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In cortical neurons HDAC3 activity suppresses RD4-dependent SMRT export.

Soriano FX, Hardingham GE - PLoS ONE (2011)

Bottom Line: Consistent with a role for HDAC3 activity in promoting SMRT nuclear localization, we found that inactivation of SMRT's DAD by deletion or point mutation triggered partial redistribution of SMRT to the cytoplasm.Collectively these data support a model whereby SMRT's RD4 region can recruit factors capable of mediating nuclear export of SMRT, but whose function and/or recruitment is suppressed by HDAC3 activity.Furthermore, they underline the fact that HDAC inhibitors can cause reorganization and redistribution of corepressor complexes.

View Article: PubMed Central - PubMed

Affiliation: Centre for Integrative Physiology, University of Edinburgh, Edinburgh, United Kingdom.

ABSTRACT
The transcriptional corepressor SMRT controls neuronal responsiveness of several transcription factors and can regulate neuroprotective and neurogenic pathways. SMRT is a multi-domain protein that complexes with HDAC3 as well as being capable of interactions with HDACs 1, 4, 5 and 7. We previously showed that in rat cortical neurons, nuclear localisation of SMRT requires histone deacetylase activity: Inhibition of class I/II HDACs by treatment with trichostatin A (TSA) causes redistribution of SMRT to the cytoplasm, and potentiates the activation of SMRT-repressed nuclear receptors. Here we have sought to identify the HDAC(s) and region(s) of SMRT responsible for anchoring it in the nucleus under normal circumstances and for mediating nuclear export following HDAC inhibition. We show that in rat cortical neurons SMRT export can be triggered by treatment with the class I-preferring HDAC inhibitor valproate and the HDAC2/3-selective inhibitor apicidin, and by HDAC3 knockdown, implicating HDAC3 activity as being required to maintain SMRT in the nucleus. HDAC3 interaction with SMRT's deacetylation activation domain (DAD) is known to be important for activation of HDAC3 deacetylase function. Consistent with a role for HDAC3 activity in promoting SMRT nuclear localization, we found that inactivation of SMRT's DAD by deletion or point mutation triggered partial redistribution of SMRT to the cytoplasm. We also investigated whether other regions of SMRT were involved in mediating nuclear export following HDAC inhibition. TSA- and valproate-induced SMRT export was strongly impaired by deletion of its repression domain-4 (RD4). Furthermore, over-expression of a region of SMRT containing the RD4 region suppressed TSA-induced export of full-length SMRT. Collectively these data support a model whereby SMRT's RD4 region can recruit factors capable of mediating nuclear export of SMRT, but whose function and/or recruitment is suppressed by HDAC3 activity. Furthermore, they underline the fact that HDAC inhibitors can cause reorganization and redistribution of corepressor complexes.

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Deletion of SMRT's HDAC3-activating domain partly mimics and occludes the effect of HDAC inhibition.A) Schematic illustrating the SMRT deletion constructs generated and used in this paper. B) Analysis of the cellular localization of GFP-SMRTFL or GFP-SMRTΔ(305–547) in transfected neurons untreated or treated with TSA. *p<0.05 (n = 4). #p<0.05 comparing control and TSA-treated conditions for each SMRT construct. C) Analysis of the cellular location of GFP-SMRTFL compared to the basal localization of GFP-SMRTF451A. *p<0.05 (n = 5).
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pone-0021056-g002: Deletion of SMRT's HDAC3-activating domain partly mimics and occludes the effect of HDAC inhibition.A) Schematic illustrating the SMRT deletion constructs generated and used in this paper. B) Analysis of the cellular localization of GFP-SMRTFL or GFP-SMRTΔ(305–547) in transfected neurons untreated or treated with TSA. *p<0.05 (n = 4). #p<0.05 comparing control and TSA-treated conditions for each SMRT construct. C) Analysis of the cellular location of GFP-SMRTFL compared to the basal localization of GFP-SMRTF451A. *p<0.05 (n = 5).

Mentions: HDAC3 forms a core complex with SMRT and is absolutely required for its function as a corepressor [6], [37]. HDAC3 has been reported to interact with SMRT at least two different regions, including the RD4 region [2] and also a region in the N-terminus referred to as the deacetylase activation domain (DAD). Interaction of HDAC3 with the DAD is both necessary and sufficient to activate the deacetylase activity of HDAC3, which is otherwise inactive [14]. As such, HDAC3 activity is restricted to complexes with SMRT or its close relative N-CoR. We therefore predicted that deletion of a portion of SMRT containing the DAD could, by inactivating HDAC3, mimic the effect of TSA treatment in promoting SMRT export. We deleted amino acids 305–547 within the context of full length 2472 amino-acid SMRT (GFP-SMRTΔ(305–547)), a schematic illustration of this and all SMRT constructs used in this study is shown in Fig. 2a. GFP-SMRTΔ(305–547) exhibited increased cytoplasmic localization compared to GFP-SMRTFL (Fig. 2b). The effect of DAD deletion in causing cytoplasmic redistribution was non-additive to the effect of TSA: TSA treatment of GFP-SMRTΔ(305–547)-expressing neurons caused a small additional export, but the total level of cytoplasmic SMRT was the same in TSA-treated neurons expressing GFP-SMRTFL as GFP-SMRTΔ(305–547). Given that deletion of DAD blocks SMRT-associated HDAC3 activity [14], this indicates that TSA is acting (at least in part) by blocking SMRT-associated HDAC3 activity. Since point mutation of the DAD at several locations can also inhibit DAD function and SMRT-associated HDAC3 activity [14], we created a DAD-inactivating mutant (GFP-SMRTF451A, [14]). As with GFP-SMRTΔ(305–547), GFP-SMRTF451A exhibited increased cytoplasmic localization compared to GFP-SMRTFL (Fig. 2c), further evidence that DAD-induced HDAC3 activity is important for SMRT nuclear localization. Note though that deletion of the DAD, or its mutation did not completely mimic the effect of TSA treatment, potentially indicating that HDAC activity other than HDAC3 activated by the DAD may contribute to SMRT nuclear localization. Alternatively, since SMRT can homodimerize [38], dimerization between SMRTΔ(305–547) or SMRTF451A and endogenous SMRT could result in recruitment of active HDAC3 to the dimer.


In cortical neurons HDAC3 activity suppresses RD4-dependent SMRT export.

Soriano FX, Hardingham GE - PLoS ONE (2011)

Deletion of SMRT's HDAC3-activating domain partly mimics and occludes the effect of HDAC inhibition.A) Schematic illustrating the SMRT deletion constructs generated and used in this paper. B) Analysis of the cellular localization of GFP-SMRTFL or GFP-SMRTΔ(305–547) in transfected neurons untreated or treated with TSA. *p<0.05 (n = 4). #p<0.05 comparing control and TSA-treated conditions for each SMRT construct. C) Analysis of the cellular location of GFP-SMRTFL compared to the basal localization of GFP-SMRTF451A. *p<0.05 (n = 5).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3111469&req=5

pone-0021056-g002: Deletion of SMRT's HDAC3-activating domain partly mimics and occludes the effect of HDAC inhibition.A) Schematic illustrating the SMRT deletion constructs generated and used in this paper. B) Analysis of the cellular localization of GFP-SMRTFL or GFP-SMRTΔ(305–547) in transfected neurons untreated or treated with TSA. *p<0.05 (n = 4). #p<0.05 comparing control and TSA-treated conditions for each SMRT construct. C) Analysis of the cellular location of GFP-SMRTFL compared to the basal localization of GFP-SMRTF451A. *p<0.05 (n = 5).
Mentions: HDAC3 forms a core complex with SMRT and is absolutely required for its function as a corepressor [6], [37]. HDAC3 has been reported to interact with SMRT at least two different regions, including the RD4 region [2] and also a region in the N-terminus referred to as the deacetylase activation domain (DAD). Interaction of HDAC3 with the DAD is both necessary and sufficient to activate the deacetylase activity of HDAC3, which is otherwise inactive [14]. As such, HDAC3 activity is restricted to complexes with SMRT or its close relative N-CoR. We therefore predicted that deletion of a portion of SMRT containing the DAD could, by inactivating HDAC3, mimic the effect of TSA treatment in promoting SMRT export. We deleted amino acids 305–547 within the context of full length 2472 amino-acid SMRT (GFP-SMRTΔ(305–547)), a schematic illustration of this and all SMRT constructs used in this study is shown in Fig. 2a. GFP-SMRTΔ(305–547) exhibited increased cytoplasmic localization compared to GFP-SMRTFL (Fig. 2b). The effect of DAD deletion in causing cytoplasmic redistribution was non-additive to the effect of TSA: TSA treatment of GFP-SMRTΔ(305–547)-expressing neurons caused a small additional export, but the total level of cytoplasmic SMRT was the same in TSA-treated neurons expressing GFP-SMRTFL as GFP-SMRTΔ(305–547). Given that deletion of DAD blocks SMRT-associated HDAC3 activity [14], this indicates that TSA is acting (at least in part) by blocking SMRT-associated HDAC3 activity. Since point mutation of the DAD at several locations can also inhibit DAD function and SMRT-associated HDAC3 activity [14], we created a DAD-inactivating mutant (GFP-SMRTF451A, [14]). As with GFP-SMRTΔ(305–547), GFP-SMRTF451A exhibited increased cytoplasmic localization compared to GFP-SMRTFL (Fig. 2c), further evidence that DAD-induced HDAC3 activity is important for SMRT nuclear localization. Note though that deletion of the DAD, or its mutation did not completely mimic the effect of TSA treatment, potentially indicating that HDAC activity other than HDAC3 activated by the DAD may contribute to SMRT nuclear localization. Alternatively, since SMRT can homodimerize [38], dimerization between SMRTΔ(305–547) or SMRTF451A and endogenous SMRT could result in recruitment of active HDAC3 to the dimer.

Bottom Line: Consistent with a role for HDAC3 activity in promoting SMRT nuclear localization, we found that inactivation of SMRT's DAD by deletion or point mutation triggered partial redistribution of SMRT to the cytoplasm.Collectively these data support a model whereby SMRT's RD4 region can recruit factors capable of mediating nuclear export of SMRT, but whose function and/or recruitment is suppressed by HDAC3 activity.Furthermore, they underline the fact that HDAC inhibitors can cause reorganization and redistribution of corepressor complexes.

View Article: PubMed Central - PubMed

Affiliation: Centre for Integrative Physiology, University of Edinburgh, Edinburgh, United Kingdom.

ABSTRACT
The transcriptional corepressor SMRT controls neuronal responsiveness of several transcription factors and can regulate neuroprotective and neurogenic pathways. SMRT is a multi-domain protein that complexes with HDAC3 as well as being capable of interactions with HDACs 1, 4, 5 and 7. We previously showed that in rat cortical neurons, nuclear localisation of SMRT requires histone deacetylase activity: Inhibition of class I/II HDACs by treatment with trichostatin A (TSA) causes redistribution of SMRT to the cytoplasm, and potentiates the activation of SMRT-repressed nuclear receptors. Here we have sought to identify the HDAC(s) and region(s) of SMRT responsible for anchoring it in the nucleus under normal circumstances and for mediating nuclear export following HDAC inhibition. We show that in rat cortical neurons SMRT export can be triggered by treatment with the class I-preferring HDAC inhibitor valproate and the HDAC2/3-selective inhibitor apicidin, and by HDAC3 knockdown, implicating HDAC3 activity as being required to maintain SMRT in the nucleus. HDAC3 interaction with SMRT's deacetylation activation domain (DAD) is known to be important for activation of HDAC3 deacetylase function. Consistent with a role for HDAC3 activity in promoting SMRT nuclear localization, we found that inactivation of SMRT's DAD by deletion or point mutation triggered partial redistribution of SMRT to the cytoplasm. We also investigated whether other regions of SMRT were involved in mediating nuclear export following HDAC inhibition. TSA- and valproate-induced SMRT export was strongly impaired by deletion of its repression domain-4 (RD4). Furthermore, over-expression of a region of SMRT containing the RD4 region suppressed TSA-induced export of full-length SMRT. Collectively these data support a model whereby SMRT's RD4 region can recruit factors capable of mediating nuclear export of SMRT, but whose function and/or recruitment is suppressed by HDAC3 activity. Furthermore, they underline the fact that HDAC inhibitors can cause reorganization and redistribution of corepressor complexes.

Show MeSH