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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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Transient overexpression of MeCP2 inhibits MHC class I induction by IFN-γ.N2A cells transfected with pCMX vectors expressing murine or human MeCP2 were treated or not with IFN-γ for 48 hrs and then double immunostained for cell surface β2-microglobulin, transferrin receptor or MHC class I and intracellular MeCP2. Panel A: Dot-plots of transfected cells analysed by flow cytometry showing the cell surface level of the MHC class I molecule Ld or β2-microglobulin (x-axis) plotted against the level of intracellular MeCP2 (y-axis). Panel B: The induction factor was calculated as the ratio of MFI of treated cells (over-expressing MeCP2 or untransfected cells) on MFI of untreated N2A cells. Values used for the histogram are the mean (±SEM) of induction factors obtained in seven independent transfection experiments. Statistical significance of difference between groups was analysed by using an unpaired t-test (*, p<0.05; **, p<0.01; ***, p<0.001).
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pone-0001354-g002: Transient overexpression of MeCP2 inhibits MHC class I induction by IFN-γ.N2A cells transfected with pCMX vectors expressing murine or human MeCP2 were treated or not with IFN-γ for 48 hrs and then double immunostained for cell surface β2-microglobulin, transferrin receptor or MHC class I and intracellular MeCP2. Panel A: Dot-plots of transfected cells analysed by flow cytometry showing the cell surface level of the MHC class I molecule Ld or β2-microglobulin (x-axis) plotted against the level of intracellular MeCP2 (y-axis). Panel B: The induction factor was calculated as the ratio of MFI of treated cells (over-expressing MeCP2 or untransfected cells) on MFI of untreated N2A cells. Values used for the histogram are the mean (±SEM) of induction factors obtained in seven independent transfection experiments. Statistical significance of difference between groups was analysed by using an unpaired t-test (*, p<0.05; **, p<0.01; ***, p<0.001).

Mentions: The cytokine IFN-γ transactivates MHC class I expression predominantly by binding to IFN regulatory factor-1, which binds, in turn, to the interferon-stimulated response element, ISRE, a GC-rich region and therefore a potential binding site also for MeCP2 [47]. Since MeCP2 appeared to act as a repressor of MHC class I expression (Figure 1), we wanted to test whether it would interfere with transactivation of MHC class I expression by IFN-γ. To do so, we transfected N2A cells with pCMX vectors driving the expression of either murine or human forms of MeCP2A. The following day, these transiently transfected cells were divided into two flasks that were either treated or not with IFN-γ for 48 hrs. The levels of cell-surface MHC class I, β-2-microglobulin and transferrin receptor, and intracellular MeCP2, were evaluated by flow cytometry as in the previous experiments (Figure 2). As can be seen by the shift of the clouds between the higher and the lower panels, treatment with IFN-γ resulted in a two- to four-fold induction of MHC class I and β2-microglobulin in both mock-transfected populations and transfected populations (Figure 2A). Individual transfected cells expressing MeCP2 at high levels, however, had little more Ld and β2-microglobulin when treated with IFN-γ than untreated MeCP2-expressing cells (compare the populations encircled in Figure 2A IFN-γ with those without IFN-γ). Cells overexpressing MeCP2 therefore appear to respond to IFN-γ by upregulating MHC class I to a much lesser extent than do cells not expressing MeCP2.


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)

Transient overexpression of MeCP2 inhibits MHC class I induction by IFN-γ.N2A cells transfected with pCMX vectors expressing murine or human MeCP2 were treated or not with IFN-γ for 48 hrs and then double immunostained for cell surface β2-microglobulin, transferrin receptor or MHC class I and intracellular MeCP2. Panel A: Dot-plots of transfected cells analysed by flow cytometry showing the cell surface level of the MHC class I molecule Ld or β2-microglobulin (x-axis) plotted against the level of intracellular MeCP2 (y-axis). Panel B: The induction factor was calculated as the ratio of MFI of treated cells (over-expressing MeCP2 or untransfected cells) on MFI of untreated N2A cells. Values used for the histogram are the mean (±SEM) of induction factors obtained in seven independent transfection experiments. Statistical significance of difference between groups was analysed by using an unpaired t-test (*, p<0.05; **, p<0.01; ***, p<0.001).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2131781&req=5

pone-0001354-g002: Transient overexpression of MeCP2 inhibits MHC class I induction by IFN-γ.N2A cells transfected with pCMX vectors expressing murine or human MeCP2 were treated or not with IFN-γ for 48 hrs and then double immunostained for cell surface β2-microglobulin, transferrin receptor or MHC class I and intracellular MeCP2. Panel A: Dot-plots of transfected cells analysed by flow cytometry showing the cell surface level of the MHC class I molecule Ld or β2-microglobulin (x-axis) plotted against the level of intracellular MeCP2 (y-axis). Panel B: The induction factor was calculated as the ratio of MFI of treated cells (over-expressing MeCP2 or untransfected cells) on MFI of untreated N2A cells. Values used for the histogram are the mean (±SEM) of induction factors obtained in seven independent transfection experiments. Statistical significance of difference between groups was analysed by using an unpaired t-test (*, p<0.05; **, p<0.01; ***, p<0.001).
Mentions: The cytokine IFN-γ transactivates MHC class I expression predominantly by binding to IFN regulatory factor-1, which binds, in turn, to the interferon-stimulated response element, ISRE, a GC-rich region and therefore a potential binding site also for MeCP2 [47]. Since MeCP2 appeared to act as a repressor of MHC class I expression (Figure 1), we wanted to test whether it would interfere with transactivation of MHC class I expression by IFN-γ. To do so, we transfected N2A cells with pCMX vectors driving the expression of either murine or human forms of MeCP2A. The following day, these transiently transfected cells were divided into two flasks that were either treated or not with IFN-γ for 48 hrs. The levels of cell-surface MHC class I, β-2-microglobulin and transferrin receptor, and intracellular MeCP2, were evaluated by flow cytometry as in the previous experiments (Figure 2). As can be seen by the shift of the clouds between the higher and the lower panels, treatment with IFN-γ resulted in a two- to four-fold induction of MHC class I and β2-microglobulin in both mock-transfected populations and transfected populations (Figure 2A). Individual transfected cells expressing MeCP2 at high levels, however, had little more Ld and β2-microglobulin when treated with IFN-γ than untreated MeCP2-expressing cells (compare the populations encircled in Figure 2A IFN-γ with those without IFN-γ). Cells overexpressing MeCP2 therefore appear to respond to IFN-γ by upregulating MHC class I to a much lesser extent than do cells not expressing MeCP2.

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