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

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TLE1 Corepressor Complex Mediates a Camkinase II␦-Dependent Neurogenic Gene Activation Pathway View metadata, citation and similar papers at core.ac.uk brought to you by CORE 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 re- ported 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
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