hdac4 shrna plasmids  (Santa Cruz Biotechnology)

Journal: The Journal of Cell Biology

Article Title: Class IIa histone deacetylases link cAMP signaling to the myelin transcriptional program of Schwann cells

doi: 10.1083/jcb.201611150

Figure Lengend Snippet: Strong and sustained cAMP signaling induces nuclear shuttling of HDAC4 in Schwann cells. (A) Kinetics of c-Jun reexpression. Immunoblot (IB) for c-Jun from extracts of cultured rat Schwann cells incubated in SATO medium for 2, 5, and 10 h after down-regulation of c-Jun by 1 mM dbcAMP. (B) Reexpression of c-Jun is cell autonomous. After incubation with 1 mM dbcAMP, Schwann cells were incubated in DMEM for 10 h (DMEM) or for the same period but replacing the DMEM every 30 min (DMEM refreshed). The same amount of protein was loaded in each lane and immunoblotted with anti–c-Jun. GAPDH was used as a loading control. (C) cAMP signaling induces shuttling of HDAC4 into the nucleus of Schwann cells. Cultured rat Schwann cells were incubated in SATO medium (control) or in SATO + 1 mM dbcAMP for 24 h, fixed, and submitted to immunofluorescence with anti-HDAC4. (D) A Tukey’s box plot of the ratio between the density of fluorescence in the nucleus and the cytoplasm of 900 cells per condition (three different experiments from three different cultures) is shown. Data were analyzed with the unpaired t test (two-sided). (E) To better determine HDAC4 response to cAMP signaling, rat Schwann cells were transfected with the HDAC4-GFP construct and incubated for 24 h in SATO medium with the indicated compounds. (F) Only prodifferentiating (but not mitogenic) concentrations of dbcAMP and forskolin (FSK) were able to efficiently induce HDAC4-GFP nuclear shuttling (>1). A Tukey’s box plot of the nuclear/cytoplasmic fluorescence intensity ratio for 900 cells per condition of three different experiments is shown. Data were analyzed with the one-way ANOVA, Tukey’s multiple comparisons test. (G) PKA phosphorylation of Ser265 and Ser266 mediates HDAC4 shuttling in response to cAMP signaling. HDAC4 S265/266A GFP–transfected rat Schwann cells were incubated in SATO medium or SATO + 1 mM dbcAMP for 24 h. Shuttling was determined as before and compared with HDAC4-GFP wild-type transfected cells. As shown, Ser265 and Ser266 elimination partially prevented cAMP-induced nuclear shuttling of HDAC4. Tukey’s box plot of the nuclear/cytoplasmic fluorescence intensity ratio for 300 cells per condition of three different experiments is shown. Data were analyzed with the one-way ANOVA, Tukey’s multiple comparisons test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Mean is plotted as a “+.” Bars, 25 µm.

Article Snippet: HDAC4 shRNAi was obtained from Addgene pENTR/U6 HDAC4 shRNA (32220; Addgene).

Techniques: Western Blot, Cell Culture, Incubation, Control, Immunofluorescence, Fluorescence, Transfection, Construct, Phospho-proteomics

Journal: The Journal of Cell Biology

Article Title: Class IIa histone deacetylases link cAMP signaling to the myelin transcriptional program of Schwann cells

doi: 10.1083/jcb.201611150

Figure Lengend Snippet: HDAC4 nuclear shuttling in vivo. (A) P4 rat pups were anesthetized, and the sciatic nerve exposed and immersed in a solution of 1 mM dbcAMP in saline for 1 h. A control with saline was also performed. Then nerves were removed fixed and submitted to immunofluorescence with anti-HDAC4 antibody. Schwann cells were identified by Sox10 expression (red). Nuclei were counterstained with Hoechst. Representative confocal images at low and high magnification are shown. (B) Distribution pattern of HDAC4 in the sciatic nerve changes during postnatal development. At P1, anti-HDAC4 immunofluorescence is widely distributed, whereas at P20, immunoractivity has mainly accumulated in the nucleus. P1 and P20 wild-type mice sciatic nerves were fixed and submitted to immunofluorescence with the anti-HDAC4 antibody. Nuclei were counterstained with Hoechst and images obtained with a confocal microscope. Bars, 25 µm.

Article Snippet: HDAC4 shRNAi was obtained from Addgene pENTR/U6 HDAC4 shRNA (32220; Addgene).

Techniques: In Vivo, Saline, Control, Immunofluorescence, Expressing, Microscopy

Journal: The Journal of Cell Biology

Article Title: Class IIa histone deacetylases link cAMP signaling to the myelin transcriptional program of Schwann cells

doi: 10.1083/jcb.201611150

Figure Lengend Snippet: Loss of HDAC4 function prevents cAMP-induced Schwann cell differentiation. (A) Cultured rat Schwann cells were infected with a Lv expressing an shRNAi for HDAC4 (Lv shHDAC4), incubated in SATO medium with 1 mM dbcAMP, and submitted to immunofluorescence with anti-HDAC4. Nuclei were counterstained with Hoechst. Infected cells were identified by GFP expression. As shown, the Lv shHDAC4 (but not the empty Lv control) blocks HDAC4 expression (arrowheads). (B) Tukey’s box plot of the HDAC4 fluorescence intensity for 300 cells per condition (three different experiments from three different cultures) is included. Data were analyzed with the unpaired t test (two-sided). (C) Immunoblot with anti-HDAC4 confirmed the results. (D) Loss of HDAC4 prevents cAMP-mediated c-Jun down-regulation. Whereas most of the Lv shHDAC4–infected cells retained c-Jun expression after 24 h of 1 mM dbcAMP treatment, most of the Lv control–infected ones were c-Jun negative. (E) A Tukey’s box plot of the nuclear c-Jun fluorescence intensity for 450 cells per condition from four different experiments is shown. Data were analyzed with the unpaired t test (two-sided). (F) We also show a Tukey’s box plot of counts of c-Jun–positive cells expressed as a percentage of the total. These data were analyzed with the Mann-Whitney U test. (G) Finally, the immunoblot with anti–c-Jun confirmed the results. GAPDH was used as a loading control. (H) Loss of HDAC4 prevents Krox20 induction. Schwann cells were infected and incubated as described in A. As is shown, Lv shHDAC4–infected Schwann cells (arrowheads) lost the capacity to induce Krox20 in response to cAMP, whereas the Lv control–infected ones did not. (I) A Tukey’s box plot of the nuclear Krox20 fluorescence intensity for 900 cells per condition, from three different experiments, is shown. Data were analyzed with the unpaired t test (two-sided). (J) Periaxin expression was also impaired in these cells. (K) A Tukey’s box plot of the Periaxin cytoplasmic fluorescence intensity for 600 cells per condition, from three different experiments, is shown. Data were analyzed with the unpaired t test (two-sided). (L) Immunoblot with anti-Periaxin confirmed the results. GAPDH was used as a loading control. *, P < 0.05; **P, < 0.01; ***P, < 0.001. Mean is plotted as a “+.” Bars, 25 µm.

Article Snippet: HDAC4 shRNAi was obtained from Addgene pENTR/U6 HDAC4 shRNA (32220; Addgene).

Techniques: Cell Differentiation, Cell Culture, Infection, Expressing, Incubation, Immunofluorescence, Control, Fluorescence, Western Blot, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: Class IIa histone deacetylases link cAMP signaling to the myelin transcriptional program of Schwann cells

doi: 10.1083/jcb.201611150

Figure Lengend Snippet: Gain of HDAC4 function down-regulates c-Jun . (A) Control experiment: the enforced expression of GFP in Schwann cells produced no significant changes in the endogenous c-Jun expression. (B) c-Jun is partially down-regulated in HDAC4-GFP–transfected Schwann cells. (C) HDAC4 ΔC-GFP, which retains the Mef2-binding domain, produced no changes in c -Jun expression. (D) In contrast, the construct HDAC4 3SA GFP dramatically down-regulated c-Jun expression and induced morphological changes in the cells. Cultured rat Schwann cells were transfected and submitted to immunofluorescence with anti–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 on 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. (E) Cultured rat Schwann cells were infected with Ad HDAC4 3SA Flag or Ad GFP and harvested 3 or 6 d later after 24 h of incubation in SATO medium. Protein extracts were immunoblotted with anti–c-Jun. Extracts from noninfected Schwann cells grown in expansion medium or treated during 24 h with 1 mM dbcAMP were also included. GAPDH was used as a loading control. Bars, 25 µm. Mef2, Mef2 binding site; S/A, serine residues mutated to alanine; NES, nuclear exportation signal.

Article Snippet: HDAC4 shRNAi was obtained from Addgene pENTR/U6 HDAC4 shRNA (32220; Addgene).

Techniques: Control, Expressing, Produced, Transfection, Binding Assay, Construct, Cell Culture, Immunofluorescence, Fluorescence, Infection, Incubation

Journal: The Journal of Cell Biology

Article Title: Class IIa histone deacetylases link cAMP signaling to the myelin transcriptional program of Schwann cells

doi: 10.1083/jcb.201611150

Figure Lengend Snippet: HDAC4 binds to the promoter region of c-Jun in a Mef2–independent way and deacetylates lysine 9 of histone 3. (A) Schwann cells infected with Ad HDAC4 3SA Flag or Ad GFP were cross-linked with PFA. Chromatin was purified and immunoprecipitated with anti-Flag monoclonal antibody or a nonspecific mouse IgG (ChIP grade). qPCR was performed with specific primers for the promoter region of c-Jun . As shown, the recovery of the c-Jun promoter region in the immunoprecipitate was clearly enhanced in HDAC4 3SA Flag–expressing Schwann cells. Nonsignificant recovery was obtained with the nonspecific IgG. Data from five different experiments are given as mean ± SE and analyzed with the paired t test (two-sided). (B) In a parallel series of experiments, chromatin was immunprecipitated with anti-H3K9Ac. Less c-Jun promoter was recovered from the HDAC4 3SA Flag–infected cells than from control cells, suggesting that HDAC4 promotes the deacetylation of lysine 9 from histone 3 in the c-Jun promoter. Data from five different experiments are given as mean ± SE and analyzed with the paired t test (two-sided). (C) To confirm these observations, cultured rat Schwann cells were treated with 1 mM dbcAMP for 24 h and cross-linked with PFA. Chromatin was purified and immunoprecipitated with anti-HDAC4 monoclonal antibody or a nonspecific mouse IgG (ChIP grade). As shown, c-Jun promoter was recovered with the anti-HDAC4 but not with the nonspecific mouse IgG. Data from five different experiments are given as mean ± SE and analyzed with the Mann-Whitney U test. (D) Mutating L175A or V179A in the HDAC4 3SA GFP construct blocks interaction with Mef2. We introduced mutations L174A or V179A in the HDAC4 3SA GFP construct and transfected the resultant constructs into the Schwannoma cell line RT4D6. Cell extracts were pulled down with a GFP-trap (Chromotek) and immunoblotted with anti-Mef2. Whereas Mef2 was efficiently pulled down by HDAC4 3SA GFP, it was not found in the HDAC4 3SA L175A GFP or HDAC4 3SA V179A GFP immunoprecipitates. Expression and immunoprecitation of the transfected constructs were checked by immunoblotting with anti-GFP antibodies. (E) HDAC4 3SA GFP L175A mutant was still able to down-regulate c-Jun, suggesting that interaction with Mef2 is dispensable. (F) The same result was obtained with the HDAC4 3SA GFP V179A. The graph shows the ratio of c-Jun fluorescence intensity in transfected relative to nontransfected cultured rat Schwann 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. Bars, 25 µm.

Article Snippet: HDAC4 shRNAi was obtained from Addgene pENTR/U6 HDAC4 shRNA (32220; Addgene).

Techniques: Infection, Purification, Immunoprecipitation, Expressing, Control, Cell Culture, MANN-WHITNEY, Construct, Transfection, Western Blot, Mutagenesis, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Class IIa histone deacetylases link cAMP signaling to the myelin transcriptional program of Schwann cells

doi: 10.1083/jcb.201611150

Figure Lengend Snippet: 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.

Article Snippet: HDAC4 shRNAi was obtained from Addgene pENTR/U6 HDAC4 shRNA (32220; Addgene).

Techniques: Histone Deacetylase Assay, Activity Assay, Infection, Expressing, Western Blot, Construct, Transfection, Blocking Assay, Mutagenesis, Cell Culture, Immunofluorescence, Fluorescence, Incubation, Control, Inhibition

Journal: The Journal of Cell Biology

Article Title: Class IIa histone deacetylases link cAMP signaling to the myelin transcriptional program of Schwann cells

doi: 10.1083/jcb.201611150

Figure Lengend Snippet: HDAC4 gain of function induces Schwann cell differentiation. (A) HDAC4 3SA blocks Schwann cell proliferation. Schwann cells were infected with Ad HDAC4 3SA Flag or Ad GFP and incubated in SATO medium. 24 h later, cells were fixed and examined by immunofluorescence with ant-Ki67 antibodies. Infected cells were identified with anti-GFP or anti-flag antibodies. Arrows indicate Ki67-postive infected cells. (B) Quantification of more than 600 cells from three different experiments is shown. Data are given as mean ± SE and analyzed with the unpaired t test (two-sided). *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) HDAC4 3SA induces profound morphological changes. Ad GFP–infected Schwann cells show the typical bipolar shape, which changes to an expanded flat epithelial-like morphology with enlarged nucleus in the Ad HDAC4 3SA Flag–infected cells. (D) To substantiate these changes, nuclear area was quantified in 668 cells per condition from three different experiments. A Tukey’s box plot of the results is shown. Data were analyzed with the unpaired t test (two-sided). *, P < 0.05; **, P < 0.01; ***, P < 0.001. (E) HDAC4 3SA induces Krox20 expression. Schwann cells were infected with Ad HDAC4 3SA or Ad GFP and incubated in SATO medium. 24 h later, cells were fixed and examined by immunofluorescence with anti-Krox20 antibody. As is shown, only HDAC4 3SA–infected cells expressed Krox20. Arrowheads indicate infected cells. (F) Quantification of mRNA levels expressed typically by nonmyelin-forming (red) and by myelin-forming Schwann cells (magenta) in Ad HDAC4 3SA Flag–infected cells normalized for controls (Ad GFP–infected Schwann cells). Sox10 , a gene that is expressed by both, is in black. As shown, HDAC4 3SA induces a clear shift toward the myelin gene expression program. As expected, no changes were observed for Sox10 . Data are given as mean ± SE and analyzed with the Kolmogorov-Smirnov test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bars, 25 µm.

Article Snippet: HDAC4 shRNAi was obtained from Addgene pENTR/U6 HDAC4 shRNA (32220; Addgene).

Techniques: Cell Differentiation, Infection, Incubation, Immunofluorescence, Expressing, Gene Expression

Journal: The Journal of Cell Biology

Article Title: Class IIa histone deacetylases link cAMP signaling to the myelin transcriptional program of Schwann cells

doi: 10.1083/jcb.201611150

Figure Lengend Snippet: HDAC4 and HDAC5 redundantly contribute to Schwann cell differentiation and myelin development in vivo. (A) Removal of HDAC4 and HDAC5 in Schwann cells delays myelin gene expression. mRNA quantification for markers of nonmyelin- and myelin-forming cells in the PNS. P0 sciatic nerves were removed and total RNA extracted. RT-qPCR with mouse-specific primers for the indicated genes was performed and normalized to 18S rRNA. Graph shows the percentage of mRNA for each gene in the HDAC4/5 ScKO ( P0-Cre (+/−) ; HDAC4 (flx/flx) ; HDAC5 (−/−) ) normalized for the control ( P0-Cre (−/−) ; HDAC4 (flx/flx) ; HDAC5 (−/−) ) littermates. At least four mice from four different litters were used per genotype. Data were analyzed with the Kolmogorov-Smirnov test. A scatter plot is shown with the results obtained in each different experiment, which include also the mean ± SE. (B) Retardation in myelin gene expression is reflected in a delay in myelin development. Transmission EM of P0, P2, and P8 sciatic nerves. Tukey’s box plots show the quantification of MS density (MS/100 µm 2 ) and the myelination index (ratio of the number of myelin profiles [MS] to the total number of 1:1 segregated axons [MS+PS]). As is shown, whereas both parameters are decreased in the HDAC4/5 ScKO at P0 and P2, no differences could be found at P8. For P0, 80 images from four different mice per genotype were quantified; for P2, 78 images from six mice; for P8, 11 images from two different mice per genotype. Data were analyzed with the Mann-Whitney U test. Mean is plotted as a “+.” *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bar, 2 µm. (C) c-Jun is up-regulated faster after nerve injury in the HDAC4/5 ScKO. The sciatic nerves of three HDAC4/5 ScKO and three control mice were injured and analyzed 16 h later. Transverse cryosections were incubated with the indicated primary antibodies. Imaging was performed on a confocal microscope. Schwann cells were identified as Sox10-positive cells. (D) A Tukey’s box plot of the percentage of c-Jun–positive cells. Six images per animal were counted using Image J software. Data were analyzed with the Mann-Whitney U test. Mean is plotted as a “+.” *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bar, 25 µm.

Article Snippet: HDAC4 shRNAi was obtained from Addgene pENTR/U6 HDAC4 shRNA (32220; Addgene).

Techniques: Cell Differentiation, In Vivo, Gene Expression, Quantitative RT-PCR, Control, Transmission Assay, MANN-WHITNEY, Incubation, Imaging, Microscopy, Software

Journal: The Journal of Cell Biology

Article Title: Class IIa histone deacetylases link cAMP signaling to the myelin transcriptional program of Schwann cells

doi: 10.1083/jcb.201611150

Figure Lengend Snippet: Proposed working model. Gpr126 activation raises intracellular cAMP-activating PKA, which phosphorylates HDAC4 in Ser265/266. This (together with another indirect mechanism) shuttles HDAC4 into the nucleus, where it binds to the promoter of c-Jun (through a not yet identified transcription factor [TF]) recruiting the NCoR1/HDAC3 complex to block gene expression.

Article Snippet: HDAC4 shRNAi was obtained from Addgene pENTR/U6 HDAC4 shRNA (32220; Addgene).

Techniques: Activation Assay, Blocking Assay, Gene Expression

Journal: The Journal of Cell Biology

Article Title: Class IIa histone deacetylases link cAMP signaling to the myelin transcriptional program of Schwann cells

doi: 10.1083/jcb.201611150

Figure Lengend Snippet: Strong and sustained cAMP signaling induces nuclear shuttling of HDAC4 in Schwann cells. (A) Kinetics of c-Jun reexpression. Immunoblot (IB) for c-Jun from extracts of cultured rat Schwann cells incubated in SATO medium for 2, 5, and 10 h after down-regulation of c-Jun by 1 mM dbcAMP. (B) Reexpression of c-Jun is cell autonomous. After incubation with 1 mM dbcAMP, Schwann cells were incubated in DMEM for 10 h (DMEM) or for the same period but replacing the DMEM every 30 min (DMEM refreshed). The same amount of protein was loaded in each lane and immunoblotted with anti–c-Jun. GAPDH was used as a loading control. (C) cAMP signaling induces shuttling of HDAC4 into the nucleus of Schwann cells. Cultured rat Schwann cells were incubated in SATO medium (control) or in SATO + 1 mM dbcAMP for 24 h, fixed, and submitted to immunofluorescence with anti-HDAC4. (D) A Tukey’s box plot of the ratio between the density of fluorescence in the nucleus and the cytoplasm of 900 cells per condition (three different experiments from three different cultures) is shown. Data were analyzed with the unpaired t test (two-sided). (E) To better determine HDAC4 response to cAMP signaling, rat Schwann cells were transfected with the HDAC4-GFP construct and incubated for 24 h in SATO medium with the indicated compounds. (F) Only prodifferentiating (but not mitogenic) concentrations of dbcAMP and forskolin (FSK) were able to efficiently induce HDAC4-GFP nuclear shuttling (>1). A Tukey’s box plot of the nuclear/cytoplasmic fluorescence intensity ratio for 900 cells per condition of three different experiments is shown. Data were analyzed with the one-way ANOVA, Tukey’s multiple comparisons test. (G) PKA phosphorylation of Ser265 and Ser266 mediates HDAC4 shuttling in response to cAMP signaling. HDAC4 S265/266A GFP–transfected rat Schwann cells were incubated in SATO medium or SATO + 1 mM dbcAMP for 24 h. Shuttling was determined as before and compared with HDAC4-GFP wild-type transfected cells. As shown, Ser265 and Ser266 elimination partially prevented cAMP-induced nuclear shuttling of HDAC4. Tukey’s box plot of the nuclear/cytoplasmic fluorescence intensity ratio for 300 cells per condition of three different experiments is shown. Data were analyzed with the one-way ANOVA, Tukey’s multiple comparisons test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Mean is plotted as a “+.” Bars, 25 µm.

Article Snippet: HDAC4 shRNAi was obtained from Addgene pENTR/U6 HDAC4 shRNA (32220; Addgene).

Techniques: Western Blot, Cell Culture, Incubation, Immunofluorescence, Fluorescence, Transfection, Construct

Journal: The Journal of Cell Biology

Article Title: Class IIa histone deacetylases link cAMP signaling to the myelin transcriptional program of Schwann cells

doi: 10.1083/jcb.201611150

Figure Lengend Snippet: HDAC4 nuclear shuttling in vivo. (A) P4 rat pups were anesthetized, and the sciatic nerve exposed and immersed in a solution of 1 mM dbcAMP in saline for 1 h. A control with saline was also performed. Then nerves were removed fixed and submitted to immunofluorescence with anti-HDAC4 antibody. Schwann cells were identified by Sox10 expression (red). Nuclei were counterstained with Hoechst. Representative confocal images at low and high magnification are shown. (B) Distribution pattern of HDAC4 in the sciatic nerve changes during postnatal development. At P1, anti-HDAC4 immunofluorescence is widely distributed, whereas at P20, immunoractivity has mainly accumulated in the nucleus. P1 and P20 wild-type mice sciatic nerves were fixed and submitted to immunofluorescence with the anti-HDAC4 antibody. Nuclei were counterstained with Hoechst and images obtained with a confocal microscope. Bars, 25 µm.

Article Snippet: HDAC4 shRNAi was obtained from Addgene pENTR/U6 HDAC4 shRNA (32220; Addgene).

Techniques: In Vivo, Immunofluorescence, Expressing, Microscopy

Journal: The Journal of Cell Biology

Article Title: Class IIa histone deacetylases link cAMP signaling to the myelin transcriptional program of Schwann cells

doi: 10.1083/jcb.201611150

Figure Lengend Snippet: Loss of HDAC4 function prevents cAMP-induced Schwann cell differentiation. (A) Cultured rat Schwann cells were infected with a Lv expressing an shRNAi for HDAC4 (Lv shHDAC4), incubated in SATO medium with 1 mM dbcAMP, and submitted to immunofluorescence with anti-HDAC4. Nuclei were counterstained with Hoechst. Infected cells were identified by GFP expression. As shown, the Lv shHDAC4 (but not the empty Lv control) blocks HDAC4 expression (arrowheads). (B) Tukey’s box plot of the HDAC4 fluorescence intensity for 300 cells per condition (three different experiments from three different cultures) is included. Data were analyzed with the unpaired t test (two-sided). (C) Immunoblot with anti-HDAC4 confirmed the results. (D) Loss of HDAC4 prevents cAMP-mediated c-Jun down-regulation. Whereas most of the Lv shHDAC4–infected cells retained c-Jun expression after 24 h of 1 mM dbcAMP treatment, most of the Lv control–infected ones were c-Jun negative. (E) A Tukey’s box plot of the nuclear c-Jun fluorescence intensity for 450 cells per condition from four different experiments is shown. Data were analyzed with the unpaired t test (two-sided). (F) We also show a Tukey’s box plot of counts of c-Jun–positive cells expressed as a percentage of the total. These data were analyzed with the Mann-Whitney U test. (G) Finally, the immunoblot with anti–c-Jun confirmed the results. GAPDH was used as a loading control. (H) Loss of HDAC4 prevents Krox20 induction. Schwann cells were infected and incubated as described in A. As is shown, Lv shHDAC4–infected Schwann cells (arrowheads) lost the capacity to induce Krox20 in response to cAMP, whereas the Lv control–infected ones did not. (I) A Tukey’s box plot of the nuclear Krox20 fluorescence intensity for 900 cells per condition, from three different experiments, is shown. Data were analyzed with the unpaired t test (two-sided). (J) Periaxin expression was also impaired in these cells. (K) A Tukey’s box plot of the Periaxin cytoplasmic fluorescence intensity for 600 cells per condition, from three different experiments, is shown. Data were analyzed with the unpaired t test (two-sided). (L) Immunoblot with anti-Periaxin confirmed the results. GAPDH was used as a loading control. *, P < 0.05; **P, < 0.01; ***P, < 0.001. Mean is plotted as a “+.” Bars, 25 µm.

Article Snippet: HDAC4 shRNAi was obtained from Addgene pENTR/U6 HDAC4 shRNA (32220; Addgene).

Techniques: Cell Differentiation, Cell Culture, Infection, Expressing, Incubation, Immunofluorescence, Fluorescence, Western Blot, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: Class IIa histone deacetylases link cAMP signaling to the myelin transcriptional program of Schwann cells

doi: 10.1083/jcb.201611150

Figure Lengend Snippet: Gain of HDAC4 function down-regulates c-Jun . (A) Control experiment: the enforced expression of GFP in Schwann cells produced no significant changes in the endogenous c-Jun expression. (B) c-Jun is partially down-regulated in HDAC4-GFP–transfected Schwann cells. (C) HDAC4 ΔC-GFP, which retains the Mef2-binding domain, produced no changes in c -Jun expression. (D) In contrast, the construct HDAC4 3SA GFP dramatically down-regulated c-Jun expression and induced morphological changes in the cells. Cultured rat Schwann cells were transfected and submitted to immunofluorescence with anti–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 on 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. (E) Cultured rat Schwann cells were infected with Ad HDAC4 3SA Flag or Ad GFP and harvested 3 or 6 d later after 24 h of incubation in SATO medium. Protein extracts were immunoblotted with anti–c-Jun. Extracts from noninfected Schwann cells grown in expansion medium or treated during 24 h with 1 mM dbcAMP were also included. GAPDH was used as a loading control. Bars, 25 µm. Mef2, Mef2 binding site; S/A, serine residues mutated to alanine; NES, nuclear exportation signal.

Article Snippet: HDAC4 shRNAi was obtained from Addgene pENTR/U6 HDAC4 shRNA (32220; Addgene).

Techniques: Expressing, Produced, Transfection, Binding Assay, Construct, Cell Culture, Immunofluorescence, Fluorescence, Infection, Incubation

Journal: The Journal of Cell Biology

Article Title: Class IIa histone deacetylases link cAMP signaling to the myelin transcriptional program of Schwann cells

doi: 10.1083/jcb.201611150

Figure Lengend Snippet: HDAC4 binds to the promoter region of c-Jun in a Mef2–independent way and deacetylates lysine 9 of histone 3. (A) Schwann cells infected with Ad HDAC4 3SA Flag or Ad GFP were cross-linked with PFA. Chromatin was purified and immunoprecipitated with anti-Flag monoclonal antibody or a nonspecific mouse IgG (ChIP grade). qPCR was performed with specific primers for the promoter region of c-Jun . As shown, the recovery of the c-Jun promoter region in the immunoprecipitate was clearly enhanced in HDAC4 3SA Flag–expressing Schwann cells. Nonsignificant recovery was obtained with the nonspecific IgG. Data from five different experiments are given as mean ± SE and analyzed with the paired t test (two-sided). (B) In a parallel series of experiments, chromatin was immunprecipitated with anti-H3K9Ac. Less c-Jun promoter was recovered from the HDAC4 3SA Flag–infected cells than from control cells, suggesting that HDAC4 promotes the deacetylation of lysine 9 from histone 3 in the c-Jun promoter. Data from five different experiments are given as mean ± SE and analyzed with the paired t test (two-sided). (C) To confirm these observations, cultured rat Schwann cells were treated with 1 mM dbcAMP for 24 h and cross-linked with PFA. Chromatin was purified and immunoprecipitated with anti-HDAC4 monoclonal antibody or a nonspecific mouse IgG (ChIP grade). As shown, c-Jun promoter was recovered with the anti-HDAC4 but not with the nonspecific mouse IgG. Data from five different experiments are given as mean ± SE and analyzed with the Mann-Whitney U test. (D) Mutating L175A or V179A in the HDAC4 3SA GFP construct blocks interaction with Mef2. We introduced mutations L174A or V179A in the HDAC4 3SA GFP construct and transfected the resultant constructs into the Schwannoma cell line RT4D6. Cell extracts were pulled down with a GFP-trap (Chromotek) and immunoblotted with anti-Mef2. Whereas Mef2 was efficiently pulled down by HDAC4 3SA GFP, it was not found in the HDAC4 3SA L175A GFP or HDAC4 3SA V179A GFP immunoprecipitates. Expression and immunoprecitation of the transfected constructs were checked by immunoblotting with anti-GFP antibodies. (E) HDAC4 3SA GFP L175A mutant was still able to down-regulate c-Jun, suggesting that interaction with Mef2 is dispensable. (F) The same result was obtained with the HDAC4 3SA GFP V179A. The graph shows the ratio of c-Jun fluorescence intensity in transfected relative to nontransfected cultured rat Schwann 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. Bars, 25 µm.

Article Snippet: HDAC4 shRNAi was obtained from Addgene pENTR/U6 HDAC4 shRNA (32220; Addgene).

Techniques: Infection, Purification, Immunoprecipitation, Expressing, Cell Culture, MANN-WHITNEY, Construct, Transfection, Western Blot, Mutagenesis, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Class IIa histone deacetylases link cAMP signaling to the myelin transcriptional program of Schwann cells

doi: 10.1083/jcb.201611150

Figure Lengend Snippet: 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.

Article Snippet: HDAC4 shRNAi was obtained from Addgene pENTR/U6 HDAC4 shRNA (32220; Addgene).

Techniques: Histone Deacetylase Assay, Activity Assay, Infection, Expressing, Western Blot, Construct, Transfection, Blocking Assay, Mutagenesis, Cell Culture, Immunofluorescence, Fluorescence, Incubation, Inhibition

Journal: The Journal of Cell Biology

Article Title: Class IIa histone deacetylases link cAMP signaling to the myelin transcriptional program of Schwann cells

doi: 10.1083/jcb.201611150

Figure Lengend Snippet: HDAC4 gain of function induces Schwann cell differentiation. (A) HDAC4 3SA blocks Schwann cell proliferation. Schwann cells were infected with Ad HDAC4 3SA Flag or Ad GFP and incubated in SATO medium. 24 h later, cells were fixed and examined by immunofluorescence with ant-Ki67 antibodies. Infected cells were identified with anti-GFP or anti-flag antibodies. Arrows indicate Ki67-postive infected cells. (B) Quantification of more than 600 cells from three different experiments is shown. Data are given as mean ± SE and analyzed with the unpaired t test (two-sided). *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) HDAC4 3SA induces profound morphological changes. Ad GFP–infected Schwann cells show the typical bipolar shape, which changes to an expanded flat epithelial-like morphology with enlarged nucleus in the Ad HDAC4 3SA Flag–infected cells. (D) To substantiate these changes, nuclear area was quantified in 668 cells per condition from three different experiments. A Tukey’s box plot of the results is shown. Data were analyzed with the unpaired t test (two-sided). *, P < 0.05; **, P < 0.01; ***, P < 0.001. (E) HDAC4 3SA induces Krox20 expression. Schwann cells were infected with Ad HDAC4 3SA or Ad GFP and incubated in SATO medium. 24 h later, cells were fixed and examined by immunofluorescence with anti-Krox20 antibody. As is shown, only HDAC4 3SA–infected cells expressed Krox20. Arrowheads indicate infected cells. (F) Quantification of mRNA levels expressed typically by nonmyelin-forming (red) and by myelin-forming Schwann cells (magenta) in Ad HDAC4 3SA Flag–infected cells normalized for controls (Ad GFP–infected Schwann cells). Sox10 , a gene that is expressed by both, is in black. As shown, HDAC4 3SA induces a clear shift toward the myelin gene expression program. As expected, no changes were observed for Sox10 . Data are given as mean ± SE and analyzed with the Kolmogorov-Smirnov test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bars, 25 µm.

Article Snippet: HDAC4 shRNAi was obtained from Addgene pENTR/U6 HDAC4 shRNA (32220; Addgene).

Techniques: Cell Differentiation, Infection, Incubation, Immunofluorescence, Expressing

Journal: The Journal of Cell Biology

Article Title: Class IIa histone deacetylases link cAMP signaling to the myelin transcriptional program of Schwann cells

doi: 10.1083/jcb.201611150

Figure Lengend Snippet: HDAC4 and HDAC5 redundantly contribute to Schwann cell differentiation and myelin development in vivo. (A) Removal of HDAC4 and HDAC5 in Schwann cells delays myelin gene expression. mRNA quantification for markers of nonmyelin- and myelin-forming cells in the PNS. P0 sciatic nerves were removed and total RNA extracted. RT-qPCR with mouse-specific primers for the indicated genes was performed and normalized to 18S rRNA. Graph shows the percentage of mRNA for each gene in the HDAC4/5 ScKO ( P0-Cre (+/−) ; HDAC4 (flx/flx) ; HDAC5 (−/−) ) normalized for the control ( P0-Cre (−/−) ; HDAC4 (flx/flx) ; HDAC5 (−/−) ) littermates. At least four mice from four different litters were used per genotype. Data were analyzed with the Kolmogorov-Smirnov test. A scatter plot is shown with the results obtained in each different experiment, which include also the mean ± SE. (B) Retardation in myelin gene expression is reflected in a delay in myelin development. Transmission EM of P0, P2, and P8 sciatic nerves. Tukey’s box plots show the quantification of MS density (MS/100 µm 2 ) and the myelination index (ratio of the number of myelin profiles [MS] to the total number of 1:1 segregated axons [MS+PS]). As is shown, whereas both parameters are decreased in the HDAC4/5 ScKO at P0 and P2, no differences could be found at P8. For P0, 80 images from four different mice per genotype were quantified; for P2, 78 images from six mice; for P8, 11 images from two different mice per genotype. Data were analyzed with the Mann-Whitney U test. Mean is plotted as a “+.” *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bar, 2 µm. (C) c-Jun is up-regulated faster after nerve injury in the HDAC4/5 ScKO. The sciatic nerves of three HDAC4/5 ScKO and three control mice were injured and analyzed 16 h later. Transverse cryosections were incubated with the indicated primary antibodies. Imaging was performed on a confocal microscope. Schwann cells were identified as Sox10-positive cells. (D) A Tukey’s box plot of the percentage of c-Jun–positive cells. Six images per animal were counted using Image J software. Data were analyzed with the Mann-Whitney U test. Mean is plotted as a “+.” *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bar, 25 µm.

Article Snippet: HDAC4 shRNAi was obtained from Addgene pENTR/U6 HDAC4 shRNA (32220; Addgene).

Techniques: Cell Differentiation, In Vivo, Expressing, Quantitative RT-PCR, Transmission Assay, MANN-WHITNEY, Incubation, Imaging, Microscopy, Software

Journal: The Journal of Cell Biology

Article Title: Class IIa histone deacetylases link cAMP signaling to the myelin transcriptional program of Schwann cells

doi: 10.1083/jcb.201611150

Figure Lengend Snippet: Proposed working model. Gpr126 activation raises intracellular cAMP-activating PKA, which phosphorylates HDAC4 in Ser265/266. This (together with another indirect mechanism) shuttles HDAC4 into the nucleus, where it binds to the promoter of c-Jun (through a not yet identified transcription factor [TF]) recruiting the NCoR1/HDAC3 complex to block gene expression.

Article Snippet: HDAC4 shRNAi was obtained from Addgene pENTR/U6 HDAC4 shRNA (32220; Addgene).

Techniques: Activation Assay, Blocking Assay, Expressing