Exp Neurobiol 2011; 20(2): 81-84
Published online June 30, 2011
© The Korean Society for Brain and Neural Sciences
Bongki Cho, Hyun Jung Kim, Hyun Kim and Woong Sun*
Department of Anatomy, BK21 Program, Korea University College of Medicine, Seoul 136-705, Korea
Correspondence to: *To whom correspondence should be addressed.
TEL: 82-2-920-6404, FAX: 82-2-929-5696
Epigenetic modification such as DNA methylation and histone acetylation plays essential roles in many aspects of cellular function and development of animals. There is an increasing amounts of evidence for dynamic changes in the histone acetylation of specific gene segments, but little attempt was made to examine global pattern changes in the histone acetylation in developing nervous system. In this study, we found that acetylated histone H3 and H4 immunoreactivities were relatively weak in neuroepithelial cells in the ventricular zone of developing rat cerebral cortex or chick spinal cord, compared to the immature young neurons in the cortical plate of a rat embryo or lateral motor column in chick spinal cord. On the other hand, adult neural stem cells in the dentate gyrus (DG) of rat hippocampal formation did not exhibit such diminished histone acetylation, compared to neuroblasts and mature DG neurons. These results suggest that the level of histone acetylation is highly dynamic and tightly linked to the neuronal types and the differentiation stages.
Keywords: histone acetylation, nervous system, development, neuroepithelium
Many biological processes are not only determined by DNA code itself, but also regulated by activity-related chromosomal remodeling (Clapier and Cairns, 2009). Since acetylation of histone modifies the basicity of histone protein which affects histone-DNA association, histone acetylation is essential event for the epigenetic control of gene expression (Allfrey et al., 1964; Hebbes et al., 1988; Riccio, 2010). The amount of histone acetylation is controlled by histone acetyltranferases (HATs) (Brownell et al., 1996) and histone deacetylases (HDACs) (Ruiz-Carrillo et al., 1975; Annunziato and Seale, 1983). During the development, changes in the histone acetylation greatly affect the differentiation of stem cells (Lee et al., 2004), neuronal maturation (Hsieh et al., 2004), and programmed cell death (Pelzel et al., 2010) via modulation of specific gene expressions which are responsible for these biological processes. It is believed that gene transcription machineries recruit histone modification enzymes into their transcriptome complex, and in this way specific gene transcription-mediated biological processes would be promoted. For instance, histone acetylation level declines during the differentiation of embryonic stem cells, which affects the gene transcription of stemness genes, such as Oct4 and Brachyury (Lee et al., 2004). Therefore, treatments of HDAC inhibitors, such as trichostatin A (TSA) and valproic acid (VPA), or deficiency of HDAC greatly affect many aspects of brain function, including differentiation of neurons and oligodendrocytes (Marin-Husstege et al., 2002; Hsieh et al., 2004), neuronal apoptosis (Pelzel et al., 2010), motor innervation (Mejat et al., 2005), and memory consolidation (Korzus et al., 2004).
While there have been extensive investigations of the histone acetylation patterns of specific gene promoter regions, little attempt was made to explore global changes in the histone acetylation during the normal embryonic development. In this study, we found that histone H3 and H4 acetylation levels were stronger in the early differentiating neurons in the rat cortex and chick spinal cord, comparing to the proliferating neuroepithelial cells in the ventricular zone. These results suggest that transient enhancement of histone acetylation may be an important event for neuronal differentiation.
Time-pregnant C57Bl/6 mice were obtained from Orient Co. (Korea), and fertilized eggs were obtained from Pulmuone (Korea). Eggs were incubated in humidified incubator at 38℃ for 3 days. Stage of chick embryo was identified according to the Hamilton-Hamburger's criteria (Hamburger and Hamilton, 1992). All experiments were carried out in accordance with the ethical guidelines of Korea University, and with the approval of the Animal Care and Use Committee of Korea University.
Immunohistochemical analyses were performed as previously reported (Sun et al., 2003). Briefly, brain or trunk tissues were isolated from embryos and fixed with 4% paraformaldehyde overnight. Tissues were then cryoprotected in 30% sucrose, sectioned (20 µm) and attached on a gelatin-coated slide glass. Acetylated Histone H3 (Upstate, #06-599, 1 : 500) and H4 (Upstate, #06-866, 1 : 1,000) antibodies were applied overnight. After several washes with PBS, secondary antibodies were applied for 30 minutes. Subsequently, sections were washed, counter-stained with Hechest33342, mounted and observed with a fluorescence microscope (Zeiss LSM510, Goettingen, Germany).
Embryonic rat cerebral cortex (E17) was immunolabeled with anti-acetylated histone H3 or H4 antibodies (Fig. 1). Interestingly, both acetylated histone H3 and H4 immunoreactivities were strong in the cortical plate (CP) where post-mitotic neurons localize, compared to the ventricular zone (VZ) and intermediate zone (IZ) enriched for mitotic/migrating neuroblasts. The concentration of histone proteins was proportional to the total DNA density, which may be affected by the size of nuclei. Therefore, we counterstained the nuclei DNA by Hoechst33342, and the relative densities of acetylated histones and DNA were measured. This analysis further confirmed the preferential histone acetylation in the CP.
To determine whether similar difference in histone acetylation levels in developing nervous system in other species appear, we also examined the developing chick (E7) spinal cord (Fig. 2). Similar to the rat cerebral cortex, the level of histone acetylations were strong in the lateral motor column (LMC) where post-mitotic motor neurons localize, whereas histone acetylation were relatively weak in the periventricular regions enriched for proliferating neuroepithelial cells. Another important aspect of histone acetylation pattern is the heterogeneity within the same population. For instance, large magnification images of LMC clearly show that the intensities of acetylated histones were different among motor neurons.
These results suggest that histone acetylation is closely associated with the mitosis and differentiation of neural stem/progenitor cells. Neural stem cells remain in the specific areas of adult brain, and they continue to produce neurons. Therefore, we next examined whether adult neural stem cells and their progenitors also showed different levels of histone H3 acetylation (Fig. 3). To identify different cell types, three specific markers were used; glial fibrary acidic protein (GFAP), a marker for astrocytes and neural stem cells, doublecortin (DCX), a marker for neuroblasts, and neuronal nuclei (NeuN), a marker for differentiated neurons. Although a considerable heterogeneity of histone H3 acetylation levels was observed among the same cell populations, there was no significant difference among cell types.
Increased acetylation of histones may promote the overall gene transcription efficiency. In this study, we found that developing neurons in the mouse cerebral cortex and chick spinal cord exhibit considerably high level of histone H3 and H4 acetylation, compared to the proliferating stem/progenitor populations. The level of histone acetylation was reported to increase during the postnatal development of rat brain (Serra et al., 1986), consistent with our current observation. Although there was little attempt to compare histone acetylation or RNA synthesis rates between stem cells and progenitor neurons, neurons appear to have higher histone acetylation level than glial cells (Hsieh et al., 2004; Shen et al., 2005; Humphrey et al., 2008). Considering that the size of neurons greatly expands during the differentiation, it appears that neurons have high demands for synthesis of many different RNA species during neuronal differentiation. However, it is noted that histone acetylation does not directly indicate the rate of RNA synthesis., Histone acetylation do not control the RNA polymerase activity, but it allows RNA polymerases to bind to the DNA. In this respect, increased histone acetylation may be linked to other biological processes.
In contrast to our
Another important aspects about global histone acetylation levels identified in this study, is the heterogeneity among the same cell population. For instance, we found that only a subset of LMC chick motoneurons exhibited very strong level of histone acetylation. We failed to correlate the levels with known MN subtypes (data not shown), suggesting that these are differences among the same type of cells. This heterogeneity may be caused by highly dynamic nature of histone acetylation. In fact, histone acetylation is mainly regulated by two enzyme systems, histone acetyltransferases (HAT) and histone deacetylases (HDAC) (Riccio, 2010). Since these enzyme activities are dynamically regulated by multiple mechanisms, it is believed that the level of histone acetylation in cells are flexible and modulated by multiple factors, which may produce the diversity of histone acetylation even in the same population of cells.
In contrast to the developmental nervous system, we failed to detect any significant difference in the global histone acetylation levels among neural stem cells, neuroblasts, and mature neurons in the adult dentate gyrus. Compared to the embryonic development, neural stem cells and their progenitor neurons are similar in size and space, thus they may experience less dramatic changes in cell-autonomous gene transcription and extracellular environment. Additional studies would clarify how these differences occur, and the mechanism/significance underlying it.