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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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Over-expression of the RD4 region of SMRT inhibits HDAC inhibition-mediated SMRT export.A) Upper: Over-expression of RD4 inhibits TSA-mediated nuclear export of SMRT. Neurons were transfected with GFP-SMRTFL plus an expression vector encoding the amino-acids 1025–1526 (RD3) or 1025–1861 (RD3–4) of SMRT or a control plasmid (encoding ß-globin). After 48 h neurons were treated with TSA and the subcellular localization of GFP-SMRTFL was analyzed. *p<0.05 (n = 4). Lower: Example pictures to illustrate the nuclear localization of SMRT1025–1526 and SMRT1025–1861. Neurons were transfected with plasmids encoding myc-tagged SMRT1025–1526 or SMRT1025–1861. After 48 h the localization of these portions of SMRT was analysed by immunofluorescence using an anti-myc antibody. Arrows point to a transfected cell in each case. B) Over-expression of the RD4 region partially reverses the cytoplasmic redistribution of SMRT caused by deletion of the DAD. Neurons were co-transfected with GFP-SMRTFL or SMRTΔ(305–547) with an expression vector encoding the amino acids 1025–1861 of SMRT or a control plasmid (encoding ß-globin) as indicated. After 48 h the cellular localization of SMRT and SMRTΔ(305–547) was analyzed. *p<0.05 (n = 3).
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pone-0021056-g004: Over-expression of the RD4 region of SMRT inhibits HDAC inhibition-mediated SMRT export.A) Upper: Over-expression of RD4 inhibits TSA-mediated nuclear export of SMRT. Neurons were transfected with GFP-SMRTFL plus an expression vector encoding the amino-acids 1025–1526 (RD3) or 1025–1861 (RD3–4) of SMRT or a control plasmid (encoding ß-globin). After 48 h neurons were treated with TSA and the subcellular localization of GFP-SMRTFL was analyzed. *p<0.05 (n = 4). Lower: Example pictures to illustrate the nuclear localization of SMRT1025–1526 and SMRT1025–1861. Neurons were transfected with plasmids encoding myc-tagged SMRT1025–1526 or SMRT1025–1861. After 48 h the localization of these portions of SMRT was analysed by immunofluorescence using an anti-myc antibody. Arrows point to a transfected cell in each case. B) Over-expression of the RD4 region partially reverses the cytoplasmic redistribution of SMRT caused by deletion of the DAD. Neurons were co-transfected with GFP-SMRTFL or SMRTΔ(305–547) with an expression vector encoding the amino acids 1025–1861 of SMRT or a control plasmid (encoding ß-globin) as indicated. After 48 h the cellular localization of SMRT and SMRTΔ(305–547) was analyzed. *p<0.05 (n = 3).

Mentions: If this were indeed the case, we predicted that over-expression of the RD4 region within the nucleus would compete with SMRT for these hypothetical factors and inhibit TSA-induced export of GFP-SMRTFL. We first expressed a portion of SMRT containing the RD4 region (SMRT1517–1861) but found it to be exclusively cytoplasmic (data not shown), consistent with our recent observations that sequences N-terminal of position 1523 are required for nuclear localization of SMRT [16]. We therefore expressed a larger portion of SMRT (SMRT1025–1861), still including the RD4 region but with additional N-terminal sequence within the RD3 region which, upon expression, revealed nuclear localization (Fig. 4a lower). We therefore investigated the effect of expressing SMRT1025–1861 on TSA-induced export of GFP-SMRTFL. We found that co-expression of SMRT1025–1861 inhibited TSA-induced export of GFP-SMRTFL (Fig. 4a, upper). To determine whether this effect could be attributed directly to the RD4 region (SMRT1523–1861), we investigated the effect of expressing SMRT1025–1523, the region N-terminal of the RD4 region (containing RD3) that we added in order to confer nuclear localization. Expression of SMRT1025–1523, which is localized to the nucleus ([16] and Fig. 4a lower), failed to inhibit TSA-induced export of SMRTFL (Fig. 4a, upper), strongly indicating that the inhibitory effect of SMRT1025–1861 is due to the presence of the RD4 region and not the RD3 region. Thus, over-expression of RD4 region interferes with TSA-induced SMRTFL export, consistent with the deletion studies and further implicating this domain as being required for interaction with the SMRT export machinery.


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

Soriano FX, Hardingham GE - PLoS ONE (2011)

Over-expression of the RD4 region of SMRT inhibits HDAC inhibition-mediated SMRT export.A) Upper: Over-expression of RD4 inhibits TSA-mediated nuclear export of SMRT. Neurons were transfected with GFP-SMRTFL plus an expression vector encoding the amino-acids 1025–1526 (RD3) or 1025–1861 (RD3–4) of SMRT or a control plasmid (encoding ß-globin). After 48 h neurons were treated with TSA and the subcellular localization of GFP-SMRTFL was analyzed. *p<0.05 (n = 4). Lower: Example pictures to illustrate the nuclear localization of SMRT1025–1526 and SMRT1025–1861. Neurons were transfected with plasmids encoding myc-tagged SMRT1025–1526 or SMRT1025–1861. After 48 h the localization of these portions of SMRT was analysed by immunofluorescence using an anti-myc antibody. Arrows point to a transfected cell in each case. B) Over-expression of the RD4 region partially reverses the cytoplasmic redistribution of SMRT caused by deletion of the DAD. Neurons were co-transfected with GFP-SMRTFL or SMRTΔ(305–547) with an expression vector encoding the amino acids 1025–1861 of SMRT or a control plasmid (encoding ß-globin) as indicated. After 48 h the cellular localization of SMRT and SMRTΔ(305–547) was analyzed. *p<0.05 (n = 3).
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pone-0021056-g004: Over-expression of the RD4 region of SMRT inhibits HDAC inhibition-mediated SMRT export.A) Upper: Over-expression of RD4 inhibits TSA-mediated nuclear export of SMRT. Neurons were transfected with GFP-SMRTFL plus an expression vector encoding the amino-acids 1025–1526 (RD3) or 1025–1861 (RD3–4) of SMRT or a control plasmid (encoding ß-globin). After 48 h neurons were treated with TSA and the subcellular localization of GFP-SMRTFL was analyzed. *p<0.05 (n = 4). Lower: Example pictures to illustrate the nuclear localization of SMRT1025–1526 and SMRT1025–1861. Neurons were transfected with plasmids encoding myc-tagged SMRT1025–1526 or SMRT1025–1861. After 48 h the localization of these portions of SMRT was analysed by immunofluorescence using an anti-myc antibody. Arrows point to a transfected cell in each case. B) Over-expression of the RD4 region partially reverses the cytoplasmic redistribution of SMRT caused by deletion of the DAD. Neurons were co-transfected with GFP-SMRTFL or SMRTΔ(305–547) with an expression vector encoding the amino acids 1025–1861 of SMRT or a control plasmid (encoding ß-globin) as indicated. After 48 h the cellular localization of SMRT and SMRTΔ(305–547) was analyzed. *p<0.05 (n = 3).
Mentions: If this were indeed the case, we predicted that over-expression of the RD4 region within the nucleus would compete with SMRT for these hypothetical factors and inhibit TSA-induced export of GFP-SMRTFL. We first expressed a portion of SMRT containing the RD4 region (SMRT1517–1861) but found it to be exclusively cytoplasmic (data not shown), consistent with our recent observations that sequences N-terminal of position 1523 are required for nuclear localization of SMRT [16]. We therefore expressed a larger portion of SMRT (SMRT1025–1861), still including the RD4 region but with additional N-terminal sequence within the RD3 region which, upon expression, revealed nuclear localization (Fig. 4a lower). We therefore investigated the effect of expressing SMRT1025–1861 on TSA-induced export of GFP-SMRTFL. We found that co-expression of SMRT1025–1861 inhibited TSA-induced export of GFP-SMRTFL (Fig. 4a, upper). To determine whether this effect could be attributed directly to the RD4 region (SMRT1523–1861), we investigated the effect of expressing SMRT1025–1523, the region N-terminal of the RD4 region (containing RD3) that we added in order to confer nuclear localization. Expression of SMRT1025–1523, which is localized to the nucleus ([16] and Fig. 4a lower), failed to inhibit TSA-induced export of SMRTFL (Fig. 4a, upper), strongly indicating that the inhibitory effect of SMRT1025–1861 is due to the presence of the RD4 region and not the RD3 region. Thus, over-expression of RD4 region interferes with TSA-induced SMRTFL export, consistent with the deletion studies and further implicating this domain as being required for interaction with the SMRT export machinery.

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