Figure 6.
Figure 6. HDAC4 recruits the deacetylase activity from other HDACs through interaction with NcoR1 to down-regulate c-Jun. (A) HDAC4 interacts with the NCoR1/HDAC3 complex in Schwann cells. Schwann cells were infected with Ad HDAC4 3SA Flag or Ad GFP, lysed, and extracts pulled down with anti-Flag agarose beads. Immunoprecipitates, inputs, and postimmunoprecipitates were immunobloted with anti-NCoR1 or anti-HDAC3. NCoR1 and HDAC3 were recovered exclusively from Ad HDAC43SA Flag–infected cells. Expression and immunoprecitation of the introduced proteins was checked by immunoblotting with anti-Flag and anti-GFP antibodies. (B) We introduced mutations D934N or H803A in the HDAC4 3SA GFP construct and transfected the resultant construct into the Schwannoma cell line RT4D6. Cell extracts were pulled down with GFP-trap (Chromotek) and immunoblotted with anti-NCoR1 and anti-HDAC3 antibodies. Whereas NCoR1 and HDAC3 were efficiently pulled down by HDAC4 3SA GFP, none of these proteins were found in the HDAC4 3SA D934N GFP or HDAC4 3SA H803A GFP immunoprecipitates. Expression and immunoprecitation of the transfected constructs were checked by immunoblotting with anti-GFP antibodies. (C) Blocking the interaction of HDAC4 3SA with the multiprotein complex NcoR1/HDAC3 by the mutation D934N interferes with its capacity to down-regulate c-Jun. Cultured Schwann cells were transfected with this mutant and submitted to immunofluorescence with the c-Jun antibody. Transfected cells were identified by GFP expression (arrowheads). The graph shows the ratio of c-Jun fluorescence intensity in transfected relative to nontransfected cells of the same coverslip obtained from 300 cells per condition in three different experiments. Data are given as mean ± SE and analyzed with the t test (two-sided). *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) A similar result was obtained with the HDAC4 3SA H803A GFP protein. Bars, 25 μm. (E) Summary statistical analysis of the effects of the different mutations introduced in HDAC4 on c-Jun expression levels normalized for GFP transfected Schwann cells. Data are given as mean ± SE and analyzed with the t test (two-sided). *, P < 0.05; **, P < 0.01; ***, P < 0.001. (F) c-Jun down-regulation by cAMP depends on a protein with deacetylase activity. Cultured rat Schwann cells were incubated with 1 mM dbcAMP for 48 h to down-regulate c-Jun. Then, and still in the presence of dbcAMP, a pan-HDAC inhibitor was added (2 µM TSA). Cells were harvested at different time points and immunoblotted for c-Jun. GAPDH was used as a loading control. As shown, inhibition of deacetylase activity reverts the down-regulation of c-Jun by dbcAMP.

HDAC4 recruits the deacetylase activity from other HDACs through interaction with NcoR1 to down-regulate c-Jun. (A) HDAC4 interacts with the NCoR1/HDAC3 complex in Schwann cells. Schwann cells were infected with Ad HDAC4 3SA Flag or Ad GFP, lysed, and extracts pulled down with anti-Flag agarose beads. Immunoprecipitates, inputs, and postimmunoprecipitates were immunobloted with anti-NCoR1 or anti-HDAC3. NCoR1 and HDAC3 were recovered exclusively from Ad HDAC43SA Flag–infected cells. Expression and immunoprecitation of the introduced proteins was checked by immunoblotting with anti-Flag and anti-GFP antibodies. (B) We introduced mutations D934N or H803A in the HDAC4 3SA GFP construct and transfected the resultant construct into the Schwannoma cell line RT4D6. Cell extracts were pulled down with GFP-trap (Chromotek) and immunoblotted with anti-NCoR1 and anti-HDAC3 antibodies. Whereas NCoR1 and HDAC3 were efficiently pulled down by HDAC4 3SA GFP, none of these proteins were found in the HDAC4 3SA D934N GFP or HDAC4 3SA H803A GFP immunoprecipitates. Expression and immunoprecitation of the transfected constructs were checked by immunoblotting with anti-GFP antibodies. (C) Blocking the interaction of HDAC4 3SA with the multiprotein complex NcoR1/HDAC3 by the mutation D934N interferes with its capacity to down-regulate c-Jun. Cultured Schwann cells were transfected with this mutant and submitted to immunofluorescence with the c-Jun antibody. Transfected cells were identified by GFP expression (arrowheads). The graph shows the ratio of c-Jun fluorescence intensity in transfected relative to nontransfected cells of the same coverslip obtained from 300 cells per condition in three different experiments. Data are given as mean ± SE and analyzed with the t test (two-sided). *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) A similar result was obtained with the HDAC4 3SA H803A GFP protein. Bars, 25 μm. (E) Summary statistical analysis of the effects of the different mutations introduced in HDAC4 on c-Jun expression levels normalized for GFP transfected Schwann cells. Data are given as mean ± SE and analyzed with the t test (two-sided). *, P < 0.05; **, P < 0.01; ***, P < 0.001. (F) c-Jun down-regulation by cAMP depends on a protein with deacetylase activity. Cultured rat Schwann cells were incubated with 1 mM dbcAMP for 48 h to down-regulate c-Jun. Then, and still in the presence of dbcAMP, a pan-HDAC inhibitor was added (2 µM TSA). Cells were harvested at different time points and immunoblotted for c-Jun. GAPDH was used as a loading control. As shown, inhibition of deacetylase activity reverts the down-regulation of c-Jun by dbcAMP.

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