It has been known that mouse Grb10
shows reciprocal imprinting depending on the tissue-specific promoters. In most tissues, Grb10
is expressed exclusively from the maternal allele, whereas in the brain, it is expressed predominantly from the paternal allele (1
). Such reciprocal imprinting of Grb10
in a tissue-specific and promoter-specific manner is a good model to elucidate how promoter-specific imprinting is epigenetically controlled in tissues. In this study, we have developed a cell culture system with which cell-type-specific imprinting of Grb10
can be characterized in the mouse brain. We demonstrated that promoter-specific and developmental stage-specific imprinting of Grb10
expression in the brain is associated with parental allele-specific epigenetic modifications in brain cell lineages.
Two previous reports described that reciprocal imprinting of Grb10
occurs in a tissue-specific and promoter-specific manner (1
). Our studies with cultured cortical cells revealed that the brain type transcript containing exon 1b was expressed in neurons but not in glial cells, while the major-type transcript containing exon 1a was expressed in all cultured cells, including neurons (Fig. ). These findings indicate that the brain-specific promoter actually implies the neuron-specific promoter and that the major-type promoter works as the common promoter in all tissues. Imprinting analysis of these transcripts clearly showed that the brain type transcript is expressed exclusively from the paternal allele and the major-type transcript is expressed exclusively from the maternal allele (Fig. ). These results in vitro can explain the previous data that the brain type transcript was not detected in whole embryo at E9.5 (17
), when neurogenesis has not yet occurred. In addition, our data on Grb10
expression, i.e., brain development-dependent switching from the major-type to the brain type transcript, can also support the previous report that Grb10
is expressed predominantly from the paternal allele in the adult brain (17
), which consists of neurons and glial cells.
In our expression analysis, we detected both brain type and major-type transcripts in cultured neurons (Fig. ). Recently, it was reported that the Pcdh
(protocadherin) gene was monoallelically expressed in individual neurons (10
). The Pcdh
gene family (Pcdha
, and Pcdhc
) has variable exons and alternative splice forms. Esumi et al. analyzed the expression of transcripts in the variable exons of Pcdha
by using a single-cell RT-PCR approach for the determination of the allelic origin for each variable exon at the individual cell level (10
). The individual cells showed monoallelic expression for each variable exon. In our analysis of Grb10
, the discrepancy between the modifications in CGI1 and the expression of the major-type transcript in neurons was recognized. Similar to a monoallelic expression pattern of variable Pcdha
exons in individual neurons, the discrepancy may be explained by the existence of two different cell populations in cultured neurons, each of which expresses either the major-type or the brain type transcript exclusively. As shown in Fig. , the brain type transcript was obviously highly expressed compared to the major-type transcript during long culture periods. The larger population of cells with the brain type transcript may affect the result of histone modifications more than the smaller population of cells with the major-type transcript.
It has been reported that histone modifications and DNA methylation are not synchronized as a transcriptionally active/silent signal in some imprinted genes, such as NDN
, and Igf2r
). Our data also showed an epigenetically unsynchronized active/silent signal between DNA methylation and histone modifications in Grb10
(Fig. ). In this study, we showed that the brain type transcript is expressed in neurons but not in glial cells (Fig. ), where both differential methylation in CGI2 and biallelic hypomethylation in CGI1 were maintained regardless of expression (Fig. ). The result that allele-specific DNA methylation is not sufficient to direct imprinted expression in brain cells implies that other epigenetic modifications may affect cell lineage-specific imprinting.
FIG. 7. Summary of epigenetic modifications across promoter regions of Grb10. M and P represent maternal and paternal chromosomes, respectively. Large and small arrows indicate expression levels. The nucleosome model shows DNA wrapping around a histone octamer (more ...)
In our analysis of histone modifications, histone acetylation status correlated with the expression status of the major-type transcript in glial cells and fibroblasts and the brain type transcript in neurons (Fig. ). Such histone acetylation status in Grb10
expression is consistent with the findings that allele-specific histone acetylation was associated with allelic gene expression in the imprinted gene, NDN
). Histone acetylation offers the best example of a direct link between tissue-specific gene expression and histone modifications.
Unlike that of histone acetylation, the status of histone methylation has been implicated as an early event for chromatin conformations. Methylation of histones H3K4 and H3K9 is associated with active chromatin and silent chromatin, respectively. According to our results, allele-specific H3K4 and H3K9 methylation in CGI1 and CGI2 did not correlate with allele-specific gene expression in each cultured cell. In glial cells, H3K4 in CGI2 was hypermethylated in the paternal chromosome, which was silent with no brain type transcript. It seems that H3mK4 is maintained during differentiation as an imprint mark with H3mK9 but is not related to promoter activity (28
), although histone modifications in oocytes remain unknown. In CGI1, H3me2K9 and H3me3K9 were hypomethylated in both parental chromosomes independent of the expression of the major-type transcript in cultured cells. It is likely that H3K9 methylation in germ cells is maintained as a stable and heritable imprint mark but may not be secondarily acquired during development.
Then, how is maternal chromosome-specific expression of the major-type transcript regulated without differential DNA methylation in CGI1? The PcG protein Eed complex is known to be a part of a memory system that maintains repression of the imprinted X chromosome (36
) and silencing of some imprinted genes (24
is reported to be one of the imprinted genes that are regulated by the PcG protein Eed complex. Interestingly, in Eed−/−
embryos, the major-type transcript was biallelically expressed without major alteration of allelic DNA methylation (24
). The Eed/Ezh2 PcG complex possesses histone methyltransferase activity on H3K27 (5
) and interacts with histone deacetylases (34
). Methylation of H3K27 is a repressive epigenetic mark regulated by the SET domain containing Ezh2/Eed complex (5
). In our analysis, H3mK27 was clearly precipitated in neurons and fibroblasts in CGI1 but not in CGI2 (Fig. ). The paternal chromosome-specific methylation of H3K27 in CGI1 was observed in fibroblasts but not in neurons (Fig. ). These data indicate that the Eed PcG complex can biallelically interact on CGI1 as a trans
-acting factor in neurons but paternally in other cells. In the absence of DNA methylation in CGI1, PcG complexes may mediate a nonpermissive chromatin state for transcription, leading to repressive histone modifications. Interestingly, other genes, Cdkn1c
, imprinting of which was reported to be regulated by Eed (24
), show tissue-specific imprinting, and their imprinted expression in trophoblasts is associated with repressive histone H3K27 methylation rather than DNA methylation (22
Figure shows the summary of our data. In CGI2, DNA methylation in a gametically methylated CpG island on the maternal allele was maintained throughout development. Allelic methylation of H3K4 and H3K9 associated with gametic DNA methylation was also stable as an epigenetic mark, independent of Grb10
expression. Histone acetylation status was correlated with the expression status of the brain type transcript: histones H3 and H4 were paternally acetylated only in neurons, where the brain type transcript was paternally expressed. H3K27 was not methylated biallelically. In CGI1, biallelic DNA hypomethylation and biallelic hypomethylation of H3K9 were observed. Acetylation of histones H3 and H4 and methylation of H3K4 and H3K27 were allelically detected, corresponding to the allelic expression of the major-type transcript, although the discordance in histone modifications and expression in neurons was detected, probably depending on maturation of neurons. Methylation of H3K9 and H3K27 is thought to be a repressive chromatin marker, but it is not completely clear whether PcG-mediated silencing involves methylation of H3K9 synchronized with H3mK27 in all PcG target genes. We did not observe coexistence of H3mK27 and H3mK9 in both CGI1 and CGI2 of Grb10
. Umlauf et al. also reported discordance between localizations of H3mK27 and H3mK9 in some imprinted genes in the Kcnq1
). Further work should determine how histone modifications, especially methylation of H3K9 and H3K27, are coordinated or uncoordinated as epigenetic determinants in tissue-specific imprinting.
These data about epigenetic modifications analyzed at the cell level, in addition to the evidence for Dnmt3Lm−/−
embryos, lead to a working model for tissue-specific reciprocal imprinting of Grb10
(Fig. ). The previous model by Hikichi et al. (17
) was modified in our model based on the data of DNA methylation and repressive histone modifications mediated by the PcG complex in brain cell lineages. In undifferentiated cells, a DNA methylation-sensitive insulator, CTCF, binds to the paternal CGI2 and blocks the paternal activity of the downstream major enhancer, resulting in silent expression of the major-type transcript on the paternal allele. On the maternal allele, the major enhancer works on the major-type promoter to recruit transcription factors. In CGI1, the Eed/Ezh2 PcG complex binds on the paternal allele, whereas it competes with transcription factors on the maternal allele. The Eed/Ezh2 PcG complex methylates H3K27 and interacts with histone deacetylases, leading to silencing of the chromatin on the paternal CGI1. In Dnmt3Lm−/−
embryos, biallelic hypomethylation in CGI2 makes CTCF bind biallelically on CGI2, resulting in null expression of the major-type transcript, regardless of the PcG complex. In Eed−/−
embryos, the silent state on the paternal CGI1 regulated by the Eed PcG complex is released to the biallelically active state without major alteration of DNA methylation in maternal CGI2. In neurons, the other molecular mechanism of imprinting works in a promoter-specific manner, different from that in other differentiated cells. During neurogenesis, expression of Grb10
shifts from the major-type to the brain type transcript by switching from the major-type promoter to the brain type promoter. The neuronal enhancer instead of the major enhancer may work on the brain type promoter, depending on DNA methylation in CGI2. The maternally active major-type promoter becomes silent without transcription factors, and consequently, the Eed/Ezh2 PcG complex binds to make the chromatin structure silent. This implies that the PcG complex is necessary to maintain cell-type-specific imprinting. It remains unknown how neuron-specific imprinting is regulated by DNA methylation and/or histone modifications mediated by the PcG complex, because Dnmt3Lm−/−
embryos are lethal by E10.5 (4
) and E8.5 (11
), respectively, just before neurogenesis.
FIG. 8. Working models for tissue-specific reciprocal imprinting of Grb10. The previous enhancer/insulator model by Hikichi et al. was modified based on the analysis of DNA methylation and histone modifications mediated by the PcG complex containing Eed (17). (more ...)
As far as we know, this is the first report of an epigenetic analysis of cultured cells where DNA methylation and chromatin remodeling by PcG proteins establish and maintain cell-type-specific imprinting at one gene locus. Although allelic DNA methylation established in the gamete contributes primarily to tissue-specific imprinting, tissue-specific Grb10 imprinting is directly regulated by the repressive chromatin mediated by the PcG complex during development. Our analysis of promoter-specific and cell-type-specific imprinting of Grb10 gives an important clue for understanding the mechanism of tissue-specific imprinting.