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High levels of MeCP2 depress MHC class I expression in neuronal cells.

Miralvès J, Magdeleine E, Kaddoum L, Brun H, Peries S, Joly E - PLoS ONE (2007)

Bottom Line: The molecular basis of this regulation is poorly understood, but the genes are particularly rich in CpG islands.We show here that transiently transfected cells expressing high levels of MeCP2 specifically downregulate cell-surface expression of MHC class I molecules in the neuronal cell line N2A and they prevent the induction of MHC class I expression in response to interferon in these cells, supporting our first hypothesis.Immunohistological analyses of brain slices from MECP2 knockout mice (the MeCP2(tm1.1Bird) strain) demonstrated a small but reproducible increase in MHC class I when compared to their wild type littermates, but we found no difference in MHC class I expression in primary cultures of mixed glial cells (mainly neurons and astrocytes) from the knockout and wild-type mice.

View Article: PubMed Central - PubMed

Affiliation: Institut de Pharmacologie et Biologie Structurale, Centre National de Recherche Scientifique (CNRS), Toulouse, France.

ABSTRACT

Background: The expression of MHC class I genes is repressed in mature neurons. The molecular basis of this regulation is poorly understood, but the genes are particularly rich in CpG islands. MeCP2 is a transcriptional repressor that binds to methylated CpG dinucleotides; mutations in this protein also cause the neurodevelopmental disease called Rett syndrome. Because MHC class I molecules play a role in neuronal connectivity, we hypothesised that MeCP2 might repress MHC class I expression in the CNS and that this might play a role in the pathology of Rett syndrome.

Methodology: We show here that transiently transfected cells expressing high levels of MeCP2 specifically downregulate cell-surface expression of MHC class I molecules in the neuronal cell line N2A and they prevent the induction of MHC class I expression in response to interferon in these cells, supporting our first hypothesis. Surprisingly, however, overexpression of the mutated forms of MeCP2 that cause Rett syndrome had a similar effect on MHC class I expression as the wild-type protein. Immunohistological analyses of brain slices from MECP2 knockout mice (the MeCP2(tm1.1Bird) strain) demonstrated a small but reproducible increase in MHC class I when compared to their wild type littermates, but we found no difference in MHC class I expression in primary cultures of mixed glial cells (mainly neurons and astrocytes) from the knockout and wild-type mice.

Conclusion: These data suggest that high levels of MeCP2, such as those found in mature neurons, may contribute to the repression of MHC expression, but we find no evidence that MeCP2 regulation of MHC class I is important for the pathogenesis of Rett syndrome.

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Deletion of MECP2 does not affect basal or IFN-γ -induced MHC class I expression in primary cultures of mixed glial cells.Mixed glial cell cultures established from two-day old wild-type, MeCP2+/− and MeCP2−/y mice (10, 6 and 8 animals per group, respectively) were treated or not with IFN-γ on the second day of culture and analysed two days later by flow cytometry for MHC class I expression. Neurons were identified by their intracellular staining with an anti-β-III-tubulin antibody (inset). Large cells, containing mainly astrocytes, were analysed separately by an appropriate forward/side scatter gate. Primary spleen fibroblasts from the same mice were also subjected or not to IFN-γ treatment and stained for their MHC class I expression. Panel A: Representative histograms showing cell surface staining (x axis) against cell number (y axis), obtained with cells from wild-type and MeCP2−/y male littermates. White-filled curves represent background staining, gray-filled curves represent MHC I-specific staining. Panel B: MHC class I fold-induction in response to IFN-γ was calculated as the ratio of MFI of treated cells (induced MHC I level) on MFI of untreated cells (basal MHC I level). Grey-filled squares show MHC class I fold-induction for individual mice and for each cell type. White-filled squares represent the group's mean of fold-inductions (±SD).
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pone-0001354-g005: Deletion of MECP2 does not affect basal or IFN-γ -induced MHC class I expression in primary cultures of mixed glial cells.Mixed glial cell cultures established from two-day old wild-type, MeCP2+/− and MeCP2−/y mice (10, 6 and 8 animals per group, respectively) were treated or not with IFN-γ on the second day of culture and analysed two days later by flow cytometry for MHC class I expression. Neurons were identified by their intracellular staining with an anti-β-III-tubulin antibody (inset). Large cells, containing mainly astrocytes, were analysed separately by an appropriate forward/side scatter gate. Primary spleen fibroblasts from the same mice were also subjected or not to IFN-γ treatment and stained for their MHC class I expression. Panel A: Representative histograms showing cell surface staining (x axis) against cell number (y axis), obtained with cells from wild-type and MeCP2−/y male littermates. White-filled curves represent background staining, gray-filled curves represent MHC I-specific staining. Panel B: MHC class I fold-induction in response to IFN-γ was calculated as the ratio of MFI of treated cells (induced MHC I level) on MFI of untreated cells (basal MHC I level). Grey-filled squares show MHC class I fold-induction for individual mice and for each cell type. White-filled squares represent the group's mean of fold-inductions (±SD).

Mentions: The immunohistochemistry approach did not allow us to quantify the small variations we observed in MHC class I expression between MeCP2 knockout and wild-type mice or to identify the cell types that expressed MHC class I in the absence of MeCP2 (neurons, astrocytes, oligodendrocytes or endothelial cells). We therefore decided to look at primary cultures of brain cells (called mixed glial cells; MGCs) and fibroblasts from spleen taken from individual 2-day-old mice born from crossing a heterozygous female (MeCP2tm1.1Bird +/−) with a wild-type male. The tail DNA from each newborn mouse used to prepare the cell lines was analysed to establish the genotype of each culture. On the second day of culture, the MGCs were treated with IFN-γ or not and then analysed two days later for expression of MHC class I on the cell surface by flow cytometry (Figure 5). The fibroblast cultures, which took a few more days to establish, were similarly treated with IFN-γ after five days and analysed on the seventh day. Figure 5A shows typical examples of histograms obtained with wild-type and MeCP2tm1.1Bird −/y male littermates. Similar data were obtained for all four genotypes: wild-type female (+/+) and male (+/y), heterozygous mutant female (MeCP2tm1.1Bird −/+) and hemizygous mutant male (MeCP2tm1.1Bird −/y).


High levels of MeCP2 depress MHC class I expression in neuronal cells.

Miralvès J, Magdeleine E, Kaddoum L, Brun H, Peries S, Joly E - PLoS ONE (2007)

Deletion of MECP2 does not affect basal or IFN-γ -induced MHC class I expression in primary cultures of mixed glial cells.Mixed glial cell cultures established from two-day old wild-type, MeCP2+/− and MeCP2−/y mice (10, 6 and 8 animals per group, respectively) were treated or not with IFN-γ on the second day of culture and analysed two days later by flow cytometry for MHC class I expression. Neurons were identified by their intracellular staining with an anti-β-III-tubulin antibody (inset). Large cells, containing mainly astrocytes, were analysed separately by an appropriate forward/side scatter gate. Primary spleen fibroblasts from the same mice were also subjected or not to IFN-γ treatment and stained for their MHC class I expression. Panel A: Representative histograms showing cell surface staining (x axis) against cell number (y axis), obtained with cells from wild-type and MeCP2−/y male littermates. White-filled curves represent background staining, gray-filled curves represent MHC I-specific staining. Panel B: MHC class I fold-induction in response to IFN-γ was calculated as the ratio of MFI of treated cells (induced MHC I level) on MFI of untreated cells (basal MHC I level). Grey-filled squares show MHC class I fold-induction for individual mice and for each cell type. White-filled squares represent the group's mean of fold-inductions (±SD).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2131781&req=5

pone-0001354-g005: Deletion of MECP2 does not affect basal or IFN-γ -induced MHC class I expression in primary cultures of mixed glial cells.Mixed glial cell cultures established from two-day old wild-type, MeCP2+/− and MeCP2−/y mice (10, 6 and 8 animals per group, respectively) were treated or not with IFN-γ on the second day of culture and analysed two days later by flow cytometry for MHC class I expression. Neurons were identified by their intracellular staining with an anti-β-III-tubulin antibody (inset). Large cells, containing mainly astrocytes, were analysed separately by an appropriate forward/side scatter gate. Primary spleen fibroblasts from the same mice were also subjected or not to IFN-γ treatment and stained for their MHC class I expression. Panel A: Representative histograms showing cell surface staining (x axis) against cell number (y axis), obtained with cells from wild-type and MeCP2−/y male littermates. White-filled curves represent background staining, gray-filled curves represent MHC I-specific staining. Panel B: MHC class I fold-induction in response to IFN-γ was calculated as the ratio of MFI of treated cells (induced MHC I level) on MFI of untreated cells (basal MHC I level). Grey-filled squares show MHC class I fold-induction for individual mice and for each cell type. White-filled squares represent the group's mean of fold-inductions (±SD).
Mentions: The immunohistochemistry approach did not allow us to quantify the small variations we observed in MHC class I expression between MeCP2 knockout and wild-type mice or to identify the cell types that expressed MHC class I in the absence of MeCP2 (neurons, astrocytes, oligodendrocytes or endothelial cells). We therefore decided to look at primary cultures of brain cells (called mixed glial cells; MGCs) and fibroblasts from spleen taken from individual 2-day-old mice born from crossing a heterozygous female (MeCP2tm1.1Bird +/−) with a wild-type male. The tail DNA from each newborn mouse used to prepare the cell lines was analysed to establish the genotype of each culture. On the second day of culture, the MGCs were treated with IFN-γ or not and then analysed two days later for expression of MHC class I on the cell surface by flow cytometry (Figure 5). The fibroblast cultures, which took a few more days to establish, were similarly treated with IFN-γ after five days and analysed on the seventh day. Figure 5A shows typical examples of histograms obtained with wild-type and MeCP2tm1.1Bird −/y male littermates. Similar data were obtained for all four genotypes: wild-type female (+/+) and male (+/y), heterozygous mutant female (MeCP2tm1.1Bird −/+) and hemizygous mutant male (MeCP2tm1.1Bird −/y).

Bottom Line: The molecular basis of this regulation is poorly understood, but the genes are particularly rich in CpG islands.We show here that transiently transfected cells expressing high levels of MeCP2 specifically downregulate cell-surface expression of MHC class I molecules in the neuronal cell line N2A and they prevent the induction of MHC class I expression in response to interferon in these cells, supporting our first hypothesis.Immunohistological analyses of brain slices from MECP2 knockout mice (the MeCP2(tm1.1Bird) strain) demonstrated a small but reproducible increase in MHC class I when compared to their wild type littermates, but we found no difference in MHC class I expression in primary cultures of mixed glial cells (mainly neurons and astrocytes) from the knockout and wild-type mice.

View Article: PubMed Central - PubMed

Affiliation: Institut de Pharmacologie et Biologie Structurale, Centre National de Recherche Scientifique (CNRS), Toulouse, France.

ABSTRACT

Background: The expression of MHC class I genes is repressed in mature neurons. The molecular basis of this regulation is poorly understood, but the genes are particularly rich in CpG islands. MeCP2 is a transcriptional repressor that binds to methylated CpG dinucleotides; mutations in this protein also cause the neurodevelopmental disease called Rett syndrome. Because MHC class I molecules play a role in neuronal connectivity, we hypothesised that MeCP2 might repress MHC class I expression in the CNS and that this might play a role in the pathology of Rett syndrome.

Methodology: We show here that transiently transfected cells expressing high levels of MeCP2 specifically downregulate cell-surface expression of MHC class I molecules in the neuronal cell line N2A and they prevent the induction of MHC class I expression in response to interferon in these cells, supporting our first hypothesis. Surprisingly, however, overexpression of the mutated forms of MeCP2 that cause Rett syndrome had a similar effect on MHC class I expression as the wild-type protein. Immunohistological analyses of brain slices from MECP2 knockout mice (the MeCP2(tm1.1Bird) strain) demonstrated a small but reproducible increase in MHC class I when compared to their wild type littermates, but we found no difference in MHC class I expression in primary cultures of mixed glial cells (mainly neurons and astrocytes) from the knockout and wild-type mice.

Conclusion: These data suggest that high levels of MeCP2, such as those found in mature neurons, may contribute to the repression of MHC expression, but we find no evidence that MeCP2 regulation of MHC class I is important for the pathogenesis of Rett syndrome.

Show MeSH
Related in: MedlinePlus