First published online November 18, 2003
doi: 10.1242/10.1242/jcs.00804
Chromatin remodeling and neuronal response: multiple signaling pathways induce specific histone H3 modifications and early gene expression in hippocampal neurons
Claudia Crosio1,
Estelle Heitz1,
C. David Allis2,
Emiliana Borrelli3 and
Paolo Sassone-Corsi1,*
1 Department of Gene Expression, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS – INSERM – Université Louis Pasteur, 1 rue Laurent Fries, 67404 Illkirch, Strasbourg, France
2 University of Virginia H. S. C., Department of Biochemistry and Molecular Genetics, Box 800733, Charlottesville, VA 22908-0733, USA
3 Department of Neuroscience, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS – INSERM – Université Louis Pasteur, 1 rue Laurent Fries, 67404 Illkirch, Strasbourg, France
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Fig. 1. Phosphorylation of histone H3 is induced in the hippocampus by stimulation of DA, mACh and GLU receptors. (A) Schematic representation of the histone H3 N-terminal tail. The key residues within the H3 tail where covalent regulatory modifications occurs are indicated. These include acetylation at lysines and phosphorylation at conserved serine residues. (B) Immunohistochemistry on mouse hippocampal cryosections using P.H3 antibody. In order to stimulate DA, mACh or GLU receptors, mice were injected with SKF82958 (5 mg kg–1), pilocarpine (300 mg kg–1) or kainic acid (35 mg kg–1), respectively, and sacrificed after15 minutes, 1 hour or 3 hour. Control animals, indicated with the symbol (–), were injected with saline solution. (C) Phosphorylation of histone H3 in the DG and in the CA3 after stimulation of DA, mACh and mGLU receptors. (D) Quantification of the data in (A,B). A total of six animals were analysed from three independent experiments. Cell counts were performed under the light microscope. Statistical comparisons were performed with one-way ANOVA on the total number of positive cells (independently from the intensity of the staining that is indicated on the graph in gray and black) followed by Bonferroni's post hoc test. ***, P<0.001; **, P<0.01. Scale bars, 300 µM (B); 70 µM (C).
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Fig. 2. Dose-dependent histone H3 phosphorylation. Mice were injected with 5 mg kg–1, 1 mg kg–1 or 0.2 mg kg–1 SKF82958, 300 mg kg–1, 100 mg kg–1 or 30 mg kg–1 pilocarpine, or 35 mg kg–1, 20 mg kg–1 or 10 mg kg–1 kainic acid, and sacrificed after 1 hour. Control animals, indicated with the symbol (–) were injected with saline solution. A portion of the DG is shown. Scale bar, 70 µM.
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Fig. 3. Histone H3 phosphorylation and ERK phosphorylation occur in the same hippocampal neurons. (A) Immunohistochemistry using P.ERK antibody on mouse hippocampal cryosections obtained from mice sacrificed 1 hour after saline, SKF82958, pilocarpine or kainic acid injection. (B) IHC was performed using anti-P.H3 (NBT/BCIP staining, top), anti-P.ERK (DAB staining, middle) and a mix of these two antibodies (bottom). The DG regions are shown. (C) Quantification of the data in (B). Cell counts were performed under the light microscope. Statistical comparisons were performed with one-way ANOVA followed by Bonferroni's post hoc test. ***, P<0.001. (D) High magnifications of CA1, CA2 and CA3 regions revealed with both anti-P.H3 and anti-P.ERK antibodies. Scale bars, 300 µM (A); 20 µM (B,D).
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Fig. 4. Correlation of histone H3 phosphorylation and IEG induction in the hippocampus. Cryosections obtained from mice sacrificed 1 hour after saline, SKF82958, pilocarpine or kainic-acid injection were used for in situ hybridization experiments using c-fos (A), MKP-1 (C, top) and MKP-3 (C, bottom), S35-labeled riboprobes or in situ-immuno hybridization (B), using c-fos DIG-labeled riboprobe (FITC staining) and P.H3 antibody (Cy3 staining). Nuclear localization was assessed by DAPI staining. Scale bars, 300 µM (A,C); 20 µM (B).
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Fig. 5. Phospho-acetylation of histone H3 after stimulation of DA, mACh and GLU receptors. IHC was performed using (from top to bottom) anti-P.H3 (P), anti-Ac14.H3 (Ac14), anti-Ac9.H3 (Ac9), anti-Ac9/14.H3 (Ac9/14), anti-P-Ac9.H3 (P-Ac9) and anti-P-Ac14.H3 (P-Ac14) antibodies on cryosections obtained from mice treated as in Fig. 1B. (A) High magnifications of the DG revealed with all these antibodies. (B) High magnifications of the CA1, CA2 and CA3 regions revealed with anti-P-Ac14.H3 (P-Ac14) antibody. Scale bar, 15 µM.
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Fig. 6. (A) Western blot analysis on total protein extracts obtained from mice sacrificed 15 minutes, 1 hour or 3 hour after saline, SKF82958, pilocarpine or kainic-acid injection. 15 µg of proteins were separated on SDS/PAGE gels and revealed using anti-P.ERK, anti-panERK (ERK), anti-P.H3 (P), anti-Ac14.H3 (Ac14) and anti-P-Ac14.H3 (P-Ac14) antibodies. (B) Phosphorylation-acetylation of histone H3 on c-fos and MKP-1 chromatin. ChIP assays were performed on chromatin obtained from hippocampi of mice injected with 35 mg kg–1 kainic acid (GLU) or saline treated (–), using a preimmune serum (no Ab), anti-P.H3 (P), anti-Ac14.H3 (Ac14) or anti-P-Ac14.H3 (P-Ac14). The DNA recovered from the antibody-bound fraction and the DNA from the input chromatin were analysed by PCR with oligonucleotides specific for the indicated gene.
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© The Company of Biologists Ltd 2003
