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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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Inhibition of class I HDAC and knockdown of HDAC3 is sufficient to promote SMRT export.A) GFP-SMRTFL export is insensitive to Leptomycin B and to mutation of a leucine-rich potential nuclear export sequence. Neurons were transfected with plasmids encoding GFP-SMRTFL or GFP-SMRTΔLeu and treated, where indicated with Leptomycin B (20 ng/ml) for 1 h prior to treatment with TSA. Note that although redistribution was observed after 1–2 h treatment with TSA and other HDAC inhibitors, the effect observed was greater after 6–8 h and so all data presented in this manuscript relates to treatment with the indicated drugs for this time. *p<0.05 (n = 4). B) Neurons were transfected with plasmids encoding GFP-SMRTFL and 48 h after transfection the neurons were treated with TSA, VPA or Apicidin and the cellular localization of GFP-SMRTFL was analyzed. *p<0.05 (n = 3). C) Examples of the cellular localization of GFP-SMRTFL after treatments with the indicated HDAC inhibitors. Scale bar is 20 µm here and throughout the manuscript. D) Example pictures to demonstrate the efficacy of HDAC3-directed siRNA in knocking down endogenous HDAC3 expression in rat cortical neurons. Neurons were transfected with the siRNAs as indicated, plus peGFP to identify transfected cells. After 72 h, cells were fixed and HDAC3 expression analysed by immunofluorescence. White arrows point to transfected neurons. E) HDAC3 siRNA causes redistribution of SMRT to the cytoplasm. Neurons were transfected with plasmids encoding GFP-SMRTFL plus siRNA as indicated. SMRT localization was studied 72 h post-transfection. *p<0.05 (n = 5).
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pone-0021056-g001: Inhibition of class I HDAC and knockdown of HDAC3 is sufficient to promote SMRT export.A) GFP-SMRTFL export is insensitive to Leptomycin B and to mutation of a leucine-rich potential nuclear export sequence. Neurons were transfected with plasmids encoding GFP-SMRTFL or GFP-SMRTΔLeu and treated, where indicated with Leptomycin B (20 ng/ml) for 1 h prior to treatment with TSA. Note that although redistribution was observed after 1–2 h treatment with TSA and other HDAC inhibitors, the effect observed was greater after 6–8 h and so all data presented in this manuscript relates to treatment with the indicated drugs for this time. *p<0.05 (n = 4). B) Neurons were transfected with plasmids encoding GFP-SMRTFL and 48 h after transfection the neurons were treated with TSA, VPA or Apicidin and the cellular localization of GFP-SMRTFL was analyzed. *p<0.05 (n = 3). C) Examples of the cellular localization of GFP-SMRTFL after treatments with the indicated HDAC inhibitors. Scale bar is 20 µm here and throughout the manuscript. D) Example pictures to demonstrate the efficacy of HDAC3-directed siRNA in knocking down endogenous HDAC3 expression in rat cortical neurons. Neurons were transfected with the siRNAs as indicated, plus peGFP to identify transfected cells. After 72 h, cells were fixed and HDAC3 expression analysed by immunofluorescence. White arrows point to transfected neurons. E) HDAC3 siRNA causes redistribution of SMRT to the cytoplasm. Neurons were transfected with plasmids encoding GFP-SMRTFL plus siRNA as indicated. SMRT localization was studied 72 h post-transfection. *p<0.05 (n = 5).

Mentions: We previously reported that HDAC inhibition achieved by treatment with TSA promotes nuclear export of SMRT [26], prompting us to further investigate the mechanism and basis for this export. We confirmed our previous observation that treatment of cortical neurons with TSA caused the export of full length GFP-SMRTα (GFP-SMRTFL, Fig. 1a). Many proteins are exported via a CRM1-dependent association with a leucine-rich nuclear export site (NES), although many are not [35]. Search for a classical leucine-rich nuclear export site [33] revealed only one potential site at position 1985 (QELELRSL), which when mutated to QEAEARSL (GFP-SMRTΔLeu), had no effect on TSA-induced export (Fig. 1a), or indeed, export by synaptic activity (data not shown). Moreover, TSA-induced SMRT export was found to be insensitive to leptomycin B (Fig. 1a), and thus joins a lengthening list of proteins (that include many nuclear hormone receptors) whose export is independent of the CRM1/leucine-rich NES pathway.


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

Soriano FX, Hardingham GE - PLoS ONE (2011)

Inhibition of class I HDAC and knockdown of HDAC3 is sufficient to promote SMRT export.A) GFP-SMRTFL export is insensitive to Leptomycin B and to mutation of a leucine-rich potential nuclear export sequence. Neurons were transfected with plasmids encoding GFP-SMRTFL or GFP-SMRTΔLeu and treated, where indicated with Leptomycin B (20 ng/ml) for 1 h prior to treatment with TSA. Note that although redistribution was observed after 1–2 h treatment with TSA and other HDAC inhibitors, the effect observed was greater after 6–8 h and so all data presented in this manuscript relates to treatment with the indicated drugs for this time. *p<0.05 (n = 4). B) Neurons were transfected with plasmids encoding GFP-SMRTFL and 48 h after transfection the neurons were treated with TSA, VPA or Apicidin and the cellular localization of GFP-SMRTFL was analyzed. *p<0.05 (n = 3). C) Examples of the cellular localization of GFP-SMRTFL after treatments with the indicated HDAC inhibitors. Scale bar is 20 µm here and throughout the manuscript. D) Example pictures to demonstrate the efficacy of HDAC3-directed siRNA in knocking down endogenous HDAC3 expression in rat cortical neurons. Neurons were transfected with the siRNAs as indicated, plus peGFP to identify transfected cells. After 72 h, cells were fixed and HDAC3 expression analysed by immunofluorescence. White arrows point to transfected neurons. E) HDAC3 siRNA causes redistribution of SMRT to the cytoplasm. Neurons were transfected with plasmids encoding GFP-SMRTFL plus siRNA as indicated. SMRT localization was studied 72 h post-transfection. *p<0.05 (n = 5).
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Related In: Results  -  Collection

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pone-0021056-g001: Inhibition of class I HDAC and knockdown of HDAC3 is sufficient to promote SMRT export.A) GFP-SMRTFL export is insensitive to Leptomycin B and to mutation of a leucine-rich potential nuclear export sequence. Neurons were transfected with plasmids encoding GFP-SMRTFL or GFP-SMRTΔLeu and treated, where indicated with Leptomycin B (20 ng/ml) for 1 h prior to treatment with TSA. Note that although redistribution was observed after 1–2 h treatment with TSA and other HDAC inhibitors, the effect observed was greater after 6–8 h and so all data presented in this manuscript relates to treatment with the indicated drugs for this time. *p<0.05 (n = 4). B) Neurons were transfected with plasmids encoding GFP-SMRTFL and 48 h after transfection the neurons were treated with TSA, VPA or Apicidin and the cellular localization of GFP-SMRTFL was analyzed. *p<0.05 (n = 3). C) Examples of the cellular localization of GFP-SMRTFL after treatments with the indicated HDAC inhibitors. Scale bar is 20 µm here and throughout the manuscript. D) Example pictures to demonstrate the efficacy of HDAC3-directed siRNA in knocking down endogenous HDAC3 expression in rat cortical neurons. Neurons were transfected with the siRNAs as indicated, plus peGFP to identify transfected cells. After 72 h, cells were fixed and HDAC3 expression analysed by immunofluorescence. White arrows point to transfected neurons. E) HDAC3 siRNA causes redistribution of SMRT to the cytoplasm. Neurons were transfected with plasmids encoding GFP-SMRTFL plus siRNA as indicated. SMRT localization was studied 72 h post-transfection. *p<0.05 (n = 5).
Mentions: We previously reported that HDAC inhibition achieved by treatment with TSA promotes nuclear export of SMRT [26], prompting us to further investigate the mechanism and basis for this export. We confirmed our previous observation that treatment of cortical neurons with TSA caused the export of full length GFP-SMRTα (GFP-SMRTFL, Fig. 1a). Many proteins are exported via a CRM1-dependent association with a leucine-rich nuclear export site (NES), although many are not [35]. Search for a classical leucine-rich nuclear export site [33] revealed only one potential site at position 1985 (QELELRSL), which when mutated to QEAEARSL (GFP-SMRTΔLeu), had no effect on TSA-induced export (Fig. 1a), or indeed, export by synaptic activity (data not shown). Moreover, TSA-induced SMRT export was found to be insensitive to leptomycin B (Fig. 1a), and thus joins a lengthening list of proteins (that include many nuclear hormone receptors) whose export is independent of the CRM1/leucine-rich NES pathway.

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