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Astrocyte-specific genes are generally demethylated in neural precursor cells prior to astrocytic differentiation.

Hatada I, Namihira M, Morita S, Kimura M, Horii T, Nakashima K - PLoS ONE (2008)

Bottom Line: This differentiation-potential switch could be explained by epigenetic changes, since the promoters of astrocyte-specific marker genes, glial fibrillary acidic protein (Gfap) and S100beta, have been shown to become demethylated in late-stage NPCs prior to the onset of astrocyte differentiation; however, whether demethylation occurs generally in other astrocyctic genes remains unknown.Although these genes are already demethylated in late-stage NPCs, they are not expressed until cells differentiate into astrocytes.Thus, late-stage NPCs have epigenetic potential which can be realized in their expression after astrocyte differentiation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan. ihatada@showa.gunma-u.ac.jp

ABSTRACT
Epigenetic changes are thought to lead to alterations in the property of cells, such as differentiation potential. Neural precursor cells (NPCs) differentiate only into neurons in the midgestational brain, yet they become able to generate astrocytes in the late stage of development. This differentiation-potential switch could be explained by epigenetic changes, since the promoters of astrocyte-specific marker genes, glial fibrillary acidic protein (Gfap) and S100beta, have been shown to become demethylated in late-stage NPCs prior to the onset of astrocyte differentiation; however, whether demethylation occurs generally in other astrocyctic genes remains unknown. Here we analyzed DNA methylation changes in mouse NPCs between the mid-(E11.5) and late (E14.5) stage of development by a genome-wide DNA methylation profiling method using microarrays and found that many astrocytic genes are demethylated in late-stage NPCs, enabling the cell to become competent to express these genes. Although these genes are already demethylated in late-stage NPCs, they are not expressed until cells differentiate into astrocytes. Thus, late-stage NPCs have epigenetic potential which can be realized in their expression after astrocyte differentiation.

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DNA methylation of Gfap and Stat3 gene.DNA methylation was analyzed by bisulfite sequencing. Closed circles indicate methylated CpG sites and open circles indicate unmethylated CpG sites. STAT3 binding sites are indicated by arrows.
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pone-0003189-g001: DNA methylation of Gfap and Stat3 gene.DNA methylation was analyzed by bisulfite sequencing. Closed circles indicate methylated CpG sites and open circles indicate unmethylated CpG sites. STAT3 binding sites are indicated by arrows.

Mentions: Methylation changes in the differentiation potential switch of NPCs were analyzed by comparing mid-(E11.5) and late (E14.5)-stage NPCs using the MIAMI method [16]. We also compared the methylation status with that of postnatal day 1 (P1) astrocytes. Before analysis, the key genes involved in astrocyte differentiation, such as Gfap and Stat3, were analyzed for DNA methylation in the sample we used (Fig. 1). As described previously [15], the STAT3-binding site of Gfap gene was demethylated in E14.5 NPCs prior to astrocyte differentiation. On the other hand, the promoter of Stat3 gene was unmethylated throughout differentiation. The microarray used consisted of probes chosen from the Agilent promoter array using an eArray system (http://earray.chem.agilent.com/earray/). The probes are located on Hpa II fragments of less than 1 kilobasepair (kb) and cover 14,543 genes. Probes which showed methylation changes at least in E14.5 NPCs or astrocytes compared to E11.5 NPCs are presented in Fig. 2A and Table S1 (Name of the probes and genes were indicated in Table S1). As shown in Fig. 2B, E14.5 NPCs are hypomethylated in 85 probes (80 genes) and hypermethylated in 15 probes (15 genes). On the other hand, astrocytes are hypomethylated in 275 probes (256 genes) and hypermethylated in 170 probes (152 genes). The reliability of the analysis was confirmed by bisulfite sequencing analysis of eight genes (Fig. 3). The methylation ratio analyzed by MIAMI (Fig. 3A) had good correlation with the methylation of two Hpa II sites adjacent to the probes (Fig. 3B). Interestingly 80% of the probes hypomethylated in E14.5 NPCs are also hypomethylated in astrocytes. (Fig. 2C). If we extend the hypomethylation change criteria to the threshold level (D10<0.5, usually we use D10<0.2 for hypomethylation), 89% of the probes hypomethylated in E14.5 NPCs are also hypomethylated in astrocytes. In other words, 48% of the hypomethylated probes in astrocytes are also hypomethylated in E14.5 NPCs. These include a probe for an astrocyte-specific marker gene, Gfap, which was previously shown to be hypomethylated both in E14.5 NPCs and astrocytes [15]. However, another hypomethylated astrocyte marker, S100β [18], was not detected by the MIAMI method because the DNA sequence with methylation change does not contain any Hpa II sites, leading to underestimation of the total number of genes actually demethylated in E14.5 NPCs. Nevertheless, including the genes undetectable by MIAMI, many hypomethylated sequences in astrocytes are already demethylated in E14.5 NPCs, which are competent to differentiate into astrocytes. These include important genes for astrocyte-specific function or phenotype in addition to an astrocyte-specific marker gene, Gfap. For example, Aldolase C encodes a member of the class I fructose-biphosphate aldolase gene family specific to astrocytes (Aldoc, 19). The demethylated region of Aldoc is located on exon 1. We further confirmed this result by bisulfite sequencing and found that Aldoc was methylated in E11.5 NPCs and demethylated in E14.5 NPCs and astrocytes (Fig. 3B). Another example, Kcnj10 (Kir4.1), is widely expressed in astrocytes throughout the brain [20]. The product of this gene is absent in immature proliferating cells, and progressive expression of the genes correlates with astrocyte differentiation, which is characterized by the establishment of a negative membrane potential and exit from the cell cycle. Kcnj10 (Kir4.1) encodes a member of the inward rectifier-type potassium channel family, characterized by having a greater tendency to allow potassium to flow into, rather than out of, a cell, resulting in negative membrane potential. The encoded protein is responsible for potassium buffering action, which is a major function of astrocytes and is also responsible for promoting differentiation and inhibiting cell growth [20], [21]. Mutations in this gene have been associated with seizure susceptibility of common idiopathic generalized epilepsy syndromes [22], [23]. Sparcl1 is known as an astrocyte marker colocalized with Gfap [24] and is also known as a candidate gene for multiple sclerosis [25]. Cbs and BC055107 are also known as astrocyte markers [26], [27]. Thus, we found that demethylation occurred not only in Gfap but also in the other genes involved in astrogenesis prior to astrocyte differentiation.


Astrocyte-specific genes are generally demethylated in neural precursor cells prior to astrocytic differentiation.

Hatada I, Namihira M, Morita S, Kimura M, Horii T, Nakashima K - PLoS ONE (2008)

DNA methylation of Gfap and Stat3 gene.DNA methylation was analyzed by bisulfite sequencing. Closed circles indicate methylated CpG sites and open circles indicate unmethylated CpG sites. STAT3 binding sites are indicated by arrows.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2527128&req=5

pone-0003189-g001: DNA methylation of Gfap and Stat3 gene.DNA methylation was analyzed by bisulfite sequencing. Closed circles indicate methylated CpG sites and open circles indicate unmethylated CpG sites. STAT3 binding sites are indicated by arrows.
Mentions: Methylation changes in the differentiation potential switch of NPCs were analyzed by comparing mid-(E11.5) and late (E14.5)-stage NPCs using the MIAMI method [16]. We also compared the methylation status with that of postnatal day 1 (P1) astrocytes. Before analysis, the key genes involved in astrocyte differentiation, such as Gfap and Stat3, were analyzed for DNA methylation in the sample we used (Fig. 1). As described previously [15], the STAT3-binding site of Gfap gene was demethylated in E14.5 NPCs prior to astrocyte differentiation. On the other hand, the promoter of Stat3 gene was unmethylated throughout differentiation. The microarray used consisted of probes chosen from the Agilent promoter array using an eArray system (http://earray.chem.agilent.com/earray/). The probes are located on Hpa II fragments of less than 1 kilobasepair (kb) and cover 14,543 genes. Probes which showed methylation changes at least in E14.5 NPCs or astrocytes compared to E11.5 NPCs are presented in Fig. 2A and Table S1 (Name of the probes and genes were indicated in Table S1). As shown in Fig. 2B, E14.5 NPCs are hypomethylated in 85 probes (80 genes) and hypermethylated in 15 probes (15 genes). On the other hand, astrocytes are hypomethylated in 275 probes (256 genes) and hypermethylated in 170 probes (152 genes). The reliability of the analysis was confirmed by bisulfite sequencing analysis of eight genes (Fig. 3). The methylation ratio analyzed by MIAMI (Fig. 3A) had good correlation with the methylation of two Hpa II sites adjacent to the probes (Fig. 3B). Interestingly 80% of the probes hypomethylated in E14.5 NPCs are also hypomethylated in astrocytes. (Fig. 2C). If we extend the hypomethylation change criteria to the threshold level (D10<0.5, usually we use D10<0.2 for hypomethylation), 89% of the probes hypomethylated in E14.5 NPCs are also hypomethylated in astrocytes. In other words, 48% of the hypomethylated probes in astrocytes are also hypomethylated in E14.5 NPCs. These include a probe for an astrocyte-specific marker gene, Gfap, which was previously shown to be hypomethylated both in E14.5 NPCs and astrocytes [15]. However, another hypomethylated astrocyte marker, S100β [18], was not detected by the MIAMI method because the DNA sequence with methylation change does not contain any Hpa II sites, leading to underestimation of the total number of genes actually demethylated in E14.5 NPCs. Nevertheless, including the genes undetectable by MIAMI, many hypomethylated sequences in astrocytes are already demethylated in E14.5 NPCs, which are competent to differentiate into astrocytes. These include important genes for astrocyte-specific function or phenotype in addition to an astrocyte-specific marker gene, Gfap. For example, Aldolase C encodes a member of the class I fructose-biphosphate aldolase gene family specific to astrocytes (Aldoc, 19). The demethylated region of Aldoc is located on exon 1. We further confirmed this result by bisulfite sequencing and found that Aldoc was methylated in E11.5 NPCs and demethylated in E14.5 NPCs and astrocytes (Fig. 3B). Another example, Kcnj10 (Kir4.1), is widely expressed in astrocytes throughout the brain [20]. The product of this gene is absent in immature proliferating cells, and progressive expression of the genes correlates with astrocyte differentiation, which is characterized by the establishment of a negative membrane potential and exit from the cell cycle. Kcnj10 (Kir4.1) encodes a member of the inward rectifier-type potassium channel family, characterized by having a greater tendency to allow potassium to flow into, rather than out of, a cell, resulting in negative membrane potential. The encoded protein is responsible for potassium buffering action, which is a major function of astrocytes and is also responsible for promoting differentiation and inhibiting cell growth [20], [21]. Mutations in this gene have been associated with seizure susceptibility of common idiopathic generalized epilepsy syndromes [22], [23]. Sparcl1 is known as an astrocyte marker colocalized with Gfap [24] and is also known as a candidate gene for multiple sclerosis [25]. Cbs and BC055107 are also known as astrocyte markers [26], [27]. Thus, we found that demethylation occurred not only in Gfap but also in the other genes involved in astrogenesis prior to astrocyte differentiation.

Bottom Line: This differentiation-potential switch could be explained by epigenetic changes, since the promoters of astrocyte-specific marker genes, glial fibrillary acidic protein (Gfap) and S100beta, have been shown to become demethylated in late-stage NPCs prior to the onset of astrocyte differentiation; however, whether demethylation occurs generally in other astrocyctic genes remains unknown.Although these genes are already demethylated in late-stage NPCs, they are not expressed until cells differentiate into astrocytes.Thus, late-stage NPCs have epigenetic potential which can be realized in their expression after astrocyte differentiation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan. ihatada@showa.gunma-u.ac.jp

ABSTRACT
Epigenetic changes are thought to lead to alterations in the property of cells, such as differentiation potential. Neural precursor cells (NPCs) differentiate only into neurons in the midgestational brain, yet they become able to generate astrocytes in the late stage of development. This differentiation-potential switch could be explained by epigenetic changes, since the promoters of astrocyte-specific marker genes, glial fibrillary acidic protein (Gfap) and S100beta, have been shown to become demethylated in late-stage NPCs prior to the onset of astrocyte differentiation; however, whether demethylation occurs generally in other astrocyctic genes remains unknown. Here we analyzed DNA methylation changes in mouse NPCs between the mid-(E11.5) and late (E14.5) stage of development by a genome-wide DNA methylation profiling method using microarrays and found that many astrocytic genes are demethylated in late-stage NPCs, enabling the cell to become competent to express these genes. Although these genes are already demethylated in late-stage NPCs, they are not expressed until cells differentiate into astrocytes. Thus, late-stage NPCs have epigenetic potential which can be realized in their expression after astrocyte differentiation.

Show MeSH