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Cell cycle-linked MeCP2 phosphorylation modulates adult neurogenesis involving the Notch signalling pathway.

Li H, Zhong X, Chau KF, Santistevan NJ, Guo W, Kong G, Li X, Kadakia M, Masliah J, Chi J, Jin P, Zhang J, Zhao X, Chang Q - Nat Commun (2014)

Bottom Line: Neuronal activity regulates the phosphorylation states at multiple sites on MeCP2 in postmitotic neurons.However, it is unknown whether phosphorylation at any of the previously identified sites on MeCP2 can be induced by signals other than neuronal activity in other cell types, and what functions MeCP2 phosphorylation may have in those contexts.Our findings suggest MeCP2 S421 phosphorylation may function as a general epigenetic switch accessible by different extracellular stimuli through different signalling pathways for regulating diverse biological functions in different cell types.

View Article: PubMed Central - PubMed

Affiliation: 1] Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA [2] Genetics Training Program, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA.

ABSTRACT
Neuronal activity regulates the phosphorylation states at multiple sites on MeCP2 in postmitotic neurons. The precise control of the phosphorylation status of MeCP2 in neurons is critical for the normal development and function of the mammalian brain. However, it is unknown whether phosphorylation at any of the previously identified sites on MeCP2 can be induced by signals other than neuronal activity in other cell types, and what functions MeCP2 phosphorylation may have in those contexts. Here we show that in neural progenitor cells isolated from the adult mouse hippocampus, cell cycle-linked phosphorylation at serine 421 on MeCP2 is directly regulated by aurora kinase B and modulates the balance between proliferation and neural differentiation through the Notch signalling pathway. Our findings suggest MeCP2 S421 phosphorylation may function as a general epigenetic switch accessible by different extracellular stimuli through different signalling pathways for regulating diverse biological functions in different cell types.

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Reduced Notch signaling in MeCP2 phosphor-mutant aNPCs and phenotypic rescue by NICD overexpression(a) RT-qPCR analysis of the relative mRNA level of Dll1 and Notch1 in WT and Mecp2S421A;S424A/y aNPCs. (n=5 in each group for Dll1, n=8 in each group for Notch1) (b) RT-qPCR analysis of the relative mRNA level of Notch target gene Hes5 and Hey1 in WT and Mecp2S421A;S424A/y aNPCs. (n=5 in each group) (c,d) Western blot analysis and quantification of the relative protein level of DLL1 in WT and Mecp2S421A;S424A/y aNPCs. (n=4 in each group) (e, f) Western blot analysis and quantification of NICD level in WT and Mecp2S421A;S424A/y aNPCs (n=5 in each group) (g) RT-qPCR analysis of the relative mRNA level of Hes5 and Hey1 in WT and Mecp2S421A;S424A/y aNPCs infected with GFP- or NICD-lentivirus. (n=3 in each group) (h, i) Representative images and quantification of BrdU-labeled cells in WT and Mecp2S421A;S424A/y aNPCs infected with GFP- or NICD-lentivirus, followed by BrdU pulse labeling. (n=3 in each group) (j, k) Representative images and quantification of Tuj1+ neurons differentiated from WT and Mecp2S421A;S424A/y aNPCs infected with GFP- or NICD-lentivirus. (n=3 in each group) (l) Relative mRNA level of neuronal marker (Tuj1 and NeuroD1) and astrocyte marker (GFAP) in WT and Mecp2S421A;S424A/y aNPCs, which are infected with GFP- or NICD-lentivirus and then cultured in differentiation condition. (n=3 in each group) Scale bar 50 μm. (m) ChIP-qPCR analysis of the promoter occupancy of WT and phosphor-mutant MeCP2 on Dll1 and Notch1 promoters. (n=4 in each group) Numbers next to Western blots are molecular weight markers. The bar graphs in this figure show the mean ± s.e.m * p<0.05 ** p<0.01
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Figure 4: Reduced Notch signaling in MeCP2 phosphor-mutant aNPCs and phenotypic rescue by NICD overexpression(a) RT-qPCR analysis of the relative mRNA level of Dll1 and Notch1 in WT and Mecp2S421A;S424A/y aNPCs. (n=5 in each group for Dll1, n=8 in each group for Notch1) (b) RT-qPCR analysis of the relative mRNA level of Notch target gene Hes5 and Hey1 in WT and Mecp2S421A;S424A/y aNPCs. (n=5 in each group) (c,d) Western blot analysis and quantification of the relative protein level of DLL1 in WT and Mecp2S421A;S424A/y aNPCs. (n=4 in each group) (e, f) Western blot analysis and quantification of NICD level in WT and Mecp2S421A;S424A/y aNPCs (n=5 in each group) (g) RT-qPCR analysis of the relative mRNA level of Hes5 and Hey1 in WT and Mecp2S421A;S424A/y aNPCs infected with GFP- or NICD-lentivirus. (n=3 in each group) (h, i) Representative images and quantification of BrdU-labeled cells in WT and Mecp2S421A;S424A/y aNPCs infected with GFP- or NICD-lentivirus, followed by BrdU pulse labeling. (n=3 in each group) (j, k) Representative images and quantification of Tuj1+ neurons differentiated from WT and Mecp2S421A;S424A/y aNPCs infected with GFP- or NICD-lentivirus. (n=3 in each group) (l) Relative mRNA level of neuronal marker (Tuj1 and NeuroD1) and astrocyte marker (GFAP) in WT and Mecp2S421A;S424A/y aNPCs, which are infected with GFP- or NICD-lentivirus and then cultured in differentiation condition. (n=3 in each group) Scale bar 50 μm. (m) ChIP-qPCR analysis of the promoter occupancy of WT and phosphor-mutant MeCP2 on Dll1 and Notch1 promoters. (n=4 in each group) Numbers next to Western blots are molecular weight markers. The bar graphs in this figure show the mean ± s.e.m * p<0.05 ** p<0.01

Mentions: To elucidate the molecular mechanism underlying the effect of S421 phosphorylation on balancing aNPC proliferation/neural differentiation, we performed transcription profiling in WT and phosphor-mutant aNPCs using the Neurogenesis and Neural Stem Cells PCR Array (Qiagen). Among the significant transcription changes in phosphor-mutant aNPCs, several belonged to the well-studied Notch signaling pathway (Supplementary Fig. 4a). Thus we expanded our analysis to examine RNA levels of additional components of the Notch pathway, and found consistent decrease in the ligand (Dll1 and Jag2), receptor (Notch1) and several target genes (Hes3, Hes5, Heyl, and Hey1) of this pathway in phosphor-mutant aNPCs (Fig. 4a–b and Supplementary Fig. 4b). In addition, Western blot results confirmed the significant decrease in DLL1 protein level in phosphor-mutant aNPCs (Fig. 4c–d). As the link between ligand/receptor interaction on the cell membrane and target gene activation in the nucleus20, the intracellular domain of Notch (NICD) was found to be significantly lower in the phosphor-mutant aNPCs (Fig. 4e–f). Consistent with the idea that decreased Notch signaling in phosphor-mutant aNPC may underlie the observed changes in aNPC proliferation/differentiation, treatment of WT aNPCs with a Notch inhibitor led to decreased proliferation and increased neural differentiation similar to those observed in phosphor-mutant aNPCs (Supplementary Fig. 4c–d). Furthermore, we attempted to rescue the proliferation/differentiation phenotypes by overexpressing NICD in these cells. GFP- or NICD-expressing lentiviruses were produced and used to infect either WT or phosphor-mutant aNPCs. While the NICD level in GFP-lentivirus-infected phosphor-mutant aNPCs remained much lower than that in GFP-lentivirus-infected WT aNPCs, the NICD level was similar between NICD-lentivirus-infected phosphor-mutant aNPCs and GFP-lentivirus-infected WT aNPCs (Supplementary Fig. 4e). In addition, overexpression of NICD, but not overexpression of GFP, was sufficient to reverse the reduced transcription of Notch signaling target genes in the phosphor-mutant aNPCs (Fig. 4g). Finally, the key phenotypes of reduced proliferation and increased neural differentiation in phosphor-mutant aNPCs were both rescued by the overexpression of NICD, as determined by comparing the number of BrdU labeled aNPCs (Fig. 4h–i), the number of Tuj1 positive cells (Fig. 4j–k) and the expression level of Tuj1 and NeuroD1 RNA (Fig. 4l) upon induction of differentiation in WT and phosphor-mutant aNPCs infected with either NICD- or GFP-lentiviruses. NICD expression did not lead to any significant change in glial differentiation in our aNPCs (Supplementary Fig. 4f). Interestingly, NICD overexpression in WT aNPCs didn’t alter either the cell cycle-linked S421 phosphorylation or the MeCP2 occupancy on the Notch1 promoter, further suggesting that Notch signaling is downstream of MeCP2 phosphorylation in aNPCs (Supplementary Fig. 4g–h). In order to distinguish the role of S421 phosphorylation regulation from that of overall MeCP2 expression regulation, we overexpressed MeCP2 in both the WT and phosphor-mutant aNPCs using a lentivirus encoding MeCP2. MeCP2 overexpression in WT aNPCs did not alter either Notch target gene transcription or the proliferation and neural differentiation of these cells (Supplementary Fig. 4i–k), nor did MeCP2 overexpression in the phosphor-mutant aNPCs rescue either the phenotype of decreased Notch target gene transcription or the phenotypes of decreased proliferation/increased neural differentiation in these cells (Supplementary Fig. 4i–k). Results from this series of experiments suggest that, in this particular cell type, the epigenetic regulatory mechanism is at the level of phosphorylation but not the overall expression level of MeCP2. As the first step to reveal how MeCP2 phosphorylation may regulate Notch signaling, we conducted chromatin immunoprecipitation (ChIP) experiment to determine whether the phosphor-mutant MeCP2 protein has altered promoter occupancy at the promoters of Dll1 and Notch1. We found increased binding of the phosphor-mutant protein at the promoters of both Dll1 and Notch1 (Fig. 4m), but not the promoter of Crh, a well known MeCP2 target gene, or the major satellite repeats (Supplementary Fig. l). Similar promoter binding changes were observed when S421 phosphorylation was blocked by the aurora kinase B inhibitor hesperidin or by knocking down aurora kinase B (Supplementary Fig. 4m–n), suggesting aurora kinase B regulated S421 phosphorylation may change MeCP2 binding to gene promoters. Together, these results indicate cell cycle-linked MeCP2 phosphorylation may directly regulate the transcription of both the ligand and receptor of Notch signaling pathway to influence aNPC proliferation and differentiation.


Cell cycle-linked MeCP2 phosphorylation modulates adult neurogenesis involving the Notch signalling pathway.

Li H, Zhong X, Chau KF, Santistevan NJ, Guo W, Kong G, Li X, Kadakia M, Masliah J, Chi J, Jin P, Zhang J, Zhao X, Chang Q - Nat Commun (2014)

Reduced Notch signaling in MeCP2 phosphor-mutant aNPCs and phenotypic rescue by NICD overexpression(a) RT-qPCR analysis of the relative mRNA level of Dll1 and Notch1 in WT and Mecp2S421A;S424A/y aNPCs. (n=5 in each group for Dll1, n=8 in each group for Notch1) (b) RT-qPCR analysis of the relative mRNA level of Notch target gene Hes5 and Hey1 in WT and Mecp2S421A;S424A/y aNPCs. (n=5 in each group) (c,d) Western blot analysis and quantification of the relative protein level of DLL1 in WT and Mecp2S421A;S424A/y aNPCs. (n=4 in each group) (e, f) Western blot analysis and quantification of NICD level in WT and Mecp2S421A;S424A/y aNPCs (n=5 in each group) (g) RT-qPCR analysis of the relative mRNA level of Hes5 and Hey1 in WT and Mecp2S421A;S424A/y aNPCs infected with GFP- or NICD-lentivirus. (n=3 in each group) (h, i) Representative images and quantification of BrdU-labeled cells in WT and Mecp2S421A;S424A/y aNPCs infected with GFP- or NICD-lentivirus, followed by BrdU pulse labeling. (n=3 in each group) (j, k) Representative images and quantification of Tuj1+ neurons differentiated from WT and Mecp2S421A;S424A/y aNPCs infected with GFP- or NICD-lentivirus. (n=3 in each group) (l) Relative mRNA level of neuronal marker (Tuj1 and NeuroD1) and astrocyte marker (GFAP) in WT and Mecp2S421A;S424A/y aNPCs, which are infected with GFP- or NICD-lentivirus and then cultured in differentiation condition. (n=3 in each group) Scale bar 50 μm. (m) ChIP-qPCR analysis of the promoter occupancy of WT and phosphor-mutant MeCP2 on Dll1 and Notch1 promoters. (n=4 in each group) Numbers next to Western blots are molecular weight markers. The bar graphs in this figure show the mean ± s.e.m * p<0.05 ** p<0.01
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Figure 4: Reduced Notch signaling in MeCP2 phosphor-mutant aNPCs and phenotypic rescue by NICD overexpression(a) RT-qPCR analysis of the relative mRNA level of Dll1 and Notch1 in WT and Mecp2S421A;S424A/y aNPCs. (n=5 in each group for Dll1, n=8 in each group for Notch1) (b) RT-qPCR analysis of the relative mRNA level of Notch target gene Hes5 and Hey1 in WT and Mecp2S421A;S424A/y aNPCs. (n=5 in each group) (c,d) Western blot analysis and quantification of the relative protein level of DLL1 in WT and Mecp2S421A;S424A/y aNPCs. (n=4 in each group) (e, f) Western blot analysis and quantification of NICD level in WT and Mecp2S421A;S424A/y aNPCs (n=5 in each group) (g) RT-qPCR analysis of the relative mRNA level of Hes5 and Hey1 in WT and Mecp2S421A;S424A/y aNPCs infected with GFP- or NICD-lentivirus. (n=3 in each group) (h, i) Representative images and quantification of BrdU-labeled cells in WT and Mecp2S421A;S424A/y aNPCs infected with GFP- or NICD-lentivirus, followed by BrdU pulse labeling. (n=3 in each group) (j, k) Representative images and quantification of Tuj1+ neurons differentiated from WT and Mecp2S421A;S424A/y aNPCs infected with GFP- or NICD-lentivirus. (n=3 in each group) (l) Relative mRNA level of neuronal marker (Tuj1 and NeuroD1) and astrocyte marker (GFAP) in WT and Mecp2S421A;S424A/y aNPCs, which are infected with GFP- or NICD-lentivirus and then cultured in differentiation condition. (n=3 in each group) Scale bar 50 μm. (m) ChIP-qPCR analysis of the promoter occupancy of WT and phosphor-mutant MeCP2 on Dll1 and Notch1 promoters. (n=4 in each group) Numbers next to Western blots are molecular weight markers. The bar graphs in this figure show the mean ± s.e.m * p<0.05 ** p<0.01
Mentions: To elucidate the molecular mechanism underlying the effect of S421 phosphorylation on balancing aNPC proliferation/neural differentiation, we performed transcription profiling in WT and phosphor-mutant aNPCs using the Neurogenesis and Neural Stem Cells PCR Array (Qiagen). Among the significant transcription changes in phosphor-mutant aNPCs, several belonged to the well-studied Notch signaling pathway (Supplementary Fig. 4a). Thus we expanded our analysis to examine RNA levels of additional components of the Notch pathway, and found consistent decrease in the ligand (Dll1 and Jag2), receptor (Notch1) and several target genes (Hes3, Hes5, Heyl, and Hey1) of this pathway in phosphor-mutant aNPCs (Fig. 4a–b and Supplementary Fig. 4b). In addition, Western blot results confirmed the significant decrease in DLL1 protein level in phosphor-mutant aNPCs (Fig. 4c–d). As the link between ligand/receptor interaction on the cell membrane and target gene activation in the nucleus20, the intracellular domain of Notch (NICD) was found to be significantly lower in the phosphor-mutant aNPCs (Fig. 4e–f). Consistent with the idea that decreased Notch signaling in phosphor-mutant aNPC may underlie the observed changes in aNPC proliferation/differentiation, treatment of WT aNPCs with a Notch inhibitor led to decreased proliferation and increased neural differentiation similar to those observed in phosphor-mutant aNPCs (Supplementary Fig. 4c–d). Furthermore, we attempted to rescue the proliferation/differentiation phenotypes by overexpressing NICD in these cells. GFP- or NICD-expressing lentiviruses were produced and used to infect either WT or phosphor-mutant aNPCs. While the NICD level in GFP-lentivirus-infected phosphor-mutant aNPCs remained much lower than that in GFP-lentivirus-infected WT aNPCs, the NICD level was similar between NICD-lentivirus-infected phosphor-mutant aNPCs and GFP-lentivirus-infected WT aNPCs (Supplementary Fig. 4e). In addition, overexpression of NICD, but not overexpression of GFP, was sufficient to reverse the reduced transcription of Notch signaling target genes in the phosphor-mutant aNPCs (Fig. 4g). Finally, the key phenotypes of reduced proliferation and increased neural differentiation in phosphor-mutant aNPCs were both rescued by the overexpression of NICD, as determined by comparing the number of BrdU labeled aNPCs (Fig. 4h–i), the number of Tuj1 positive cells (Fig. 4j–k) and the expression level of Tuj1 and NeuroD1 RNA (Fig. 4l) upon induction of differentiation in WT and phosphor-mutant aNPCs infected with either NICD- or GFP-lentiviruses. NICD expression did not lead to any significant change in glial differentiation in our aNPCs (Supplementary Fig. 4f). Interestingly, NICD overexpression in WT aNPCs didn’t alter either the cell cycle-linked S421 phosphorylation or the MeCP2 occupancy on the Notch1 promoter, further suggesting that Notch signaling is downstream of MeCP2 phosphorylation in aNPCs (Supplementary Fig. 4g–h). In order to distinguish the role of S421 phosphorylation regulation from that of overall MeCP2 expression regulation, we overexpressed MeCP2 in both the WT and phosphor-mutant aNPCs using a lentivirus encoding MeCP2. MeCP2 overexpression in WT aNPCs did not alter either Notch target gene transcription or the proliferation and neural differentiation of these cells (Supplementary Fig. 4i–k), nor did MeCP2 overexpression in the phosphor-mutant aNPCs rescue either the phenotype of decreased Notch target gene transcription or the phenotypes of decreased proliferation/increased neural differentiation in these cells (Supplementary Fig. 4i–k). Results from this series of experiments suggest that, in this particular cell type, the epigenetic regulatory mechanism is at the level of phosphorylation but not the overall expression level of MeCP2. As the first step to reveal how MeCP2 phosphorylation may regulate Notch signaling, we conducted chromatin immunoprecipitation (ChIP) experiment to determine whether the phosphor-mutant MeCP2 protein has altered promoter occupancy at the promoters of Dll1 and Notch1. We found increased binding of the phosphor-mutant protein at the promoters of both Dll1 and Notch1 (Fig. 4m), but not the promoter of Crh, a well known MeCP2 target gene, or the major satellite repeats (Supplementary Fig. l). Similar promoter binding changes were observed when S421 phosphorylation was blocked by the aurora kinase B inhibitor hesperidin or by knocking down aurora kinase B (Supplementary Fig. 4m–n), suggesting aurora kinase B regulated S421 phosphorylation may change MeCP2 binding to gene promoters. Together, these results indicate cell cycle-linked MeCP2 phosphorylation may directly regulate the transcription of both the ligand and receptor of Notch signaling pathway to influence aNPC proliferation and differentiation.

Bottom Line: Neuronal activity regulates the phosphorylation states at multiple sites on MeCP2 in postmitotic neurons.However, it is unknown whether phosphorylation at any of the previously identified sites on MeCP2 can be induced by signals other than neuronal activity in other cell types, and what functions MeCP2 phosphorylation may have in those contexts.Our findings suggest MeCP2 S421 phosphorylation may function as a general epigenetic switch accessible by different extracellular stimuli through different signalling pathways for regulating diverse biological functions in different cell types.

View Article: PubMed Central - PubMed

Affiliation: 1] Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA [2] Genetics Training Program, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA.

ABSTRACT
Neuronal activity regulates the phosphorylation states at multiple sites on MeCP2 in postmitotic neurons. The precise control of the phosphorylation status of MeCP2 in neurons is critical for the normal development and function of the mammalian brain. However, it is unknown whether phosphorylation at any of the previously identified sites on MeCP2 can be induced by signals other than neuronal activity in other cell types, and what functions MeCP2 phosphorylation may have in those contexts. Here we show that in neural progenitor cells isolated from the adult mouse hippocampus, cell cycle-linked phosphorylation at serine 421 on MeCP2 is directly regulated by aurora kinase B and modulates the balance between proliferation and neural differentiation through the Notch signalling pathway. Our findings suggest MeCP2 S421 phosphorylation may function as a general epigenetic switch accessible by different extracellular stimuli through different signalling pathways for regulating diverse biological functions in different cell types.

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