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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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Mutant forms of MeCP2 that cause RTT retain their repressive effect on MHC class I expression.Panel A: Schematic representation of the MeCP2 protein. Red and orange arrows indicate the positions of the mutations introduced in MeCP2 by site-directed mutagenesis (MBD: methyl-CpG binding domain, TRD: transcription repression domain, WW: group II WW-domain-binding region). Panel B: N2A cells transfected with empty pcDNA3.1 (mock cells), with pcDNA3.1 expressing Myc-tagged MeCP2A (pMeCP2A-myc) or with pcDNA3.1 expressing Myc-tagged MeCP2A with the R133C point mutation (pMeCP2A-R133C-myc) were stained with mouse anti-Myc 9E10 monoclonal antibody, and FITC-labelled anti-mouse IgG antibody. Coverslips were mounted in DAPI-containing ProLong Gold antifade reagent (Molecular Probes) before observation by fluorescence microscopy. Panel C: N2A cells transfected as in panel B were double immunostained for cell surface MHC class I and intracellular Myc-tagged MeCP2, then analysed by flow cytometry. Similar data were obtained for all four mutated forms of MeCP2A and MeCP2B (not shown), and these observations were reproduced in three independent transfection experiments.
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pone-0001354-g003: Mutant forms of MeCP2 that cause RTT retain their repressive effect on MHC class I expression.Panel A: Schematic representation of the MeCP2 protein. Red and orange arrows indicate the positions of the mutations introduced in MeCP2 by site-directed mutagenesis (MBD: methyl-CpG binding domain, TRD: transcription repression domain, WW: group II WW-domain-binding region). Panel B: N2A cells transfected with empty pcDNA3.1 (mock cells), with pcDNA3.1 expressing Myc-tagged MeCP2A (pMeCP2A-myc) or with pcDNA3.1 expressing Myc-tagged MeCP2A with the R133C point mutation (pMeCP2A-R133C-myc) were stained with mouse anti-Myc 9E10 monoclonal antibody, and FITC-labelled anti-mouse IgG antibody. Coverslips were mounted in DAPI-containing ProLong Gold antifade reagent (Molecular Probes) before observation by fluorescence microscopy. Panel C: N2A cells transfected as in panel B were double immunostained for cell surface MHC class I and intracellular Myc-tagged MeCP2, then analysed by flow cytometry. Similar data were obtained for all four mutated forms of MeCP2A and MeCP2B (not shown), and these observations were reproduced in three independent transfection experiments.

Mentions: Many disease-causing mutations of MECP2 have been described [48]. Among them, some occur more frequently than others, and/or have been more thoroughly characterised. To investigate the effect of these MECP2 mutations on MHC class I expression, we transiently transfected the N2A cell line with plasmids expressing well-characterised mutants of both the A and B isoforms of MeCP2 (T158M, R133C, R306C and R308*). The point mutations T158M, R133C and R306C are located in the functional MBD and TRD domains of the protein (Figure 3A). The mutant form that is truncated after the R308 residue corresponds to the form of MeCP2 found in the mouse model of RTT generated by Dr. Zoghbi's group [13]. We performed mutagenesis on vectors expressing either the A or B form of Myc-tagged MeCP2. All the mutated plasmids were sequenced and checked for functional expression and intracellular localisation of the wild-type and mutated MeCP2 proteins by anti-Myc immunofluorescence on transiently transfected N2A cells (Figure 3B). All the mutant forms of Myc-taged MeCP2 were located in intranuclear punctate structures typical of the wild-type protein, which forms foci on heterochromatin [22], [49] (data is shown for the R133C MeCP2A-Myc mutant and the wild-type pMeCP2A-Myc protein only). Subsequently, we evaluated the cell-surface expression level of MHC class I in the transiently transfected N2A cells by flow cytometry using the rat anti-pan-MHC I antibody M1/42, as before. The intracellular MeCP2 level was evaluated based on the intensity of immunostaining for the Myc tag, and was found to be similar to the wild-type for both the A and B forms of the four mutants. The R133C MeCP2A-Myc mutant had the same effect as its wild-type counterpart on the cell-surface level of MHC class I (Figure 3C), whereas neither mutant nor wild type had a significant effect on expression of the transferrin receptor (not shown). Similar effects were found for both A and B isoforms of all four mutants tested (Data for T158M R306C and R308* are not shown). Thus, these mutations responsible for RTT do not abolish the repressive effect of MeCP2 on MHC class I in cells in culture. These results strongly suggest that the repressive function of MeCP2 on the levels of MHC molecules expressed by transiently transfected cells in vitro is unlikely to be directly related to the pathogenesis of Rett syndrome.


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)

Mutant forms of MeCP2 that cause RTT retain their repressive effect on MHC class I expression.Panel A: Schematic representation of the MeCP2 protein. Red and orange arrows indicate the positions of the mutations introduced in MeCP2 by site-directed mutagenesis (MBD: methyl-CpG binding domain, TRD: transcription repression domain, WW: group II WW-domain-binding region). Panel B: N2A cells transfected with empty pcDNA3.1 (mock cells), with pcDNA3.1 expressing Myc-tagged MeCP2A (pMeCP2A-myc) or with pcDNA3.1 expressing Myc-tagged MeCP2A with the R133C point mutation (pMeCP2A-R133C-myc) were stained with mouse anti-Myc 9E10 monoclonal antibody, and FITC-labelled anti-mouse IgG antibody. Coverslips were mounted in DAPI-containing ProLong Gold antifade reagent (Molecular Probes) before observation by fluorescence microscopy. Panel C: N2A cells transfected as in panel B were double immunostained for cell surface MHC class I and intracellular Myc-tagged MeCP2, then analysed by flow cytometry. Similar data were obtained for all four mutated forms of MeCP2A and MeCP2B (not shown), and these observations were reproduced in three independent transfection experiments.
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Related In: Results  -  Collection

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

pone-0001354-g003: Mutant forms of MeCP2 that cause RTT retain their repressive effect on MHC class I expression.Panel A: Schematic representation of the MeCP2 protein. Red and orange arrows indicate the positions of the mutations introduced in MeCP2 by site-directed mutagenesis (MBD: methyl-CpG binding domain, TRD: transcription repression domain, WW: group II WW-domain-binding region). Panel B: N2A cells transfected with empty pcDNA3.1 (mock cells), with pcDNA3.1 expressing Myc-tagged MeCP2A (pMeCP2A-myc) or with pcDNA3.1 expressing Myc-tagged MeCP2A with the R133C point mutation (pMeCP2A-R133C-myc) were stained with mouse anti-Myc 9E10 monoclonal antibody, and FITC-labelled anti-mouse IgG antibody. Coverslips were mounted in DAPI-containing ProLong Gold antifade reagent (Molecular Probes) before observation by fluorescence microscopy. Panel C: N2A cells transfected as in panel B were double immunostained for cell surface MHC class I and intracellular Myc-tagged MeCP2, then analysed by flow cytometry. Similar data were obtained for all four mutated forms of MeCP2A and MeCP2B (not shown), and these observations were reproduced in three independent transfection experiments.
Mentions: Many disease-causing mutations of MECP2 have been described [48]. Among them, some occur more frequently than others, and/or have been more thoroughly characterised. To investigate the effect of these MECP2 mutations on MHC class I expression, we transiently transfected the N2A cell line with plasmids expressing well-characterised mutants of both the A and B isoforms of MeCP2 (T158M, R133C, R306C and R308*). The point mutations T158M, R133C and R306C are located in the functional MBD and TRD domains of the protein (Figure 3A). The mutant form that is truncated after the R308 residue corresponds to the form of MeCP2 found in the mouse model of RTT generated by Dr. Zoghbi's group [13]. We performed mutagenesis on vectors expressing either the A or B form of Myc-tagged MeCP2. All the mutated plasmids were sequenced and checked for functional expression and intracellular localisation of the wild-type and mutated MeCP2 proteins by anti-Myc immunofluorescence on transiently transfected N2A cells (Figure 3B). All the mutant forms of Myc-taged MeCP2 were located in intranuclear punctate structures typical of the wild-type protein, which forms foci on heterochromatin [22], [49] (data is shown for the R133C MeCP2A-Myc mutant and the wild-type pMeCP2A-Myc protein only). Subsequently, we evaluated the cell-surface expression level of MHC class I in the transiently transfected N2A cells by flow cytometry using the rat anti-pan-MHC I antibody M1/42, as before. The intracellular MeCP2 level was evaluated based on the intensity of immunostaining for the Myc tag, and was found to be similar to the wild-type for both the A and B forms of the four mutants. The R133C MeCP2A-Myc mutant had the same effect as its wild-type counterpart on the cell-surface level of MHC class I (Figure 3C), whereas neither mutant nor wild type had a significant effect on expression of the transferrin receptor (not shown). Similar effects were found for both A and B isoforms of all four mutants tested (Data for T158M R306C and R308* are not shown). Thus, these mutations responsible for RTT do not abolish the repressive effect of MeCP2 on MHC class I in cells in culture. These results strongly suggest that the repressive function of MeCP2 on the levels of MHC molecules expressed by transiently transfected cells in vitro is unlikely to be directly related to the pathogenesis of Rett syndrome.

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