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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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Related in: MedlinePlus

MeCP2 overexpression diminishes MHC class I expression.N2A cells transfected with pCMX vectors expressing either murine or human MeCP2 were immunostained for one of the three MHC class I molecules (Kk, Ld or Dd), β2-microglobulin or the transferrin receptor on the cell surface as well as for intracellular MeCP2. The level of staining was then analysed by fluorescence-activated cell sorting. Panel A: Dot plots of the amount of surface antigen (x-axis) against the amount of intracellular MeCP2 (y-axis) in cells transiently transfected with pCMX expressing human MeCP2 and analysed 48 hrs later. Panel B: For each kind of staining, the variation in expression level was calculated as the ratio of MFI of MeCP2 overexpressing cells over MFI of mock-transfected cells. The histograms summarise the mean (±SEM) of the variation in cell surface levels from 15 independent transfections with vectors expressing mouse MeCP2A (grey fill) and 12 independent transfections with vectors expressing human MeCP2A (black fill). Panel C: N2A and NIH3T3 cells were transfected with empty pcDNA3.1(+) (mock cells) or expressing Myc-tagged human MeCP2A or B isoforms. 48 h after transfection, cells were subjected to double staining against cell surface MHC class I molecules and intracellular Myc-tagged MeCP2, then analyzed by flow cytometry. The variation in expression level of MHC class I molecules was calculated as the ratio of MFI of MeCP2 over-expressing cells over the MFI of mock cells. The histograms represent the mean (±SEM) of the cell surface level variation from 4 independent transfections with each of the vectors. Panel D: A representative example of dot-plots obtained for double immunostaining of transiently transfected N2A cells with anti-Myc 9E10 and rat-anti-mouse-MHC I M1/42 monoclonal antibodies. Dotted and continuous circles indicate the different populations expressing high and intermediate levels of MeCP2, respectively. Statistical significance of difference between groups was analysed by using an unpaired t-test (**, p<0.01 ; ***, p<0.001).
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pone-0001354-g001: MeCP2 overexpression diminishes MHC class I expression.N2A cells transfected with pCMX vectors expressing either murine or human MeCP2 were immunostained for one of the three MHC class I molecules (Kk, Ld or Dd), β2-microglobulin or the transferrin receptor on the cell surface as well as for intracellular MeCP2. The level of staining was then analysed by fluorescence-activated cell sorting. Panel A: Dot plots of the amount of surface antigen (x-axis) against the amount of intracellular MeCP2 (y-axis) in cells transiently transfected with pCMX expressing human MeCP2 and analysed 48 hrs later. Panel B: For each kind of staining, the variation in expression level was calculated as the ratio of MFI of MeCP2 overexpressing cells over MFI of mock-transfected cells. The histograms summarise the mean (±SEM) of the variation in cell surface levels from 15 independent transfections with vectors expressing mouse MeCP2A (grey fill) and 12 independent transfections with vectors expressing human MeCP2A (black fill). Panel C: N2A and NIH3T3 cells were transfected with empty pcDNA3.1(+) (mock cells) or expressing Myc-tagged human MeCP2A or B isoforms. 48 h after transfection, cells were subjected to double staining against cell surface MHC class I molecules and intracellular Myc-tagged MeCP2, then analyzed by flow cytometry. The variation in expression level of MHC class I molecules was calculated as the ratio of MFI of MeCP2 over-expressing cells over the MFI of mock cells. The histograms represent the mean (±SEM) of the cell surface level variation from 4 independent transfections with each of the vectors. Panel D: A representative example of dot-plots obtained for double immunostaining of transiently transfected N2A cells with anti-Myc 9E10 and rat-anti-mouse-MHC I M1/42 monoclonal antibodies. Dotted and continuous circles indicate the different populations expressing high and intermediate levels of MeCP2, respectively. Statistical significance of difference between groups was analysed by using an unpaired t-test (**, p<0.01 ; ***, p<0.001).

Mentions: To investigate whether MeCP2 represses MHC class I expression, we transfected the murine neuronal cell line N2A with pCMX plasmids that transiently express either human or murine MeCP2A. Forty-eight hours after transfection, we evaluated by flow cytometry the cell surface levels of Kk, Ld and Dd (the three ‘classical’ MHC class I molecule heavy chains expressed by N2A), β-2-microglobulin (the light chain subunit of MHC class I) and, as control, the transferrin receptor, a cell surface glycoprotein of similar size and half-life to MHC molecules (Figure 1). The expression of MeCP2 in transfected cells was detected by a rabbit anti-MeCP2 polyclonal antibody on cells fixed and permeabilised after they had been stained for MHC class I cell surface expression. Dot plot representative examples of these analyses are shown on Figure 1A, where each dot represents an individual cell, and an decrease in MHC staining results in a shift to the left, and an increase in MeCP2 in an upward shift. Because these are transient transfections, MeCP2 overexpression is detected in only a certain percentage (typically 20–30%) of the cells. As can be seen for all MHC class I molecules ( Kk, Ld, Dd ) and β-2-microglobulin, in the cloud of cells overexpressing MeCP2, we detected a clear shift to the left compared to the lower cloud of untransfected cells and to mock-transfected cells, indicating a reduction of the cell surface expression of all these molecules in the cells that overexpress MeCP2. This effect appears to be specific for these molecules since the level of transferrin receptor was basically unaffected by MeCP2 overexpression. The reproducibility and statistical significance of these observations was ascertained by repeating this experiment many times, which also allowed us to conclude that MeCP2 overexpression results in a similar 40% decrease of all three MHC class I molecules and of β-2-microglobulin (Figure 1B). In the experiment shown, the mock-transfected cells were simply treated with the transfection reagent, but similar results were obtained when the negative control consisted of empty plasmids, or plasmids expressing other proteins such as GFP (not shown).


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)

MeCP2 overexpression diminishes MHC class I expression.N2A cells transfected with pCMX vectors expressing either murine or human MeCP2 were immunostained for one of the three MHC class I molecules (Kk, Ld or Dd), β2-microglobulin or the transferrin receptor on the cell surface as well as for intracellular MeCP2. The level of staining was then analysed by fluorescence-activated cell sorting. Panel A: Dot plots of the amount of surface antigen (x-axis) against the amount of intracellular MeCP2 (y-axis) in cells transiently transfected with pCMX expressing human MeCP2 and analysed 48 hrs later. Panel B: For each kind of staining, the variation in expression level was calculated as the ratio of MFI of MeCP2 overexpressing cells over MFI of mock-transfected cells. The histograms summarise the mean (±SEM) of the variation in cell surface levels from 15 independent transfections with vectors expressing mouse MeCP2A (grey fill) and 12 independent transfections with vectors expressing human MeCP2A (black fill). Panel C: N2A and NIH3T3 cells were transfected with empty pcDNA3.1(+) (mock cells) or expressing Myc-tagged human MeCP2A or B isoforms. 48 h after transfection, cells were subjected to double staining against cell surface MHC class I molecules and intracellular Myc-tagged MeCP2, then analyzed by flow cytometry. The variation in expression level of MHC class I molecules was calculated as the ratio of MFI of MeCP2 over-expressing cells over the MFI of mock cells. The histograms represent the mean (±SEM) of the cell surface level variation from 4 independent transfections with each of the vectors. Panel D: A representative example of dot-plots obtained for double immunostaining of transiently transfected N2A cells with anti-Myc 9E10 and rat-anti-mouse-MHC I M1/42 monoclonal antibodies. Dotted and continuous circles indicate the different populations expressing high and intermediate levels of MeCP2, respectively. Statistical significance of difference between groups was analysed by using an unpaired t-test (**, p<0.01 ; ***, p<0.001).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2131781&req=5

pone-0001354-g001: MeCP2 overexpression diminishes MHC class I expression.N2A cells transfected with pCMX vectors expressing either murine or human MeCP2 were immunostained for one of the three MHC class I molecules (Kk, Ld or Dd), β2-microglobulin or the transferrin receptor on the cell surface as well as for intracellular MeCP2. The level of staining was then analysed by fluorescence-activated cell sorting. Panel A: Dot plots of the amount of surface antigen (x-axis) against the amount of intracellular MeCP2 (y-axis) in cells transiently transfected with pCMX expressing human MeCP2 and analysed 48 hrs later. Panel B: For each kind of staining, the variation in expression level was calculated as the ratio of MFI of MeCP2 overexpressing cells over MFI of mock-transfected cells. The histograms summarise the mean (±SEM) of the variation in cell surface levels from 15 independent transfections with vectors expressing mouse MeCP2A (grey fill) and 12 independent transfections with vectors expressing human MeCP2A (black fill). Panel C: N2A and NIH3T3 cells were transfected with empty pcDNA3.1(+) (mock cells) or expressing Myc-tagged human MeCP2A or B isoforms. 48 h after transfection, cells were subjected to double staining against cell surface MHC class I molecules and intracellular Myc-tagged MeCP2, then analyzed by flow cytometry. The variation in expression level of MHC class I molecules was calculated as the ratio of MFI of MeCP2 over-expressing cells over the MFI of mock cells. The histograms represent the mean (±SEM) of the cell surface level variation from 4 independent transfections with each of the vectors. Panel D: A representative example of dot-plots obtained for double immunostaining of transiently transfected N2A cells with anti-Myc 9E10 and rat-anti-mouse-MHC I M1/42 monoclonal antibodies. Dotted and continuous circles indicate the different populations expressing high and intermediate levels of MeCP2, respectively. Statistical significance of difference between groups was analysed by using an unpaired t-test (**, p<0.01 ; ***, p<0.001).
Mentions: To investigate whether MeCP2 represses MHC class I expression, we transfected the murine neuronal cell line N2A with pCMX plasmids that transiently express either human or murine MeCP2A. Forty-eight hours after transfection, we evaluated by flow cytometry the cell surface levels of Kk, Ld and Dd (the three ‘classical’ MHC class I molecule heavy chains expressed by N2A), β-2-microglobulin (the light chain subunit of MHC class I) and, as control, the transferrin receptor, a cell surface glycoprotein of similar size and half-life to MHC molecules (Figure 1). The expression of MeCP2 in transfected cells was detected by a rabbit anti-MeCP2 polyclonal antibody on cells fixed and permeabilised after they had been stained for MHC class I cell surface expression. Dot plot representative examples of these analyses are shown on Figure 1A, where each dot represents an individual cell, and an decrease in MHC staining results in a shift to the left, and an increase in MeCP2 in an upward shift. Because these are transient transfections, MeCP2 overexpression is detected in only a certain percentage (typically 20–30%) of the cells. As can be seen for all MHC class I molecules ( Kk, Ld, Dd ) and β-2-microglobulin, in the cloud of cells overexpressing MeCP2, we detected a clear shift to the left compared to the lower cloud of untransfected cells and to mock-transfected cells, indicating a reduction of the cell surface expression of all these molecules in the cells that overexpress MeCP2. This effect appears to be specific for these molecules since the level of transferrin receptor was basically unaffected by MeCP2 overexpression. The reproducibility and statistical significance of these observations was ascertained by repeating this experiment many times, which also allowed us to conclude that MeCP2 overexpression results in a similar 40% decrease of all three MHC class I molecules and of β-2-microglobulin (Figure 1B). In the experiment shown, the mock-transfected cells were simply treated with the transfection reagent, but similar results were obtained when the negative control consisted of empty plasmids, or plasmids expressing other proteins such as GFP (not shown).

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