking ubiquitination and degradation of cyclin-dependent ki-nase 1 (CDK1). These data reveal previously unidentified insightsregarding the key role of HDAC3 in controlling adult neurogenesisby regulating cell proliferation and cell cycle progression.

ResultsHDAC3 Is Required to Promote the Proliferation of Adult NSPCs inVitro. To rapidly determine whether HDAC3 is required foradult NSPC proliferation in vitro, we first designed a shRNAplasmid to knockdown HDAC3 (Fig. S1A). The HDAC3 shRNAwas packaged into a lentivirus vector expressing internal ribo-some entry site (IRES)-EGFP and used to infect a line of pre-viously characterized adult rat hippocampal neural progenitorcells (HCN cell line) (15). Expression of HDAC3 shRNA-IRES-EGFP resulted in fewer GFP+ HCN cells incorporating BrdU,compared with control IRES-EGFP infected cells (Fig. 1A),suggesting HDAC3 is required for NSPC proliferation.As a complementary approach, we obtained an HDAC3-

selective inhibitor (HDAC3i) to test in HCN cells (16). HDAC3ishows 10 times higher selectivity against HDAC3 relative toHDAC1 and HDAC2 (16). It has also been used to investigatethe role of HDAC3 in cocaine-seeking behavior in mice (10). Todetermine the optimum dosage for HDAC3i in HCN cells, we per-formed the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay to determine its cytotoxicity

Significance

Cell cycle regulation is one of the most fundamental mecha-nisms to control various biological processes, including theproliferation of neural stem/progenitor cells (NSPCs) in adultmouse brain. This study shows that histone deacetylase 3(HDAC3), a well-studied epigenetic factor, is required for theproliferation of neural stem cells. We also demonstrate thatHDAC3 controls gap 2 and mitosis phase of cell cycle throughstabilization of cell cycle protein cyclin-dependent kinase 1.These findings suggest an important role for HDAC3 in NSPCproliferation, and modulation of HDAC3 activity pharma-cologically may be beneficial for tissue regeneration andcancer therapy.

Author contributions: Y.J. and J.H. designed research, Y.J. performed research, and Y.J.and J.H. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. Email: jenny.hsieh@utsouthwestern.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1411939111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1411939111 PNAS | September 16, 2014 | vol. 111 | no. 37 | 13541–13546

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as well as performed Western blotting against acetyl-histoneH4 lysine 12 at different concentrations as a biological readout ofits activity (Fig. S1 B and C). We found that 10 μM minimizedcytotoxicity and maximized biological activity. Thus, 10 μMHDAC3i was used to treat HCN cells for 1 d (Fig. 1B). Weperformed immunostaining of BrdU after adding BrdU for 1 hbefore fixing the cells. Consistent with the effects of knockingdown HDAC3 with an shRNA, HCN cells treated with anHDAC3i also showed significant defects in proliferation as in-dicated by the decreased number of BrdU+ cells.To confirm that HDAC3 is required for NSPC proliferation in

multiple rodent species, we also used established primary NSPCs(neurospheres) from the hippocampus and subventricular zoneof 1-mo-old Hdac3 WT (HDAC3 WT) or floxed (HDAC3 F/F)mice (17). First, we confirmed that adenovirus (Ad) Cre-GFPinfection of Hdac3 floxed neurospheres could efficiently KOHDAC3 levels (Fig. S1D). Using BrdU exposure (1 h) after 2 dof Ad-Cre-GFP infection, we observed a reduction of pro-liferation in Hdac3 floxed neurospheres as indicated by fewerBrdU and GFP double-positive cells (Fig. 1C). Moreover, we didnot observe a significant effect on differentiation, at least underneuronal differentiation conditions (Fig. S1E). Taken together,we showed in primary mouse NSPC cultures and in an adult rat

hippocampal NSPC line that the major effect of deletion or in-hibition of HDAC3 is a proliferation defect in vitro.

Adult NSPCs Lacking HDAC3 Display Specific G2/M Phase Defects.Previous studies showed that HDAC3 could regulate the G1/Sphase transition in HeLa cells (14), but was only required forS–phase progression in mouse embryonic fibroblast (MEF) cells(18), suggesting that HDAC3 has distinct roles on cell cycleprogression in different cell types. To gain further insight intothe role of HDAC3 in NSPC proliferation, specifically, in reg-ulating cell cycle progression, we performed cell cycle analysisfollowing propidium iodide staining of WT and Hdac3 floxedneurospheres. After infection with Ad-Cre-GFP, HDAC3 de-leted neurospheres showed decreased percentage of cells inS phase and increased percentage of cells in G2/M phase com-pared with WT neurospheres (Fig. 2 A and B and Fig. S2 Aand B).To further confirm the role of HDAC3 in cell cycle pro-

gression in adult NSPCs, we treated HCN cells with HDAC3i for1 d before cell cycle analysis. Compared with cells treated withvehicle (DMSO), HDAC3i-treated cells exhibited a decrease inS phase and an increase in G2/M phase (Fig. 2C). To determinewhether decreased S phase and increased G2/M phase was a

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Fig. 1. Loss of HDAC3 results in decreased proliferation in vitro. (A) Im-munocytochemical staining of BrdU followed by 1 h BrdU labeling in HCNcells infected with control (Cont) or HDAC3-shRNA-IRES-EGFP lentivirus for3 d. Quantification of GFP+BrdU+ cells out of the total GFP+ cells is shown. (B)BrdU staining of 1 h BrdU labeling in HCN cells treated with either vehicle(DMSO) or HDAC3i for 1 d. Quantification of BrdU+ cells out of the totalDAPI+ cells is shown. (C) HDAC3 WT or F/F neurospheres were infected withAd-Cre-GFP virus for 2 d. Immunocytochemical staining of GFP and BrdUfollowing 1 h BrdU labeling revealed a decrease in the percentage ofBrdU+GFP+ double-positive cells out of the total GFP+ cells in HDAC3 F/Fneurospheres. In A–C, *P < 0.05. (Scale bars: 200 μm in A and B; 100 μm in C.)All experiments were performed at least three times independently.

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Fig. 2. HDAC3 controls G2/M phase progression. (A and B) Flow cytometryanalysis of HDAC3 WT and F/F neurospheres infected with Ad-Cre-GFP virusfor 2 d before being harvested and fixed in 70% ethanol. Quantification ofthe percentage of cells in S and G2/M phase is shown. (C) Cell cycle analysisof HCN cells treated with HDAC3i or VPA for 1 d. The percentage of each cellcycle stage from one representative experiment is shown in the bar graph.(D) HCN cells were synchronized in the G1/G0 phase by adding BMP4 to theproliferation medium for 1 d before switching to proliferation mediumcontaining either DMSO or HDAC3i. Cells were collected at 6 and 12 h andanalyzed by flow cytometry. The percentage of each cell cycle stage fromone representative experiment is shown in the bar graph. (E) HCN cells weresynchronized in M phase by addition of nocodazole in proliferation mediumfor 16 h before switching to proliferation medium containing either DMSOor HDAC3i. Cells were collected at 6 and 12 h and analyzed by flowcytometry. The percentage of each cell cycle stage from one representativeexperiment is shown in the bar graph. In A and B, *P < 0.05. All cell cycleexperiments were replicated at least three times independently.

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general property of blocking HDACs, we treated HCN cellswith the broad HDACi valproic acid (VPA) for 1 d. Interestingly,we observed an increase in G1/G0 phase and a decrease in S phase,with no change in G2/M phase (Fig. 2C), suggesting that in-dividual HDACs have divergent effects on cell cycle progression.To determine the primary cause of the observed cell cycle

defects after HDAC3 inhibition, we performed cell cycle syn-chronization in HCN cells using various molecules to enrich cellsin either G1/G0 or G2/M phase. We synchronized HCN cells inG1/G0 phase after addition of bone morphogenic protein 4(BMP4) to proliferation medium (fibroblast growth factor 2) for1 d (19) (Fig. 2D and Fig. S2C). To release HCN cells fromG1/G0, BMP4 was withdrawn and cells were treated with eitherHDAC3i or DMSO in proliferation medium. To confirm HCNcells were effectively enriched in G1/G0 and could be releasedback into cell cycle, we performed Western blotting with knowncell cycle proteins (Fig. S2C). We observed that both DMSO-and HDAC3i-treated HCN cells entered into S phase with no lagat 12 h after withdrawal of BMP4, indicating that HDAC3 isnot required for G1/S phase transition and entry into S phase(Fig. 2D).To examine whether HDAC3 is required for M-phase pro-

gression, we enriched HCN cells in M phase with nocodazoletreatment for 16 h before releasing them into HDAC3i orDMSO containing proliferation medium (Fig. 2E and Fig. S2D).To confirm HCN cells were effectively enriched in G2/M andcould be released back into cell cycle, we performed Westernblotting with known cell cycle proteins (Fig. S2D). Twelve hoursafter release from nocodazole, most of the cells treated withHDAC3i were still in G2/M phase, whereas control-treated cellshad entered into G1/G0 phase, similar to cells in proliferationconditions (Fig. 2E). These data suggest that the proliferationdefect observed after HDAC3 inhibition is primarily due to animpairment of G2/M phase progression.

CDK1 Is Selectively Degraded After Loss or Inhibition of HDAC3 inAdult NSPCs. To understand the mechanism by which HDAC3controls G2/M phase progression, we performed Western blot-ting to detect G2/M phase-enriched cyclins and CDKs in bothprimary neurospheres and HCN cells. First, we observed de-creased HDAC3 levels and increased acetylated histone H4 asexpected after Ad-Cre–mediated deletion of HDAC3 in HDAC3F/F neurospheres (Fig. 3A). We also observed a decrease in thelevels of G2/M CDKs and cyclins, such as CDK1, phospho-CDK1, and cyclin B1, but no change in the levels of the G1/Scyclin A (Fig. 3A). Treatment with HCN cells with HDAC3i alsoresulted in decreased CDK1, phospho-CDK1, and cyclin B1(Fig. 3B). Interestingly, in both HDAC3 F/F neurospheres andHCN cells, we found an increase in the mitosis marker phos-phohistone H3 serine 10 (p-H3S10) after HDAC3 deletion orHDAC3i treatment (Fig. 3 A and B), consistent with the in-creased percentage of cells in G2/M phase.To gain further mechanistic insight into HDAC3, we next

examined whether the decline of CDK1 and cyclin B1 levels afterHDAC3 deletion or inactivation was due to its roles as an epi-genetic repressor of gene transcription. Thus, we used quanti-tative PCR (qPCR) to investigate a panel of cell cycle genes andfound that only cyclin B1 mRNA levels were reduced after de-letion of HDAC3 in neurospheres (Fig. 3C). Besides deacetyla-tion of nuclear histone tails, HDAC3 has been described toshuttle between the nucleus and cytoplasm and may also havenonhistone protein targets (20). To examine whether reducedCDK1 levels after loss of HDAC3 occurred through post-translational mechanisms to control protein stability, we mea-sured CDK1 levels by treating HCN cells with HDAC3i anda protein synthesis inhibitor cycloheximide (CHX) (Fig. 3 D andE). Compared with CDK1 levels in cells treated with HDAC3ialone which decreased by 1 d, we observed decreased CDK1levels at 12 h, suggesting that CDK1 has a shorter half-life afterHDAC3 inhibition and that control of CDK1 levels by HDAC3occurred at the posttranslational level.

Ubiquitination is the most common posttranslational modifi-cation to control protein stability (21). To explore whetherCDK1 levels in adult NSPCs is subjected to ubiquitin-mediatedprotein degradation, we first tried to block the proteasomepathway by treating HCN cells with a proteasome inhibitorcarbobenzoxy-Leu-Leu-leucinal (MG132) together with HDAC3ifor 1 d. We observed selective loss of CDK1 after HDAC3itreatment in HCN cells, which was prevented by MG132 treat-ment, without a change in cyclin A or CDK4 levels (Fig. 3F).Interestingly, in non-NSPCs such as human HEK 293T and

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Fig. 3. G2/M phase-specific protein CDK1 is down-regulated after HDAC3deletion. (A) Western blotting against G2/M phase cell cycle proteins in WTand HDAC3 F/F neurospheres infected with Ad-Cre-GFP virus for 2 d and in(B) HCN cells treated with DMSO, VPA, or HDAC3i for 1 d. Levels of CDK1,p-CDK1, and cyclin B1 were quantified. (C) qPCR of several cell cycle genes inWT and HDAC3 F/F neurospheres infected with Ad-Cre-GFP virus. These qPCRassays were repeated three times independently. (D and E) Western blottingagainst CDK1 in HCN cells treated with DMSO or HDAC3i together withprotein synthesis inhibitor CHX for different lengths of time. (F) Westernblot of lysates from HCN cells treated with vehicle (DMSO), MG132, HDAC3i,or HDAC3i+MG132. (G) Immunoprecipitation of CDK1 and Western blottingagainst ubiquitin in HCN cells treated with MG132 and DMSO or HDAC3i.Western blotting of CDK1 from cell lysate is shown (Lower). (H, Left) West-ern blotting against Flag in HCN cells coelectroporated with HA-CDK1, Flag-ubiquitin (Ub), and HDAC3. Western blotting against HDAC3 from cell lysateis shown (H, Lower Left). (H, Right) Immunoprecipitation of CDK1 andWestern blotting against ubiquitin in HCN cells coelectroporated withHA-CDK1, Flag-Ub, and HDAC3 overexpression plasmids. Levels of ubiquiti-nated CDK1 were quantified. The asterisk denotes a nonspecific band. AllWestern blot experiments were replicated three times independently.

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Ink4a/Arf KO mouse glioma cells (SS05 cell line) (22), we didnot observe down-regulation of CDK1 after HDAC3i treatment(Fig. S3A), suggesting that the HDAC3/CDK1 ubiquitination path-way may be selective for NSPCs. To examine CDK1 ubiquitination inNSPCs, we first treated HCN cells with MG132 and DMSO orHDAC3i, followed by immunoprecipitation of CDK1 and Westernblotting against ubiquitin. We observed heavily ubiquitinated bandsin HDAC3i-treated cells, indicating that CDK1 can be ubiquitinatedin HCN cells (Fig. 3G). These results suggested that CDK1 might bedegraded through the ubiquitination pathway after loss or inhibitionof HDAC3 in NSPCs.To further examine whether HDAC3 controls ubiquitination

of CDK1, we overexpressed HA-tagged CDK1 and Flag-taggedubiquitin with or without a cDNA overexpressing full-lengthHDAC3 in HCN cells (Fig. 3H). Following immunoprecipitationof CDK1 and Western blotting against ubiquitin, we observedreduced CDK1 ubiquitination after HDAC3 overexpression,indicating that HDAC3 is sufficient to prevent ubiquitinationof CDK1 in adult NSPCs.Two possible mechanisms by which HDAC3 regulates ubiq-

uitination and stability of CDK1 are through regulation ofubiquitin-related genes (23) or through physical interaction anddeacetylation of CDK1. To examine the first possibility, we ex-amined the levels of Ubqln2, Ube2e3, Ube2K, Usp18, Usp28, andSumo2 and did not detect any differences in gene expressionbetween Hdac3 WT and KO neurospheres (Fig. S3B). Next, toexamine a possible interaction between HDAC3 and CDK1, weimmunoprecipitated CDK1 and performed Western blottingwith HDAC3 in HCN cells (Fig. S3C). Because HDAC3 couldinteract with CDK1, we hypothesized that CDK1 may bedeacetylated by HDAC3. To examine this, we immunoprecipi-tated CDK1 and performed Western blotting with the pan–acetyl-lysine antibody (Fig. S3D). We observed comparablelevels of CDK1 acetylation after HDAC3i treatment or over-expression of HDAC3 compared with DMSO treatment (control)(Fig. S3D), suggesting that HDAC3 is not necessary for CDK1acetylation. Taken together, these results suggest that HDAC3plays a role in stabilizing CDK1 by regulating ubiquitin levels forproper G2/M phase progression in adult NSPCs.

HDAC3 Is Broadly Expressed in Adult NSPC and Neuronal Populations.We have shown that HDAC3 is required for the proliferation ofNSPCs in vitro. To begin to investigate the role of HDAC3during the course of adult neurogenesis in vivo, we determinedthe expression pattern of HDAC3 in adult mouse brain, partic-ularly within the SGZ of the dentate gyrus. We performed im-munohistochemical (IHC) staining using an antibody recognizingHDAC3 together with stage-specific markers. RGLs can be iden-tified using a set of immunohistological markers and morphologicalcriteria (24). We observed HDAC3 expression within glial fibrillaryacidic protein (GFAP)+Sox2+ RGL and GFAP−Sox2+ TAP stem/progenitor cells (Fig. 4A). We quantified the percentage ofGFAP+Sox2+ RGL cells that expressed HDAC3 and foundHDAC3 is expressed in a large proportion of RGLs (94.1 ± 3.6%).We also detected HDAC3 in doublecortin (DCX)+ immaturedentate granule cells as well as in postmitotic NeuN+ neuronsthroughout the hippocampus and cortex (Fig. 4B and Fig. S4).Moreover, HDAC3 was found in MCM2+-proliferating cells inSGZ, consistent with its possible role in regulating proliferationin vivo as it does in vitro (Fig. 4C). Due to the broad expressionof HDAC3 in adult NSPC and neuronal populations and be-cause HDAC3 null mice are reported to die at embryonic stage9.5 due to gastrulation defects (17), we decided to use an in-ducible conditional KO (cKO) strategy to determine the cell-autonomous requirement of HDAC3 in adult NSPCs in vivo.

cKO of HDAC3 Results in Reduced Proliferation of Adult NSPCs andDecreased Neurogenesis. To evaluate the impact of deletingHDAC3 in adult NSPCs and their progeny in vivo, we crossedHdac3 floxed mice with a tamoxifen (TAM)-inducible Nestin-CreERT2 allele (Hdac3 cKO mice) and killed mice at various

time points after TAM (Fig. 5A). These mice were also bred toa R26R-YFP reporter allele so YFP+-recombined cells can beused as surrogate markers for cells recombined and deleted forHDAC3 (25). To validate the efficiency of HDAC3 recombinationafter TAM treatment in adult Hdac3 cKO mice, we performedPCR of the Hdac3 genomic region from primary hippocampalneurospheres isolated from TAM-injected adult Hdac3 floxedmice and were unable to detect Hdac3 gene expression consis-tent with an efficient recombination and deletion of Hdac3(Fig. S5A).To examine the potential ability of HDAC3 to control pro-

liferation in vivo, we killed Hdac3 cKO at an early time point,such as 10 d post-TAM (dpt). We observed a significant re-duction in the number of YFP+Ki67+- and YFP+MCM2+-pro-liferating cells in the SGZ of Hdac3 cKO mice compared withWT littermates (Fig. 5B and Fig. S5B). Moreover, further anal-ysis of YFP

+

cells with cell type-specific markers revealed thatboth the percentage of proliferating YFP+GFAP+Ki67+ RGL cellsout of the total YFP+ cells and the number of YFP+Ki67+DCX−

TAPs were reduced in Hdac3 cKO mice compared with WTmice at 10 dpt (Fig. 5 B and C), indicating that HDAC3 is re-quired for the proliferation of RGL cells as well as TAPs.Finally, one prediction of a defect in NSPC proliferation and

cell cycle progression due to conditional deletion of Hdac3 isa change in neurogenesis over time. Thus, we examined thenumber of immature and mature dentate neurons in Hdac3cKO mice between 30 and 90 dpt by quantification of immatureYFP+DCX+ and mature YFP+NeuN+ cells. We observed a de-creasing trend in the number of YFP+DCX+ cells at 60 dpt, al-though this was not significant (P = 0.062) (Fig. S5C). Consistentwith the reduction in YFP+Ki67+ cells at 10 dpt in HDAC3 cKOmice, we observed a decrease in the number of YFP+NeuN+

adult-born neurons at 60 and 90 dpt (Fig. 5D). To determine if thedecreased generation of adult-born neurons after Hdac3 deletionwas due to a failure to maintain the neural stem cell pool, wequantified the number of YFP+GFAP+Sox2+RGLs at 60 and 90 dpt.

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Fig. 4. HDAC3 is broadly expressed in adult dentate gyrus. (A) Immuno-staining of HDAC3, GFAP (a marker of RGLs), and Sox2 (a marker of RGLsand TAPs) of brain sections from P30 WT mice. HDAC3 is expressed inGFAP+Sox2+ RGL cells (arrow). (B) Immunostaining of HDAC3, DCX (a markerof immature neurons), and NeuN (a marker of mature neurons). HDAC3 isexpressed in both DCX+NeuN− immature neurons (arrows) and DCX−NeuN+

mature neurons. (C) Immunostaining of HDAC3 and MCM2 (a marker ofproliferation). Arrows indicate colocalization of HDAC3 and MCM2. (Scalebar: 50 μm.) One representative image from three independent WT miceis shown.

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Interestingly, although the number of YFP+GFAP+Sox2 RGLsdecreased between 60 and 90 dpt in WT mice as expected, wedid not observe a significant difference in the number of YFP+

GFAP+Sox2+ RGLs in Hdac3 cKO mice compared with WT at60 and 90 dpt (Fig. S5D). These results suggest that the de-creased neurogenesis found in Hdac3 cKO mice could be dueto the decreased proliferation of RGLs and TAPs and notfrom a change in the total number of RGLs. Together theseresults support HDAC3’s major role in regulating adult NSPCproliferation and cell cycle progression to ensure the continu-ous generation of adult-born neurons over time.

DiscussionThe immense cellular heterogeneity within the brain representsa formidable challenge for the development of HDAC inhibitorsas potential therapeutics to treat brain disorders. Moreover, thefunction of various HDAC members in different progenitor, neu-ronal, or glial cell types—some having redundant properties—pro-vides rationale for uncovering the roles of specific HDACs in thenervous system. In this study, we showed that HDAC3 is requiredfor the proliferation of adult NSPCs (both RGLs and TAPs), ulti-mately resulting in decreased generation of mature dentateneurons. Surprisingly, we did not observe a significant difference,although there was a trend toward reduction of immatureYFP+DCX+ cells at 60 dpt, suggesting that DCX+ neuroblasts mayincrease their proliferation to compensate for the early reductionof YFP+Ki67+ cells. We also demonstrated that HDAC3 is

important in regulating CDK1 levels for proper G2/M phaseprogression. We propose that in WT NSPCs HDAC3 stabilizesCDK1 to promote normal cell cycle progression, whereas inNSPCs lacking or inactive for HDAC3, CDK1 is degradedthrough the ubiquitin–proteasome pathway, resulting in G2/Mphase progression defects. Our results suggest that inhibitionof HDAC3 may be beneficial for preventing brain malignan-cies as well as treating certain neurological diseases, such asepilepsy, which may arise from abnormal proliferation of NSPCsafter seizures.

HDAC3 Plays a Role in Adult NSPC Proliferation, Specifically in G2/MPhase Progression. Previously, we and others showed that appli-cation of pharmacological inhibitors of class I HDACs leads toenhanced neuronal differentiation within the adult hippocampusand developing forebrain (6, 26). However, some reports in-dicate that neurogenesis in certain brain regions is decreasedafter HDAC inhibitor treatment (27). These studies indicatethere may be context-dependent differences based on the de-velopment stage and/or brain region and provide a rationale foradditional studies of the individual roles of HDACs in neuro-genesis. HDAC1 and HDAC2 appear to function redundantlyduring neurodevelopment to promote cell survival and neuronalmigration (7), whereas deletion of HDAC2 alone in adult brainis sufficient to mediate cell death and defective neuronal maturation(8). In contrast to HDAC1 and HDAC2—which are enriched inneural progenitors and mature neurons, respectively (8, 28)—we observed that HDAC3 is broadly expressed in adult brain,including hippocampal NSPCs, neuroblasts, and mature neurons.Deletion of HDAC1 and HDAC2 in fibroblasts blocks the cell

cycle in G1 phase by increasing the mRNA levels of CDKinhibitors p21 and p57 (29). In adult NSPCs, p21 does not in-crease after removal of HDAC3, which is consistent with ourobservation that G1/S phase progression is not primarily af-fected. We have shown that HDAC3 specifically controls G2/Mphase progression in adult NSPCs. In other cell types, suchas hematopoietic progenitor cells and MEFs, loss of HDAC3results in S-phase progression and DNA replication defects (13).The discrepancies in cell cycle defects after loss of HDAC3 mayreflect alterations in the level of HDAC3 at different stages ofthe cell cycle. For instance, in HeLa cells, the levels of HDAC3are reduced in metaphase due to proteasome degradation (14).However, in NSPCs, we observed consistently high levels ofHDAC3 throughout the cell cycle (Fig. S2B). Therefore, wedo not believe that HDAC3 control of cell cycle progression inmainly due to changes in HDAC3 levels, at least in adult NSPCs.

HDAC3 Mediates G2/M Progression Through Posttranslational Controlof CDK1 in Adult NSPCs. How does HDAC3 specifically controlG2/M progression? Because HDACs do not bind to chromatinduring M phase (30), one possibility is that HDAC3 may havenonhistone substrates in adult NSPCs. Indeed, HDAC3 waspreviously reported to control mitosis by targeting A-kinase–anchoring proteins AKAP96 and aurora B kinase in 293T cells(31). However, when HDAC3 is deleted in 293T cells, an in-crease in he percentage of G2/M phase is correlated with a de-crease in levels of the aurora B kinase substrate, p-H3S10, whichis in contrast to what we observed in NSPCs lacking HDAC3(Fig. 3A). Thus, it is reasonable to postulate that HDAC3 con-trols G2/M phase in adult NSPCs through targets other thanAKAP96 and aurora B, and depending on the cell type, HDAC3may have different targets to control the cell cycle.Based on our observation that HDAC3 is required for main-

taining CDK1 levels, we postulate that HDAC3 regulates thestability of CDK1, thus controlling G2/M progression. Our resultsalso indicate that HDAC3 is required for preventing CDK1ubiquitination. The exact mechanism by which HDAC3 regulatesubiquitination of CDK1 is unknown. In yeast, CDK1 can beacetylated at lysine 33, which is essential for proper growth (32).Moreover, a recent study using HeLa cells showed that HDAC3deacetylates and prevents the ubiquitination of cyclin A (14).

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Fig. 5. Loss of HDAC3 results in decreased proliferation in vivo. (A) Sche-matic of TAM injection and collection of brain tissue. (B) Immunostaining ofYFP, Ki67, and GFAP in Hdac3 WT and cKO mice 10 dpt and quantification ofYFP+Ki67+ proliferating cells (arrows) and YFP+Ki67+GFAP+ RGL cells. (C)Immunostaining of YFP, Ki67, and DCX in WT and cKO mice 10 dpt andquantification of YFP+Ki67+DCX− TAPs (arrows) at 10 and 30 dpt. (D)Immunostaining of YFP and NeuN in WT and cKO mice 60 dpt and quanti-fication of YFP+NeuN+ cells (arrows) at 30, 60, and 90 dpt. In B–D, *P < 0.05.(Scale bars: 50 μm in B–D.) In B–D, at least six mice of each genotypewere used. For the 90 dpt time point in D, three HDAC3 WT mice were used.*P < 0.05. DG, dentate gyrus.

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However, in our study, although we detected acetylated CDK1in HCN cells, we did not observe any differences in CDK1acetylation levels after HDAC3 inhibition or overexpression,possibly due to regulation by other HDACs. It would be in-teresting in future studies to examine the detailed mechanisms bywhich HDAC3 controls CDK1 ubiquitination.

Therapeutic Implications of HDAC3i. Broad HDAC inhibitors (suchas VPA) can mediate proneurogenic differentiation of NSPCsas well as spinal cord graft improvement (6, 33). Here we haveshown that an HDAC3i specifically blocks adult NSPC pro-liferation, instead of inducing neuronal differentiation. Onepotential advantage of HDAC3-selective inhibitors over broadHDAC inhibitors is their ability to pharmacologically regulatethe proliferation of adult NSPCs, such as in postseizure aberrantneurogenesis, which is a fundamental problem in epilepsy (34).Moreover, hyperproliferation of cancer cells is correlated withoverexpression of HDAC3 (12), and it has been suggested thatquiescent adult NSPCs are a cellular source of brain tumors (35).Thus, we speculate that inhibition of HDAC3 may decrease theproliferation of proliferating cancer cells as well as activatedbrain cancer stem cells, which may be a more promising strategythan targeting proliferating cells alone.On the other hand, activation of HDAC3 may promote the

proliferation of adult NSPCs. Consistent with this idea, over-expression of HDAC3 in transgenic mice was sufficient to inducecardiomyocyte proliferation (36). Our results suggest that de-velopment of HDAC3 activators may help in the pharmaco-therapy of neurodegenerative diseases. Sequential use of anHDAC3 activator to boost the endogenous NSPC pool and thentreatment with a broad HDAC inhibitor to promote neuronaldifferentiation may be more effective in spinal cord repairwhile avoiding possible immunological complications from

exogenous stem cell transplantation. Thus, combined use ofHDAC-selective activators and broad-acting inhibitors mayextend the therapeutic applications of HDAC inhibitors.In summary, we have identified a crucial role for HDAC3 in

adult NSPC proliferation. Mechanistically, HDAC3 is essentialfor proper G2/M progression, at least in part through post-translational regulation of CDK1. Our work confirms that a de-tailed analysis of individual HDACs is important for translatingHDAC inhibitors in the clinic.

Materials and MethodsAll mouse experimental procedures were approved by the Institutional AnimalCare andUse Committee at theUniversity of Texas SouthwesternMedical Center(UTSW). Mice were housed in a facility accredited by the Association for As-sessment and Accreditation of Laboratory Animal Care at UTSW on a 12 h light–dark cycle. Nestin-CreERT2, R26R-YFP, and Hdac3 floxed mice, and their geno-typing have been described previously (17, 25). Hdac3 cKO mice carryingHDAC3flox/flox, Nestin-CreERT2, and R26R-YFP alleles were given TAM i.p. dailyfor 6 d [150 mg/kg dissolved in 10% (vol/vol) ethanol and 90% (vol/vol) sun-flower oil] and then killed for analysis at indicated time points after the lastinjection. Additional experimental procedures are provided in SI Materialsand Methods and Table S1.

ACKNOWLEDGMENTS. We thank Michael Buszczak for critically reading themanuscript and Jose Cabrera for support with graphics. We also thank EricOlson [University of Texas Southwestern Medical Center (UTSW)] for Hdac3floxed mice, Amelia Eisch (UTSW) for Nestin-CreERT2 and Rosa-YFP mice,and Joel Gottesfeld (The Scripps Research Institute) for HDAC3i. This workwas supported by US National Institutes of Health Grants R01AG032383,R01NS076775, R21MH094715, K02AG041815, NS038572, and NS081203; Can-cer Prevention and Research Institute of Texas Grant RP100674; Welch Foun-dation Grant I-1660 (to J.H.); and Texas Institute for Brain Injury and Repairat UT Southwestern (J.H.).

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