TLE1 Corepressor Complex Mediates a Camkinase II␦-Dependent Neurogenic Gene Activation Pathway

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provided by Elsevier - Publisher Connector Cell, Vol. 119, 815–829, December 17, 2004, Copyright ©2004 by Cell Press ActivatingthePARP-1SensorComponentoftheGroucho/ TLE1 Corepressor Complex Mediates a CaMKinase II␦-Dependent Neurogenic Gene Activation Pathway

Bong-Gun Ju,1 Derek Solum,1 Eun Joo Song,4 entiate into neurons in response to both extrinsic and Kong-Joo Lee,4 David W. Rose,2 intrinsic cues (reviewed in Bally-Cuif and Hammer- Christopher K. Glass,3 and Michael G. Rosenfeld1,* schmidt [2003]). The commitment of neural stem cells 1Howard Hughes Medical Institute to the neuronal fate and their subsequent differentiation 2 Department of Medicine into neurons are controlled, in part, by mechanisms in- Division of Endocrinology volving the opposing activities of transcription factor 3 Department of Cellular and Molecular Medicine families containing the basic helix-loop-helix (bHLH) do- University of California, San Diego main, the Hairy/Enhancer of split (HES) family proteins Department and School of Medicine that negatively regulate neural differentiation, and other La Jolla, California 92093 bHLH family proteins belonging to the Neurogenin, Ash, 4 Center for Cell Signaling Research and NeuroD subgroups, collectively referred to as Division of Molecular Life Sciences and College proneural proteins (reviewed in Bertrand et al. [2002]; of Pharmacy Ross et al. [2003]). Ewha Womans University The inhibitory bHLH protein, HES1, interacts with Seoul 120-750 groucho (Gro)/transducin like enhancer of split (TLE) Korea corepressor in a manner dependent on its C-terminal WRPW motif and serves in a well-described repressor of MASH1 transcription preventing neuronal differentiation Summary (reviewed in Chen and Courey [2000]). Groucho can in- teract with HDAC1, and at least two additional compo- Switching specific patterns of gene repression and nents of the Sin3 complex, Sin3 and RbAP48 (Chen et al. activation in response to precise temporal/spatial sig- 1999; Choi et al., 1999). However, association between nals is critical for normal development. Here we report TLE1 and the mammalian HDAC1-HDAC6 and the mam- a pathway in which induction of CaMKII␦ triggers an malian counterparts of the highly conserved RNA poly- unexpected switch in the function of the HES1 tran- merase-associated proteins are not consistently ob- scription factor from a TLE-dependent repressor to served (Dasen et al., 2001), perhaps because the GP an activator required for neuronal differentiation. domain is poorly conserved in vertebrate TLEs. These events are based on activation of the poly(ADP- A wide variety of enzymatic activities are critical for ribose) polymerase1 (PARP-1) sensor component of actions of these corepressors and coactivators, includ- the groucho/TLE-corepressor complex mediating dis- ing histone acetylation and deacetylation, phosphoryla- missal of the corepressor complex from HES1-regu- tion, ubiquitylation, and sumoylation (reviewed in Glass lated promoters. In parallel, CaMKII␦ mediates a re- and Rosenfeld [2000]; McKenna and O’Malley [2002]). quired phosphorylation of HES1 to permit neurogenic Indeed, an ubiquitylation/proteasome pathway is widely gene activation, revealing the ability of a specific sig- used in transcriptional regulation (Muratani and Tansey, naling pathway to modulate both the derepression and 2003). In the case of liganded nuclear regulators, and the subsequent coactivator recruitment events re- several other classes of regulated transcription factors, TBLR1 and TBL1 components of an N-CoR repressor quired for transcriptional activation of a neurogenic complex serve as ligand-dependent adaptors for a re- program. The identification of PARP-1 as a regulated quired specific ubiquitin-conjugating/19S proteasomal promoter-specific exchange factor required for acti- complex, with TBLR1 mediating the exchange of the vation of specific neurogenic gene programs is likely nuclear corepressor N-CoR/SMRT for coactivators that to be prototypic of similar molecular mechanisms. are required for target gene activation (Perissi et al., 2004). Introduction An additional covalent modification recently linked to transcription is poly(ADP-ribosyl)ation of proteins medi- Precise spatial and temporal switches in patterns of ated by the poly(ADP-ribose) polymerase1 (PARP-1) en- gene activation and repression are critical for proper zyme. PARP-1 catalyzes the transfer of ADP-ribose organ development. Although a regulated exchange of chains onto glutamic acid residues of acceptor proteins, activating and repressing DNA binding factors is one including itself (automodification), histones, transcrip- mechanism for such events, alternative strategies can tion factors, and DNA repair proteins using NADϩ as a be employed, exemplified by ligand-induced exchange substrate involved in chromatin decondensation, DNA of corepressor for coactivator complexes in the case replication, and DNA repair. Therefore, poly(ADP-ribo- of nuclear receptors (reviewed in Glass and Rosenfeld syl)ation by PARP-1 affects cellular processes such as [2000]; McKenna and O’Malley [2002]). These switches apoptosis, necrosis, cellular differentiation, malignant are of particular importance in neural development. In transformation (reviewed in D’Amours et al. [1999]), and the developing mammalian central nervous system, neu- modulations activities of transcription factors (Akiyama ral stem cells located in the ventricular zone undergo et al., 2001; Butler and Ordahl, 1999; Hassa and Hottiger, proliferation and ultimately exit the cell cycle and differ- 2002; Plaza et al., 1999). While it has been recently reported that PARP influences both the expression and *Correspondence: [email protected] silencing at diverse times during Drosophila develop- Cell 816

ment (Tulin et al., 2002), it has been demonstrated that performed a biochemical purification of a TLE1 complex high PARP enzymatic activity is observed in areas of using the TAP (tandem affinity purification) method, a high transcriptional activity and chromatin decondensa- powerful approach used for functional proteomics stud- tion on the polytene chromatin (Tulin and Spradling, ies in yeast and mammalian cells (Puig et al., 2001). 2003). Together these observations suggest that PARP-1 Nuclear extracts were prepared from stably transformed may exert its function in transcription through direct 293 cells expressing TLE1 tagged with IgG binding do- binding to the gene-regulating sequences and through mains, TEV cleavage site, and the calmodulin binding modification of transcription factors by poly(ADP-ribo- peptide at its carboxyl terminus and were subjected to syl)ation. sequential purification with IgG and calmodulin affinity In this manuscript, we report an unsuspected reg- resins. About 13 polypeptides were found to be consis- ulatory pathway required for activation of neuro- tently and specifically associated with the TAP-tagged genic genes, involving both derepression and activation TLE1 (compare TLE1 lane with control lane; Figure 1E). events based on induction of a specific calcium-depen- MALDI-TOF mass spectrometry analyses identified nine dent protein kinase, CaMKII␦, and derepressed genes of these bands as nonmuscle myosin II heavy chain, regulated by the PARP-1-containing groucho/TLE1 co- TopoII␤, Rad50, PARP-1, nucleolin, HSP70, p54nrb, repressor complex. PARP-1 serves as a component of ␤-actin, and nucleophosmin. The TLE1 complex exhib- the corepressor complex, but its catalytic function ited significant stability, even in solutions containing 300 proves to be required for subsequent MASH1 gene acti- mM NaCl during purification (Figure 1F). In gel-filtration vation events. Induction of the CamKII␦-dependent sig- experiments using a Superose 6 column, most of the naling pathway in proliferating cortical progenitor (neu- polypeptides listed above migrated together with TLE1 ral stem) cells not only causes phosphorylation of in fractions 14–19, except for ␤-actin and myosin II (Fig- PARP-1 and activates its enzymatic activity resulting ure 1G and data not shown). To confirm these TLE1 in dissociation of poly(ADP-ribosyl)ated components of interactions, we performed reciprocal immunoprecipita- the corepressor complex, but it also unexpectedly pro- tion assays using 293 cells transiently expressing HA- motes site-specific phosphorylation of HES1 required tagged TLE1 and individual components of TLE1 com- for recruitment of coactivators, providing an intriguing plex (Figures 2A–2C and see Supplemental Figure S1 at promoter-specific strategy for progression of neuronal http://www.cell.com/cgi/content/full/119/6/815/ differentiation and defining a previously unknown role DC1/). Western blot analysis revealed that all compo- for PARP-1 in gene regulation. nents of TLE1 complex were coimmunoprecipitated with TLE1, but also with PARP-1 and nucleolin (Figures 2A– 2C). As a negative control, neither TBL1 nor ␤-tubulin Results showed significant binding affinity for the TLE1 complex. Together, these results reveal the PARP-1-containing A TLE1-Dependent Complex Mediates complex associated with TLE1 and show that its bio- HES1-Dependent MASH1 Repression chemically identified members are stably associated in Neural Stem Cells with TLE1 in vivo. Cortical progenitor cells (neural stem cells [NSCs]) are All components of the TLE1 complex were expressed maintained in a pluripotent state until signaling path- in the proliferating NSCs, as well as in brain (Figure ways involving Ca2ϩ-dependent gene activation events 2D). ChIP analysis of the MASH1 promoter revealed the initiate development along a neuronal pathway (Ghosh presence of all components of the TLE1 corepressor et al., 1994; Carey and Matsumoto, 1999; Ciccolini et al., complex (Figure 2E). PARP-1 s RNA, and s RNA for 2003). Thus, upon PDGF treatment nestin-expressing h h other individual components caused a 1.5- to 3-fold NSCs become TuJ1ϩ and express the proneural bHLH “derepression,” whereas unrelated, control shRNAs ex- protein, MASH1 (Figure 1A). Inspection of the promoter erted little effect on MASH1 promoter reporter activity region of rat MASH1 revealed three class C hairy binding in 293 cells or NSCs (Figures 2G and 2H and see Supple- sites (CACGCG) (Figure 1B), similar in sequence to that mental Figure S2B on the Cell website). Consistent with in the Drosophila achaete gene (Van Doren et al., 1994). these findings, we could detect little derepression of To investigate the process by which the Notch/Delta- endogenous MASH1 expression in proliferating NSCs induced bHLH repressor of HES1 blocks expression of microinjected with shRNA against TLE1 and components MASH1 in proliferating NSCs, we used chromatin immu- of the TLE1 complex (data not shown). Taken together, noprecipitation (ChIP) assays, finding that both HES1 these data indicate that the TLE1 holocomplex is re- and TLE1 were present on the MASH1 promoter in rat quired for HES1-mediated repression, but that individual NSCs (Figure 1B). A role for TLE1 in mediating repression components of the TLE1 complex exert quantitatively of the MASH1 promoter was validated by identifying smaller effects on repression of MASH1 transcription. a plasmid-based RNA-mediated interference (shRNA) To evaluate whether the enzymatic activity of PARP-1 against TLE1 that caused significant inhibition of HES1- plays a role in the HES1-mediated MASH1 repression, dependent repression of MASH1, contingent upon the we used the PARP-1 enzyme inhibitor, 3AB (3 amino- presence of the three HES1 binding sites (Figures 1C benzamide). Interestingly, no significant change in re- and 1D). No similar repression was observed when the pression of MASH1 in 293 cells was observed (Figure MASH1 promoter harboring mutations (Chen et al., 1997) 2I), suggesting that PARP-1 enzymatic activity is not in each of the three HES1 binding sites was used in required for repression. Addition of 3AB (100 ␮M) did not similar transfection experiments (Figure 1D). itself induce MASH1 transcription in FGF2-maintained In order to better understand the molecular mecha- NSC cultures (See Supplemental Figure S3 online), fur- nisms of TLE1-dependent neurogenic gene repression ther showing that the enzymatic activity of PARP-1 is and derepression events upon NSC differentiation, we not required for active repression by the TLE1 complex. CaMKII␦/PARP-1-Mediated Derepression in Neurogenesis 817

Figure 1. HES1/TLE1-Dependent Repression of MASH1 in Neural Stem Cells (NSCs) and Identification of TLE1 Complex (A) Expression of Nestin and Tuj1, in rat NSC cultures treated with PDGF and induced expression of MASH1. (B) Recruitment of HES1 and TLE1 in the HES1 binding sites of the MASH1 promoter (1,2,3), required for repression, as analyzed by ChIP. II represents PCR amplification using primers that cover the coding region of the MASH1 gene.

(C and D) TLE1 shRNA inhibits HES1-dependent repression of MASH1 promoter-driven reporter activity in 293 cells and NSCs. MASH1 promoter mutated in (1,2,3) HES1 binding sites fail to exhibit HES1-dependent repression. (E) Purification of TLE1 complex and mass spectrometric analysis of polypeptides associated with TLE1. For control experiment, empty vector (pCDNA3-TAP) were stably transfected into 293 cells. The identities of TLE1-associated polypeptides are indicated at the right. Degraded forms of nonmuscle myosin II (*) and TLE1 (**) were identified by Western blot analysis. (F) The TLE1 complex was purified in the presence of 150 or 300 mM NaCl using TAP method. (G) Gel filtration analysis of the purified TLE1 complex. The numbers over the lanes represent the eluted fraction number.

Regulated Dismissal of TLE1 Corepressor of MASH1 activation are reported to occur after 24 hr Complex and MASH1 Activation of PDGF treatment (Enarsson et al., 2002 and data not To investigate how the TLE1 complex is dismissed and shown). As expected, PDGF treatment caused dismissal what events are required for MASH1 gene activation, of TLE1 and many components of the TLE1 complex, we analyzed MASH1 promoter occupancy by various but not HES1, PARP-1, and p54nrb (Figure 3A), consis- cofactors by performing ChIP assays in differentiating tent with activation of MASH1 gene expression. The rat NSCs treated with PDGF for 24 hr. Maximum levels MASH1 promoter is now occupied by HAT-containing Cell 818

Figure 2. TLE1 Complex Represses MASH1 in Proliferating Neural Stem Cells (A–C) Reciprocal immunoprecipitation assay. HA-tagged (HA-) TLE1 (A), HA-PARP-1 (B), and HA-nucleolin (C) were transfected into 293 cells. (D) Expression of TLE family and TLE1-associated proteins were analyzed by RT-PCR assay in the proliferating NSCs and adult mouse brain. (E) ChIP analysis on rat MASH1 promoter by components of the TLE1 complex in the proliferating rat NSCs treated with FGF2. (F) PARP-1 is efficiently knocked down by

PARP-1 shRNA.

(G and H) Effects of PARP-1 shRNA on co- transfected MASH1 promoter reporter activity in 293 cells (G) and NSCs treated with FGF2 (H). (I) PARP-1 is associated with the TLE1 complex in an enzymatically inactive form. Trans- fected cells were treated with the inhibitors for PARP-1 (3AB) for 12 hr.

coactivator complexes, including CBP, p/CAF, the p160 and H4 were present on the MASH1 promoter following factor Src1, and acetylated H3/H4 histones and PolII the replacement of the corepressor complexes by HAT- (Figure 3A). containing coactivator complexes (Figure 3B). Unex- Components of the TLE1 complex remained associ- pectedly, we continued to detect the presence of HES1 ated with the MASH1 promoter for 6–9 hr following PDGF and PARP-1, as well as p54nrb, on the MASH1 promoter treatment and were then dismissed, consistent with the throughout repression and activation events, raising in- time lag (8–10 hr) required to induce MASH1 gene ex- triguing questions concerning their potential roles in pression (Figure 3B) and the 9–10 hr to observe en- MASH1 gene activation events. hanced levels of MASH1 mRNA in response to PDGF treatment (data not shown). This result suggests that Induction of CaMKII␦ Is Linked to MASH1 PDGF-dependent event(s) must be required for MASH1 Derepression Events activation in the differentiating NSCs (vide infra). By 12 Because previous studies indicate that several protein hr following PDGF treatment, the CBP-containing co- kinase pathways are involved in neuronal differentiation activator complexes, Pol II, and acetylated histones H3 (Stro¨ m et al., 1997; Enarsson et al., 2002; Me´ nard et al., CaMKII␦/PARP-1-Mediated Derepression in Neurogenesis 819

Figure 3. Dismissal of TLE1 Corepressor Complex and HES1-Dependent Recruitment of PARP-1-Containing Coactivator Complex (A) ChIP analysis on MASH1 promoter in differentiating rat NSCs. (B) Time curve of PDGF-induced switch in occupancy of MASH1 promoter, first by the TLE1 corepressor complex and later by coactivator complexes after PDGF treatment. (C) Effects of CaMK inhibitor on MASH1 expression. (D) MASH1 activation associated with CaMKII. The time curve of occupancy of CaMKII on MASH1 promoter was analyzed by ChIP. (E) Effects of KN-62 on PDGF-dependent factor/cofactor recruitment to the MASH1 promoter in rat NSCs. (F) CaMKII activity is required for MASH1 activation. After microinjection of KN-62 with TRITC-conjugated dextran together, the NSCs were maintained in the PDGF containing media for 48 hr. Photomicrographs of MASH1 expression in the KN-62-injected NSC are shown on the right, while fold increase of MASH1 expression is shown on the left as assessed by the NIH Image Program. (G) The time induction curve of CaMKII␣, ␤, ␥, and ␦ by RT-PCR of differentiating NSCs.

(H) CaMKII␦ shRNA selectively inhibits MASH1 expression. The cells were immunostained with ␣MASH IgG.

2002; Vojtek et al., 2003), we investigated the hypothesis inhibitor of calcium/calmodulin-dependent protein ki- that specific protein kinases functionally modify, or in- nase (CaMK), KN-62 (5 ␮M), significantly blocked PDGF- teract with, HES1 and/or cofactors on the MASH1 pro- induced neuronal differentiation as determined by evalu- moter in differentiating NSCs. Initially using inhibitors ating the expression of MASH1 transcripts (Figure 3C). of specific protein kinases, we showed that a specific ChIP analysis revealed that CaMKII was recruited to the Cell 820

MASH1 promoter upon PDGF treatment in contrast to induction of CaMKII dictates the temporal aspects of other tested protein kinases including CaMKIV, PKC, MASH1 activation. and RSKs (Figure 3D). Although PKC signaling is impor- tant for NGF-induced neuronal differentiation in the Poly(ADP-ribosyl)ation Activity of PARP-1 Is PC12 pheochromocytoma cell line (Stro¨ m et al., 1997), Required for Dismissal of the TLE1 Corepressor PKC inhibitors exerted minimal effects on MASH1 ex- Complex in Neural Stem Cell Differentiation pression in the differentiating NSCs (data not shown). The PARP-1 component of the TLE1 transcriptional co- Analysis of the time course of CaMKII recruitment repressor complex in proliferating NSCs (Figure 2E) re- showed that it was initially detected on the MASH1 pro- mains bound to the MASH1 promoter in NSCs differenti- moter 7–9 hr following addition of PDGF and remained ating into neurons (Figures 3A and 3B). While PARP-1 even after recruitment of the coactivator complexes plays a critical role in chromatin structure during DNA (Figure 3D). Together, these data suggest that activation repair, its role in chromatin remodeling and transcrip- of some members of the CaMKII family represents a tional regulation has been recently reported (reviewed prerequisite for PDGF-induced MASH1 activation and in Kraus and Lis [2003]; Rouleau et al. [2004]). To investi- NSC differentiation. Addition of KN-62 in PDGF-treated gate potential functions of PARP-1 in derepression cells blocked release of the TLE1/PARP-1 corepressor events, the poly(ADP-ribosyl)ation activity of PARP-1 complex from the MASH1 promoter, as detected by was assessed by Western blot analysis (Figure 4A). Poly- ChIP (Figure 3E). To functionally test this model, we (ADP-ribosyl)ated PARP-1 was detected using anti- investigated the effects of inhibiting CaMKII by KN-62 poly(ADP-ribose) antibody (␣PAR) from nuclear extract on MASH1 gene activation in differentiating NSCs using of PDGF-treated NSCs. A significantly higher level of the single-cell nuclear microinjection assay. MASH1 ex- poly(ADP-ribosyl)ation of PARP-1 was observed follow- pression was inhibited by microinjection of KN-62 in a ing PDGF treatment of NSCs for 12 hr, after which, poly dose-dependent manner (Figure 3F), consistent with a (ADP-ribosyl)ation of PARP-1 actually declined (Figure functional role of recruitment of isoform(s) of CaMKII. 4A and data not shown). In ␣T3-1 cells treated with KCl CaMKII isoforms termed ␣, ␤, ␥, and ␦ constitute a to cause Ca2ϩ signaling, most components of the TLE1 family of multifunctional protein kinases that play a ma- complex, except p54nrb, could be poly(ADP-ribo- ϩ jor role in Ca2 -mediated signal transduction (reviewed syl)ated (See Supplemental Figures S5A and S5B), con- in Hudmon and Schulman [2002]). A dramatic increase sistent with the recent report that calcium signaling in ␣ isoforms mRNA level was observed at a time that can regulate poly(ADP-ribosyl)ation activity of PARP-1 correlates with the start of dendritic maturation and in- (Homburg et al., 2000; Kun et al., 2004). To ascertain if creased synapse formation (Sugiura and Yamauchi, similar events occurred in neural stem cells, we treated 1992; Bayer et al., 1999). Remarkably, only ␥- and ␦-CaM NSCs with PDGF and evaluated poly(ADP-ribosyl)ation kinase II are expressed during early embryonic brain of TLE1, TopoII␤, Rad50, nucleolin, and nucleophosmin development and they might be involved in the early finding that each exhibited PDGF-dependent poly(ADP- events of neurogenesis (Bayer et al., 1999). The isoforms ribosyl)ation (Figure 4B). Because it has been shown of CaMKII␦ are also the principal CaMK gene products that addition of an anionic polymer on a protein can expressed during adipocyte and cardiac differentiation prevent interaction with other anionic molecules (re- (Wang et al., 1997; Hoch et al., 1998). However, specific viewed in D’Amours et al. [1999]; Ferro and Olivera functions of CaMKII␦ and ␥ remain unknown. The NSCs [1982]), we suggest that poly(ADP-ribosyl)ation of many exhibited no detectable expression of any CaMKII iso- components of the TLE1 complex is a key event in com- form, but levels of CaMKII␦ transcripts were markedly plex dismissal. induced after 7–9 hr of PDGF treatment (Figure 3G), To independently test the hypothesis that calcium consistent with the time course of MASH1 gene activa- signaling-dependent poly(ADP-ribosyl)ation is coupled tion. CaMKII␥ exhibited similar kinetics of induction, but to dismissal of the TLE1 complex, Ca2ϩ was added fold lower, and the protein was not de- to nuclear extracts from HA/TLE1-overexpressing 293-20ف at levels tected on Western blot analysis (Figure 3G and data cells (CamKIIϩ) during immunoprecipitation procedures. not shown). These results suggested that induction of Dissociation of the TLE1 complex was observed in a CaMKII␦ is likely to provide the molecular explanation Ca2ϩ concentration-dependent manner (Figure 4C). In- for the temporal “lag” in the switch from HES1/TLE1- terestingly, most of the components we have identified dependent repression to activation of MASH1. We devel- in the TLE1-associated corepressor complex have been 2ϩ oped a CaMKII␦ shRNA in a modified pSuper vector ex- reported to be regulated by Ca (Ackerman et al., 1988; pressing EGFP that markedly reduced levels of CaMKII␦ Gilchrist et al., 2002; Herrera et al., 1995; Kun et al., protein in Western blot analysis (See Supplemental Fig- 2004). To confirm these data, we performed immunopre- ure S4 online), transfected this versus control shRNA cipitation experiments using ␣T3-1 cells, which already into rat NSCs, and evaluated expression of MASH1 48 express CaMKII␦, transfected with HA-TLE1/Flag-HES1 hr after PDGF treatment. As seen in Figure 3H, there was expression vectors. The cells were cultured in the pres- an effective block of MASH1 expression with CaMKII␦ ence or absence of KCl (50 mM) for 12 hr. In accordance shRNA but not with control shRNA, indicating that induc- with the in vitro assays, KCl-evoked calcium signaling tion of CaMKII␦ was specifically required for dismissal caused a dissociation of the TLE1 complex in vivo, even of the TLE1 corepressor complex and activation of after KCl treatment for 10 min (Figure 4D and data not MASH1. Together, these data suggest that the activity shown). These data also suggest that the “lag” in disso- of CaMKII␦ is required for PDGF-induced MASH1 activa- ciation of TLE1 complex that occurs in NSCs only after tion in differentiating rat NSCs and that the kinetics of CaMKII induction does not occur in cells already ex- CaMKII␦/PARP-1-Mediated Derepression in Neurogenesis 821

Figure 4. Activation of Poly(ADP-ribosyl)a- tion Function of PARP-1 Is Required for MASH1 Activation during Neural Stem Cell Differentiation (A) PDGF treatment induces poly(ADP-ribosyl)ation of PARP-1 in the differentiating rat NSCs. (B) Components of the TLE1 complex can be poly(ADP-ribosyl)ated by PDGF treatment. (C) Calcium induces TLE1 complex dissociation. Nuclear extracts from HA/TLE1 overexpressed 293 cells were incubated at 37ЊC for

5 min in the presence of CaCl2. Purification of the TLE1 complex using ␣HA IgG and Western blot analysis was performed. (D) KCl-evoked calcium signaling induces TLE1 complex dissociation in vivo. Incu- bation overexpressing HA-TLE1/Flag-HES1 ␣T3-1 cells with 50 mM KCl Ϯ 1mM3AB for 12 hr and purification of the TLE1/HES1 complex using ␣HA IgG and Western blot analysis were performed. (E) ChIP analysis of the MASH1 promoter in NSCs treated with PDGF Ϯ 3AB, showing inhibition of TLE1 corepressor complex dismissal.

pressing CaMKII␦. Addition of the pharmacological in- to be markedly inhibited (Figure 5A). To investigate hibitor of PARP-1 (3AB) blocked these events, as did whether PARP-1 might itself be a substrate for CaMK- addition of CaMK inhibitor, KN-62 (Figure 4D and data dependent phosphorylation, we evaluated the phos- not shown). Similarly, addition of KN-62 or 3AB inhibited phorylation and auto-poly(ADP-ribosyl)ation of PARP-1 dismissal of the TLE1 complex from the MASH1 pro- using ␣T3-1 cells, which expressed high levels of CaMKII␦, moter, as assessed by ChIP assay (Figures 3E and 4E). by transfecting HA-PARP-1 expression vectors, treating Together with previous reports that protein-protein in- with KN-62 for 12 hr, immunoprecipitating with ␣HA IgG, teractions were attenuated by poly(ADP-ribosyl)ation, and immunoblotting with ␣PARP IgG, ␣PAR IgG, or our data imply that PDGF-induced poly(ADP-ribosyl)a- ␣phosphoserine IgG. As seen in Figure 5B, addition of tion results in dismissal of the TLE1 complex from the KN-62 to inhibit CaMK activity caused a marked inhibi- MASH1 promoter of differentiating NSCs after PDGF tion of both phosphorylation of PARP-1, as well as its treatment. Thus, while our data indicated a promoter auto-poly(ADP-ribosyl)ation. Consistent with this, it has specificity to CaMKII␦-dependent PARP-1 activation, been reported that PARP-1 activity can be regulated the biochemical data do not preclude signal-induced by phosphorylation of serine residue(s) during Xenopus enzymatic function of PARP-1 off the DNA. This would oocyte maturation (Aoufouchi and Shall, 1997). There- be quite analogous to promoter-specific effects of li- fore, PARP-1 activation is consistent with the posttrans- gands on nuclear receptors, but with cofactor exchange lational modification by CaMKII and provides a molecu- also occurring off the DNA as a component of transre- lar mechanism for derepression events. pression events (reviewed in Glass and Rosenfeld In order to extend these results, we utilized a mutant- [2000]). type (E988A) PARP-1 that has been established to lack We next inquired whether poly(ADP-ribosyl)ation ac- ADP-ribose polymerase activity (Marsischky et al., tivity of PARP-1 is actually required for MASH1 activa- 1995). NSCs overexpressing either wild- or mutant-type tion in PDGF-treated, differentiating NSCs. When rat PARP-1/EGFP were maintained in PDGF containing me- NSCs were maintained in PDGF for 24 hr in the presence dia for 24 hr. In the case of wild-type PARP-1, MASH1 of 3AB, PDGF-induced MASH1 expression was found expression was easily detected in the differentiating Cell 822

Figure 5. Enzymatic Activation of PARP-1 in Derepression Events (A) Inhibition of PARP-1 activity blocked PDGF-dependent induction of MASH1 transcription in 100 ␮M 3AB-treated NSCs. (B) CaMKII-dependent phosphorylation of PARP-1 activates PARP-1 enzymatic activity in ␣T3-1 cells.

(C and D) Inactivation of activity of PARP-1 or PARP-1 shRNA suppress MASH1 expression in the differentiating rat NSCs. (E) Mutant-type (E988) PARP-1 blocks dismissal of the TLE1 corepressor complex and MASH1 gene activation. A two-step ChIP analysis using anti-Flag antibody in the first round and the second round of ChIP was performed with the indicated IgGs.

NSCs by immunostaining (Figure 5C), with 80% of EGFP- Regulated Switch of HES1 Function from Gene positive cells exhibiting high levels of MASH1 expres- Repression to Gene Activation sion (Figure 5D). However, when NSCs overexpressed Because HES1 remains bound to the MASH1 promoter the mutant-type (E988) PARP-1 instead, they no longer in differentiating NSCs by ChIP analysis (Figures 3A and responded to the PDGF treatment with decreased 3B), we considered the possibility that HES1 might func- MASH1 expression in most of the EGFP-positive cells tion as a required activator of MASH1 gene transcription (Figures 5C and 5D). To eliminate the possibility that in differentiating NSCs. To test this hypothesis, we mi- overexpression of wild- or mutant-type PARP-1 has croinjected siRNA against HES1 transcripts into the toxic effects of cell viability, we applied shRNA approach NSCs with TRITC-conjugated dextran together, main- to knock down PARP-1 transcripts in the differentiating taining cells in PDGF-containing media for 48 hr. Immu- NSCs. In concert with actions of mutant PARP-1, we nostaining the cells with specific anti-MASH1 antibody failed to detect MASH1 expression in most of shPARP-1 revealed that MASH1 expression markedly decreased transfected NSCs upon PDGF treatment for 48 hr (Fig- in the HES1 siRNA-injected NSCs, but not in control ures 5C and 5D). To independently test whether poly- siRNA-injected NSCs (Figure 6A). MASH1 expression (ADP-ribosyl)ation activity of PARP-1 is required for levels were now similar to those observed in the FGF2- MASH1 activation, two-step ChIP analysis was per- treated proliferating NSCs. These results indicate that formed after transfection of Flag-tagged wild- or mutant- HES1 is required for MASH1 activation in differentiating type (E988A) PARP-1 into NSCs following PDGF treat- cortical progenitor cells. ment for 48 hr. In contrast to events with wild-type We used an independent assay to confirm that HES1 PARP-1, we failed to detect replacement of corepressor acts as an activator during NSC differentiation by per- by coactivator in mutant-type PARP-1-overexpressing forming shRNA experiments, assessing expression of a NSCs (Figure 5E). Together, these data suggest that the reporter under control of the 1.5 kb MASH1 promoter. No poly(ADP-ribosyl)ation activity of PARP-1 is required for MASH1 promoter activation was observed after PDGF dismissal of the TLE1 complex and for MASH1 gene treatment (48 hr) in HES1 shRNA-transfected cells (Fig- activation in differentiating NSCs. ure 6B). To exclude the possibility that HES1 had lost CaMKII␦/PARP-1-Mediated Derepression in Neurogenesis 823

its DNA binding activity upon PDGF treatment, we com- crease occurring with HES1 mutated in the serine resi- pared the level of HES1-specific class C DNA binding due in the so-called “orange” domain (S126A) (Figure activity present in nuclear extracts from proliferating or 6H). These results were consistent with the possibility differentiating NSCs after PDGF treatment for 12 and that phosphorylation of specific sites on HES1 might be 24 hr. We observed that the level of endogenous binding required for MASH1 activation. In contrast to wild-type activity did not decrease after 24 hr of PDGF treatment; HES1, the mutant-type (S126A) HES1 protein showed a indeed, it actually increased (Figure 6C). Western blot significant decrease in levels of detected phosphoryla- analysis of the nuclear extracts indicated no striking tion, even in KCl (50 mM)-treated cells (Figure 6I). While decrease in the levels of expression of HES1 protein HES1 can be phosphorylated in a CaMKII-dependent between proliferating and differentiating NSCs after 24 fashion in the tested cell types, it undoubtedly also hr of PDGF treatment (Figure 6C). serves as a substrate for other protein kinases, perhaps in a cell type-specific fashion. DNA binding activity of CaMKII-Dependent Phosphorylation of HES1 purified, bacterially expressed GST wild- or mutant-type Promotes Activation Events (S126A) HES1 protein was not altered based on electro- Two-step chromatin immunoprecipitation analyses were phoretic mobility gel shift assays with a synthetic dou- performed in PDGF-treated NSCs to confirm that HES1 ble-stranded oligonucleotide corresponding to the was present on the same MASH1 promoters as PARP-1, HES1-specific class C DNA site using extracts from rat CBP, and CaMKII, as well as other coactivator com- NSCs (see Supplemental Figure S6 on the Cell website). plexes, confirming that PARP-1 was bound to activated In accord with these observations, HES1 is expressed promoters on which HES1 was present (Figure 6D). We strongly not only in the proliferating NSCs, but also in therefore further investigated the underlying mecha- most of the MASH1-positive cells following 24 hr of nisms of the “switch” in HES1 function from repression PDGF treatment (Figure 6J). Multipotent FGF2-treated to activation. First, we explored regulated interactions proliferating cortical progenitor cells failed to exhibit of HES1 with coactivators and then investigated the MASH1 expression. potential role of the enzymatic activity of CaMKII. To con- To test the functional effects of these putative phos- firm potential interactions between HES1 with CBP-con- phorylation sites, we microinjected NSCs with rat-spe- taining coactivator complexes, coimmunoprecipitation ex- cific siRNA against HES1 and with wild- or mutant-type periments were performed, revealing a strong interaction human HES1 expression vectors with TRITC-conjugated between HES1 and CBP in cotransfection experiments dextran together. While wild-type human HES1 could (Figure 6E). KN-62 treatment caused a loss of HES1/ rescue MASH1 expression in the differentiating NSCs, CBP interactions, suggesting that CaMKII-dependent HES1 proteins harboring mutations in several CaMK phosphorylation may be necessary for HES1/CBP inter- phosphorylation sites, particularly the S126A mutation, actions. Consistent with a potential activation role for could no longer rescue MASH1 expression (Figure 7A). HES1, transient transfection of a HES1 expression vec- Taken together, our data support the hypothesis that tor can actually activate MASH1 promoter-driven re- phosphorylation of HES1 by CaMKII is linked to recruit- porter in AtT20 cells, which exhibit oscillations in Ca2ϩ ment of coactivator complexes, including CBP, which flux, and this HES1-dependent activation of MASH1 is functionally converts HES1 to a DNA binding transcrip- blocked by KN-62 treatment (Figure 6F). tional activator required to induce MASH1 transcription Because of the requirement for CaMKII in activation in differentiating NSCs. events and the recruitment of CaMKII to the MASH1 promoter, and previous studies documenting that the Discussion calcium signaling-induced modifications of transcription factors can involve actions of CaMK (Shaywitz and Defining the coordinated molecular strategies that un- Greenberg, 1999), we investigated the potential role of derlie the transition from a multipotent state to differenti- CaMK-dependent phosphorylation sites (R/KXXS/T). ation along specific lineage pathways, such as the pro- We tested the hypothesis that HES1 is phosphorylated gression from a neural stem cell state to development by CaMKII␦ during NSC differentiation. HA-tagged HES1 along a neuronal pathway, remains a central issue in protein was overexpressed in ␣T3-1 cells in the presence development. Here, we have reported a pathway that or absence of KN-62 together with KCl, and Western underlies a component of these switching events in neu- blot analysis was performed. After immunoprecipitation ronal differentiation, based on regulation by two func- of nuclear extracts with anti-HA antibody, phosphoryla- tionally distinct classes of bHLH transcription factors. tion levels of HES1 were analyzed using anti-phosphoserine antibodies (Figure 6G). Significant phosphoryla- PARP-1 Is a Component of the TLE1 Corepressor tion of wild-type HES1 was detected and a marked Complex Mediating Derepression Events decrease in serine phosphorylation was observed in the during Neural Stem Cell Differentiation presence of KN-62. To investigate whether these con- We find that HES1-dependent repression of MASH1 is sensus CaMK phosphorylation sites might function in dependent upon the actions of the TLE1 corepressor coactivation, we performed coimmunoprecipitation analy- complex. We have not only provided additional insights sis with CBP and wild- or mutant-type on HES1 proteins into the molecular mechanisms of TLE1-mediated re- harboring individual mutations of each potential phos- pression but also uncovered the molecular mechanism phorylation site. We observed a significantly decreased of the switch to activation function. The composition of affinity between CBP and HES1 mutated serine residues this TLE1 complex is distinct from those of other re- in the bHLH domain (S70A) with the most dramatic de- ported corepressor complexes such as N-CoR/SMRT Cell 824 CaMKII␦/PARP-1-Mediated Derepression in Neurogenesis 825

and CtBP (reviewed in Jepsen and Rosenfeld [2002]; Dual Roles of HES1 in Neural Stem Yoon et al. [2003]; Shi et al. [2003]). Interestingly, roles Cell Development for transcriptional regulation and chromatin remodeling Our data also indicate that, in addition to Ca2ϩ-CaMKII- activities have been described for most of the compo- dependent dissociation of the TLE1 corepressor from nents of the TLE1 complex that we have identified (Brou HES1, a covalent modification of HES1 itself is required et al., 1993; Adachi et al., 1991; Inouye and Seto, 1994; to permit activation of MASH1. Thus, activation of Yang et al., 1994; Tsai et al., 2000; Okuwaki et al., 2001; MASH1 is linked to sequential CaMKII␦-dependent acti- Roger et al., 2002; Gabellini et al., 2002; Sewer and vation of PARP-1 enzymatic activity, which was pre- Waterman, 2002), but we found that no component is viously inhibited in the TLE1 holocorepressor complex, alone indispensable for at least some level of TLE1- permitting dismissal of the TLE1 complex, derepression mediated repression. and phosphorylation of HES1, and recruitment of spe- Consistent with the observation that the enzymatic cific coactivators and thus causing and maintaining de- activity of PARP-1 is not required for HES1-mediated repression of genes mediating neuronal differentiation. MASH1 repression, our data favors a model predicting The actions of PARP-1 in the TLE1-mediated events are that in the TLE1 holorepressor complex the enzymatic thus analogous to the effects of covalent modifications activity of PARP-1 is inhibited. Exposure to a signal by phosphorylation and acetylation, as mediators of inducing neuronal differentiation causes activation of switches from repressor to activator function (Mannervik CaMKII␦, which is proven to be required for activation et al., 1999). of neurogenic genes. Once sufficient levels of CaMKII␦ While HES1 is recognized to regulate tissue morpho- are achieved (5–7 hr), it will, directly or indirectly, medi- genesis by maintaining undifferentiated cells and pre- ate phosphorylation and activation of PARP-1, which venting differentiation (Ishibashi et al., 1995; Ohtsuka et then catalyzes poly(ADP-ribosyl)ation of TLE1 and most al., 2001), continued occupancy of the MASH1 promoter of the other components to the corepressor complex. by HES1 in the differentiating neural stem cells has sur- prisingly proven to be required to initiate MASH1 activa- This is consistent with the observation that calcium sig- tion events. Indeed, previous, seemingly contradictory naling evoked by extrinsic and intrinsic cues can induce reports of transient transfection assays in which HES1 auto-poly(ADP-ribosyl)ation of PARP-1 (Homburg et al., can inhibit acid ␤-glucosidase genes in HepG2 cells but 2000; Kun et al., 2004); however, CaMKII␦ may also be cause activation in fibroblasts (Yan et al., 2001, 2002) activated in a calcium-independent fashion. This cova- are likely to be explained by our findings of signal- lent modification is suggested to result in their dismissal dependent HES1 switching events. from the biochemical complex and derepression of the This cortical progenitor culture system has permitted MASH1 gene. The role of PARP-1 in derepression of identification of a regulatory pathway that may be, at MASH1 and its retention on the activated MASH1 pro- least in part, partially compensated in vivo because moter is quite consistent with reports that poly(ADP- many nestin-positive neural stem cells in the subventri- ribosyl)ation of chromatin-associated proteins induce cular zone proliferate without losing the multipotentiality major changes in chromosomal architecture (reviewed to differentiate into neurons in HES1 mutant or even in D’Amours et al. [1999]; Kraus and Lis [2003]). How- HES1/HES5 double mutant mice (Ishibashi et al., 1995; ever, in the case of MASH1, we have found that dere- Ohtsuka et al., 2001). We suggest that there may be pression alone is insufficient for induction. This is in additional HES1-like repressors or unidentified protein accord with the findings for other regulated transcription partners, including HERP and other E box binding pro- factors. For example, the loss of the N-CoR corepressor teins, that are also potentially involved in MASH1 gene is not alone sufficient to activate most AP-1-regulated activation. The identification of this unexpected mecha- genes, and only in a subset of RAR target genes does nism of HES1 action in cortical progenitor cell cultures derepression result in a signal-independent “default” suggest that, in this system, the other molecules that activation of gene targets (Ogawa et al., 2004). could assume a similar function were either not ex-

Figure 6. Conversion of the HES1 Repressor to a Required Activator during Neural Stem Cell Differentiation (A) Microinjection of HES1 siRNA suppresses MASH1 expression in the differentiating rat NSCs. Photomicrographs of MASH1 expression in the HES1 siRNA microinjected NSCs are shown on the right, while fold increase of MASH1 expression is shown on the left as assessed by the NIH Image Program.

(B) Knockdown of HES1 shRNA suppresses MASH1 promoter reporter activity. (C) HES1 DNA binding activity in nuclear extracts from proliferating or differentiating rat NSCs treated with PDGF for 12 hr and 24 hr. (D) Two-step ChIP analysis of HES1/coactivator complex; after a first round of ChIP using anti-HES1 IgG in differentiating NSCs, a second round of ChIP was performed with the indicated antibodies. (E) HES1 interactions with the CBP coactivator complex in vivo. AlphaT3-1 cells coexpressing HES1 and CBP were cultured in the absence or presence of 30 ␮M KN-62 for 12 hr. Coimmunoprecipitation assays were performed with anti-CBP or anti-HES1 IgG. (F) Expression of HES1 in AtT20 cells activates the MASH1 promoter reporter activity in response to calcium signaling. (G) HES1 can be phosphorylated by CaMKII in vivo. (H) Schematic representation of murine HES1 showing the location of consensus CaMKII phosphorylation sites, each of which was mutated as diagrammed (right panel). After cotransfection of mutant HES1 and CBP into ␣T3-1 cells, coimmunoprecipitation assays were performed. (I) Effects of KCl (50 mM) Ϯ KN-62 (30 ␮M) on phosphorylation of HA-HES1 (S126A) in an experiment performed simultaneously with that shown in (G). (J) Colocalization of HES1 and MASH1 in the differentiating rat neural stem cells. Double immunostaining was performed with anti-HES1 (green) and anti-MASH1 (red) antibodies in the proliferating or differentiating rat NSCs. Cell 826

Figure 7. Dismissal of TLE1 Complex and Recruitment of CBP Complex for MASH1 Activation during Neural Differentiation (A) The microinjected rat NSCs were maintained in PDGF-containing medium for 48 hr. Relative expression of MASH1 is shown on left as assessed by the NIH Image Program, while photomcirographs of the MASH1 expression in HES1 (S126A) experiment are shown on the right. (B) Model of the CaMKII␦/TLE1/PARP-1/HES1 pathway action in repressor switching events required for neuronal differentiation (see Dis- cussion).

pressed or required. Our finding of a requirement of ation, such as PDGF in neural stem cells, act to induce HES1 in activation of neurogenic genes is consistent the CaMKII␦ isoform, which, in turn, is required for HES1- with suggestions that HES1 might promote differentia- mediated MASH1 activation (Figure 7B). The temporal tion, in addition to its role in maintenance of the undiffer- aspects of CaMKII␦ induction appear to account for the entiated state, at multiple steps of neural stem cell devel- delay in derepression and activation of MASH1 expres- opment. sion following critical PDGF signaling. CaMKII␦-induced phosphorylation of a specific serine residue in the or- Model of PARP-1-Mediated Exchange ange domain of HES1 permits it to recruit coactivators, and HES1 Activation including CBP, and has proven to be required for activa- In summary, we suggest a pathway in which a PARP-1- tion of the MASH1 (Figure 7B). The conserved relation- containing TLE1 complex is recruited by the Notch- ship of the HES1 orange domain with the HLH domain induced bHLH factor, HES1, initially mediating repres- raises the possibility of an additional role in protein inter- sion of MASH1 in the proliferating neural stem cells. Our actions that include dimerization (Davis and Turner, data suggest that signals that induce neuronal differenti- 2001). CaMKII␦/PARP-1-Mediated Derepression in Neurogenesis 827

In a sense, the observation that a component of the Gel-Filtration Chromatography TLE1-mediated repression complex, PARP-1, is also Gel-filtration chromatography was performed with a Superose 6 HR required for derepression events and maintenance of 10/30 column (Amersham). activation parallels the requirement of TBL1/TBLR1 Immunoprecipitation and Chromatin Immunoprecipitation complex for ligand-dependent exchange of N-CoR core- Immunoprecipitation was performed by standard methods (see the pressor complexes for coactivators in the switch of nu- Supplemental Data online). Chromatin immunoprecipitation was clear receptor function from repression to activation performed on neural stem cells largely as described (Jepsen et al., (Perissi et al., 2004). TBLR1 is required for recruitment of 2000; Shang et al., 2000). the ubiquitylation/19S proteosome complex to prevent Poly(ADP-ribosyl)ation Assay N-CoR/SMRT-dependent maintenance of a repression The nuclear extract was immunoprecipitated using a variety of anti- checkpoint. We suggest that PARP-1 may achieve the bodies under highly stringent conditions (420 mM NaCl). Poly(ADP- same effects by a distinct modification strategy and ribosyl)ated proteins were detected in Western blots with anti-PAR serves as a regulated sensor of neuron-inducing signals monoclonal antibody (10H, Alexis Biochemical), which is known to based on the actions of CaMKII␦ induced by the initial be specific and reliable for poly(ADP-ribose) detection. stimulus to neuronal differentiation. Therefore, we are Single-Cell Nuclear Microinjection Assays tempted to speculate that PARP-1 and TBLR1 may be Microinjection assays were carried out as previously described (Jep- critical for the exchange events required to overcome sen et al., 2000). Results are mean Ϯ SEM Ͼ100 cells injected for the repression checkpoint for TLE- and N-CoR-regu- each data point. See the Supplemental Data for procedures and lated repressors, respectively. siRNAs design. The dual functions of PARP-1 and HES1 in the progression of neural stem cells along a neuronal pathway Immunohistochemistry indicate that while the initial Notch signal causes repres- Immunohistochemistry was performed largely as previously described (Jepsen et al., 2000). sion of neurogenic genes by induction of HES1, it simul- 2ϩ taneously arms the response to subsequent Ca - Electrophoretic Mobility-Shift Assay CaMKII signals that permit MASH1 gene activation For gel-shift assays with nuclear extracts from rat neural stem cells events. Our data illustrate how a single signaling path- or purified GST-HES1 protein, we followed protocol as previously way can mediate a sequential, two-step derepression/ described (Stro¨ m et al., 1997). See the Supplemental Data for oligo- activation process required for development in gene nucleotide sequences. activation. Induction of a Ca2ϩ/CaMKII-dependent pro- Plasmid-Based RNA Interference (shRNA) gram initiates both PARP-1 activation, which is required Plasmid-based RNA interference experiments were carried out as for dismissal of the TLE1 corepressor complex, and a previously described using pSuper vector (Brummelkamp et al., second event, covalent modification of HES1, which is 2002) or modified pSuper vector with PGK promoter-EGFP. See the required for target gene activation. The requirement for Supplemental Data for shRNAs design. both a derepression and independently mediated acti- RNA Isolation and RT-PCR Analysis vation event is likely to be prototypic of many similar RT-PCR products were prepared as described in the Supplemental functions of PARP-1 factors in development. The se- Data and were analyzed by agarose gel electrophoresis. quential calcium-regulated PARP-1-dependent switch from repression to derepression to activation function Acknowledgments of HES1 is clearly an effective strategy to maximize the amplitude of the transcriptional response of neurogenic We are very grateful to C. Nelson for excellent technical assistance; gene expression to signals and to permit temporally I. Bassets for gel filtration; V. Lunyak, P. Briata, L. Erkman, and V. Perissi for critical reading of the manuscript; and X. Zhu, R. McEvilly, precise patterns of cellular response. and the other members of the Rosenfeld lab for discussions and valuable advice during the course of this study. We also thank Dr. Experimental Procedures R. Tsien for valuable discussion; R. Kageyama, D. Maiguel, and S.L. Oei for plasmids; J. Hightower and M. Fisher for figure and Additional Experimental Procedures are available in the Supplemen- manuscript preparation; and M. Gonzalez (Santa Cruz Biotechnol- tal Data at http://www.cell.com/cgi/content/full/119/6/815/DC1/. ogy) for advice on reagents. M.G.R. is an investigator with the How- ard Hughes Medical Institute and B.J. is supported by USAMRMC (DAMD17-01-1-0184). These studies were supported by grants from Antibodies and Reagents Sander Programs for Asthma Research to M.G.R. and NIH to C.K.G, Monoclonal antibody 10H for ADP-ribose polymer was obtained D.W.R., and M.G.R. from RIKEN Cell Bank (Japan). The following commercially available reagents were used: 3AB and KN-62 (Calbiochem). Other antibodies Received: August 27, 2004 are listed in the Supplemental Experimental Procedures available Revised: October 28, 2004 online. Accepted: November 4, 2004 Published: December 16, 2004

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