Global changes in gene expression correlate with progressive histone deacetylation in the developing corpus callosum
OL differentiation in the rostral corpus callosum was characterized by the synthesis of late differentiation markers including MBP, MAG, and CGT, the progressive increase of the transcription factor Sox10 ( A), and the loss of precursor markers including tenascin, nestin, and Notch1 ( B). Because OL differentiation was associated with such generalized changes in progenitors, we hypothesized that global changes affecting chromatin components could modulate timing of myelination in the corpus callosum by modulating gene expression. Histone acetylation is one of the best-characterized mechanisms of regulating gene expression, and we therefore investigated whether it occurred in the developing corpus callosum. Several acetylated proteins ranging between 10 and 100 kD were detected in Western blot analysis of protein extracts from the rostral corpus callosum at postnatal day 1 (p1), p5, p11, and p24 ( C). Of these bands, only the 14- and the 30-kD protein displayed a distinctive temporal pattern of deacetylation. The 30-kD band lost its acetyl groups only during the third postnatal week ( C). Deacetylation of the 14-kD band, in contrast, started at p5 and continued throughout the period coincident with the onset of myelination. The acetylation level of this protein, therefore, correlated with high expression of progenitor markers and progressively decreased during OL differentiation. The occurrence of protein acetylation in OL lineage cells in the medial region of the body of the corpus callosum was confirmed by immunohistochemistry using the antibody recognizing the OL differentiation marker CC1 (, D–I). At p5, the number of double-positive CC1+/AcLys+ cells was 917 ± 29 per mm2 (n = 3), corresponding to 68.7% of the total number of CC1+ cells per mm2 (x = 1334 ± 57; n = 3). At p24, in contrast, the number of CC1+/AcLys+ double-positive cells was 384 ± 29, corresponding to 27% of the total number of CC1+ cells per mm2 (x = 1384 ± 29; n = 3) ( J). The 14-kD band corresponded to acetylated histone H3 (AcH3), as determined by Western blot analysis ( A), and the presence of AcH3 in the nuclei of OL progenitors in the developing corpus callosum was further characterized by immunofluorescence for the marker Sox10 (, B–D) and NG2 (, E–G). Remarkably, in the medial region of the body of the corpus callosum at rostral levels, the majority of the Sox10+ cells ( D) and of the NG2+ cells ( G) were also AcH3. To confirm the progressive decrease in AcH3 during OL development, we also processed p5 and p24 brain sections for CC1 and AcH3 staining (, H–M) and counted the number of double-positive cells. At p5, the total number of CC1+/AcH3+ cells was 716 ± 29 per mm2 (n = 3), whereas at p24 this number was reduced to 234 ± 29 per mm2 (n = 3), thus resulting in a 40% decrease in the number of double-positive cells ( N). Together, these data suggest that progressive deacetylation of histone H3 occurs in cells of the OL lineage, during a critical temporal window coincident with timing of OL differentiation and myelin gene expression in the developing corpus callosum.
Figure 1. Global changes of gene expression in the developing corpus callosum correlated with decreased protein acetylation. (A) RT-PCR of total RNA extracted from the anterior corpus callosum of p1, p5, p11, and p24 rats and amplified with primers for CGT, MAG, (more ...)
Figure 2. Histone H3 acetylation in OL progenitors of the medial corpus callosum progressively decreased during postnatal development. The 14-kD protein undergoing deacetylation during the first two postnatal days was identified by Western blot analysis using antibodies (more ...)
Decreased protein acetylation is due to the activity of class I HDACs
Because deacetylation is due to the removal of acetyl groups from lysine residues mediated by specific enzymes called HDACs, we tested the HDAC enzymatic activity in protein extracts from the developing corpus callosum, using a fluorimetric assay ( A). High levels of HDAC activity were detected during the first two weeks of postnatal development and progressively decreased during the third postnatal week ( A). Because the acetylation level of proteins at any time point reflects the equilibrium between the addition and the removal of acetyl groups (Lehrmann et al., 2002
), these data suggest that at p1, despite the high levels of HDAC activity, the equilibrium is in favor of acetylation, whereas starting from p5 it is in favor of deacetylation. To determine the class of HDAC responsible for the effect, the experiments were repeated in the presence of the class I and II HDAC inhibitor trichostatin A (TSA), and of the class III HDAC inhibitor sirtinol. TSA decreased the total HDAC activity in the tissue extracts of developing corpus callosum of >90% ( A), whereas sirtinol did not block the HDAC enzymatic activity and it actually induced further activation, possibly due to removal of inhibitory acetyl groups regulating class I and II HDACs.
Figure 3. Total HDAC activity in extracts of the developing rostral corpus callosum. (A) Total HDAC activity was measured in protein extracts from anterior corpus callosum at p1, p5, p8, p11, and p24 using an acetylated fluorimetric substrate (black bars). The (more ...)
Given the existence of a large number of isoforms for class I and class II HDACs, it was important to determine their expression and functional relevance in the developing corpus callosum. Western blot analysis of protein extracts from the rostral corpus callosum, dissected at distinct developmental time points, revealed the presence of all the HDACs isoforms ( B). No major change in protein expression was observed for HDAC-1 to HDAC-8, whereas HDAC-7 levels decreased around p8 ( B). Interestingly, HDAC-1 and HDAC-8 both showed the presence of additional higher molecular weight bands at p24, possibly resulting from post-translational modifications of these molecules. The temporal and cellular patterns of HDAC expression in the developing corpus callosum were further assessed using double immunohistochemistry with CC1 and antibodies specific for each HDAC isoform (). At p5, the class I isoforms HDAC-1, -2, -3, and to a lesser degree HDAC-8 were expressed in the nucleus of CC1+ cells (, A–C, H). The class II isoforms HDAC-4, -5, -6, and -7, in contrast, were weakly expressed and localized in the cytoplasm (, D–G). HDAC-4 was completely absent from CC1+ cells and its filamentous staining pattern was suggestive of axonal staining or myelinated fibers in the developing corpus callosum ( D). By p24, when myelination had ensued, the immunoreactivity for several HDAC isoforms, including HDAC-2, -3, -5, and -8, decreased in CC1+ cells (). In contrast, HDAC-1 was still expressed in the nucleus of the CC1+ cells as well as in myelinated fibers ( I), HDAC-4 displayed a strong filamentous staining similar to p5 ( L), and HDAC-6 and -7 were still in the cytosol of CC1+ cells ().
Figure 4. Cellular and temporal profile of expression of class I and II HDACs in maturing OLs. Confocal images of the medial part of the body of the corpus callosum at rostral levels, identified in coronal brain sections from p5 (A–H) and p24 (I–P) (more ...)
Due to the nuclear localization of class I HDACs in the p5 corpus callosum, we confirmed the cellular specificity of these isoforms by immunolabeling with antibodies specific for oligodendrocytic (i.e., CC1), neuronal (i.e., NeuN), and astrocytic (i.e., GFAP) markers (). The majority of the HDAC-1–positive cells were identified as OLs because they were immunoreactive predominantly for CC-1 ( A), but not for NeuN ( B) or GFAP ( C). In contrast, a large part of HDAC-2–positive cells were also NeuN+ ( E) and GFAP+ ( F). A similar pattern of expression was observed for HDAC-3 (, G–I) and HDAC-8 (, J–L), which were both expressed in all three cell types.
Figure 5. Cell specificity of class I HDAC expression during the first postnatal week of development. Immunohistochemistry of coronal brain sections from p5 rat pups stained with antibodies against HDAC-1, -2, -3, and -8 (A–L, green), CC1 (A, D, G, and (more ...)
Together, these data show that during the first week of postnatal development, only class I HDACs are present in the nuclei of the differentiating OL cells, thus suggesting that histone deacetylation in these cells is likely due to the activity of class I HDAC isoforms.
Inhibiton of HDAC activity prevents myelin gene expression in the developing corpus callosum only during a critical temporal window
To address the functional relevance of histone deacetylation on OL differentiation and myelination, we investigated the effect of in vivo administration of the pharmacological inhibitor of class I HDACs, valproic acid (VPA). The short-term experimental paradigm included three groups of neonatal pups receiving a 2-d regimen of VPA (300 mg/kg body weight) starting at distinct developmental time points ( A). The first group of neonatal rats (n = 12) was injected with PBS (n = 6) or VPA (n = 6) at p6 and p7 and then harvested at p8 (injection 1), at the beginning of myelination. The second group (n = 12) was injected at p9 and 10 and harvested at p11 (injection 2), whereas the third group (n = 12) was injected at p19 and 20 and harvested at p21 (injection 3), after myelination had ensued in the rostral corpus callosum.
Figure 6. Pharmacological blockers of HDAC activity induced hypomyelination when administered during a critical temporal window of development. (A) Schematic representation of three distinct PBS/VPA injection paradigms to the neonatal rats. Each group was composed (more ...)
The hypothesis that HDAC activity was required for changes in gene expression associated with OL differentiation predicted that in vivo treatment with HDAC inhibitors prevented OL differentiation and possibly caused hypomyelination of axonal fibers in the developing corpus callosum. In agreement with this hypothesis, the expression of OL differentiation genes was significantly down-regulated in animals that received VPA injection during the first two postnatal weeks ( B). The effect of VPA on myelin gene expression was dramatic if started during the first postnatal week, but it was ineffective if started during the third postnatal week ( B). Decreased myelin gene expression in the VPA-treated pups was associated with a decreased number of mature OL and myelinated fibers, as assessed by MAG and MBP immunoreactivity. In PBS-injected pups at p8, myelination in the corpus callosum followed the latero-medial gradient, as assessed by the large number of MAG+ and MBP+ cells and fiber in the lateral () but not in the medial () region. Several myelinated fibers could also be detected in the anterior commissure (). In VPA-injected pups, in contrast, the number of MAG+ and MBP+ cells and fibers was decreased both in the lateral () and medial () corpus callosum as well as in the anterior commissure ().
Decreased MAG and MBP immunoreactivity in the corpus callosum of VPA-treated pups was accompanied by a reduction in the number of CC1+ cells (, A–E). This decrease was not due to a toxic effect of the pharmacological inhibitor because the total number of DAPI+ cells/mm2 was quite stable (x =1488 ± 49 in VPA injected and x =1563 ± 52 in PBS control) and because there was no difference in the number of TUNEL+ (unpublished data) apoptotic cells in the two groups. The reduced number of CC1+ cells was likely due to delayed differentiation, as indicated by the increased percentage of cells expressing the bipotential progenitor marker NG2+ in VPA-treated animals compared with controls (, F–H). The increased progenitor number was not due to an effect of VPA on proliferation, because the number of proliferating NG2+ cells, identified by in vivo labeling with the thymidine analogue BrdU, was very similar in treated and control rats (, I–M). The inhibitory effect of VPA on differentiation was also supported by the detection of PSA-NCAM+ precursors in cells in the subcortical white matter of treated animals (, N–Q). Together, these data suggest that short-term inhibition of HDAC activity does not impair the ability of OL progenitors to exit the cell cycle, but arrests their differentiation at a stage characterized by the expression of early progenitor traits and lack of differentiation markers.
Figure 7. Hypomyelination in VPA-treated rats resulted from delayed differentiation of OL progenitors. Coronal brain sections from PBS- (A and C) and VPA (B and D) -injected rats (inj1) were processed for immunohistochemistry using antibodies for CC1 (A and D, (more ...)
Because a 3-d VPA treatment resulted in hypomyelination, we asked whether prolonged treatment would inhibit myelination even more. To test this hypothesis, pups were subject to a 7-d injection protocol ( A) starting at p3 and followed by the assessment of myelination at p10 (, B–M). In p10 control animals myelination was almost complete in the lateral region of the anterior corpus callosum () and in the anterior commissure (), as indicated by the presence of several CC1+ and MAG+ cells and by the intense MAG and MBP immunoreactivity of the myelinated fibers. At p10 OL differentiation and myelination were detected also in the medial corpus callosum () of PBS-injected pups. The effect of long-term VPA treatment on myelination was striking. Very few, sparse CC1+ cells and MAG+ and MBP+ fibers were detected in the lateral corpus callosum (), but not in the medial corpus callosum () and in the anterior commissure (), thus suggesting that longer suppression of HDAC activity led to more severe hypomyelination.
Figure 8. Long-term treatment with HDAC inhibitors dramatically decreases myelination in the corpus callosum and anterior commissure of the developing rat. (A) Schematic representation of the long-term PBS/VPA treatment to neonatal rats. Each group was composed (more ...)
To determine whether the delayed timing of OL differentiation caused by VPA treatment was reversible, we assessed OL differentiation after a recovery period. As expected from a reversible inhibitor, 2 d after the interruption of VPA treatment the OL lineage cells were able to resume the age-appropriate developmental pattern of gene expression characterized by decreased progenitor traits (i.e., Notch1, nestin, and tenascin), increased levels of the transcriptional activator Sox10, and of the late differentiation markers MAG and MBP ( A). The majority of the cells in the corpus callosum of VPA-treated animals after recovery were immunoreactive for CC1 ( G) and MAG ( I). However, few myelinated fibers could be detected in the lateral corpus callosum of VPA-injected animals () compared with the more extensive myelination observed in PBS-injected controls ().
Figure 9. The inhibitory effect of VPA on OL differentiation was reversible. (A) To address whether OL differentiation was delayed or irreversibly impaired by VPA treatment, we repeated protocol 1 with a recovery period of 3 d after the last injection. At p11 RNA (more ...)
Together, these data support the hypothesis that epigenetic regulation of gene expression is critical for timely differentiation of OLs in the developing corpus callosum.
After myelination onset, cells in the developing corpus callosum acquire more permanent changes in chromatin components and this renders them refractory to the effect of HDAC inhibitors
We have previously discussed that the effectiveness of VPA administration on modulating myelin gene expression in vivo was limited to a specific temporal window coincident with the onset of myelination. To understand the molecular mechanisms defining this developmental window regulated by HDAC activity, we assessed the presence of AcH3 after each protocol of VPA injection ( A). Administration of VPA during the first two postnatal weeks increased the levels of AcH3 without significantly affecting the acetylation state of other high molecular weight proteins ( A). In contrast, administration of VPA during the third postnatal week did not affect the levels of AcH3 ( A). Because protein acetylation is the result of the equilibrium between histone acetyltransferases (i.e., HATs, such as p300 and CBP) and HDACs (Lehrmann et al., 2002
; Rouaux et al., 2003
), we hypothesized that the lack of VPA in the third postnatal week was consequent to low levels of HATs. This hypothesis was confirmed by the detection of decreased protein levels of CBP and p300 during the third postnatal week of development ( B). The results obtained at p24 suggested that perhaps reversible acetylation was a mechanism of regulation of gene expression that was best suited to maintain a certain “plasticity” of gene expression during early developmental stages. At later developmental stages, however, it was likely that committed cells would adopt more stable mechanisms of regulation of gene expression that would guarantee the maintenance of the differentiated phenotype. Because histone deacetylation is often followed by the more stable methylation of lysine 9 in histone H3 (Honda et al., 1975
; Eberharter and Becker, 2002
; Boulias and Talianidis, 2004
), we asked whether in the corpus callosum the global changes in gene expression initiated by histone deacetylation were also maintained by histone methylation and chromatin compaction. To test this possibility, we stained p5 and p24 brain sections with antibodies specific for methylated histone H3 and for HP1 α, a protein that specifically binds to methylated lysine 9 on histone H3 (MeK9H3) and identifies the presence of compact chromatin (Bannister et al., 2001
; Lachner et al., 2001
). In agreement with our hypothesis, at p5 before the peak of myelination, OL progenitors did not show MeK9H3+ or HP1α immunoreactivity (, C–F). In contrast, by p24 the majority of the cells in the corpus callosum were MeK9H3+/HP-1 α+ (, G–J), thus confirming the acquisition of compact chromatin structure.
Figure 10. Global histone deacetylation occurred only during the first two weeks of postnatal development and was followed by histone methylation and chromatin compaction. (A) To confirm the inhibitory effect of VPA, Western blot analysis was performed on total (more ...)
Together, these data indicate that HDAC activity is critical during the first two weeks of postnatal development of the corpus callosum and is associated with the reversible modulation of gene expression at the onset of myelination. During the third postnatal week, however, after myelination has ensued, this reversible form of regulation of gene expression is replaced by more stable changes resulting in chromatin compaction.