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Methylation Gets SMRT
Developmental Cell, Vol. 5, 359–365, September, 2003, Copyright 2003 by Cell Press
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Methylation Gets SMRT: Functional Insights into Rett Syndrome Rett syndrome, a neurodevelopmental disorder, is caused by mutations in the methyl-CpG binding protein MeCP2. A recent report demonstrates that MeCP2 cooperates with the SMRT corepressor complex to inhibit expression of a hairy-related repressor during primary neurogenesis in Xenopus, and that this can be modulated by Notch signaling. Rett syndrome mutations that disrupt interaction with the SMRT corepressor complex also prevent regulation of MeCP2 by activated Notch. “Well-timed silence hath more eloquence than speech.” —Martin Farquhar Tupper (1810–1889) During animal development, well-timed repression of gene expression can be as important as activation. DNA methylation is one mechanism for maintaining genes in a transcriptionally silent state (Meehan, 2003). DNA methylation occurs at cytosine residues within the context of a CpG dinucleotide site, allowing these sites to be recognized by methyl-CpG binding proteins, which function as transcriptional repressors (Wade, 2001). MeCP2 was the first methyl-CpG binding protein to be purified and sequenced, and this subsequently led to the identification of other family members. It is tempting to think of methyl-CpG binding proteins as global repressors of transcription at all methylated sites. This idea was first called into question when it was determined that mutations in the human MeCP2 gene result in Rett syndrome, which is an X-linked neurological disorder characterized by reduced brain growth, regressive effects on motor, speech, and cognitive development, and stereotyped hand-wringing behavior affecting 1/10,000–1/15,000 female children (Amir et al., 1999). Disruption of the MeCP2 gene in mouse produces a Rett syndrome-like phenotype including the hallmark neurological and behavioral features (Chen et al., 2001; Guy et al., 2001). Most strikingly, an identical phenotype is obtained when MeCP2 is selectively deleted embryonically in the CNS, arguing that MeCP2 is primarily required in the nervous system (Chen et al., 2001). In fact, MeCP2 is expressed at high levels in neurons but not glia, and may play a role in regulating neuronal maturation and dendrite development (Shahbazian and Zoghbi, 2002). How does MeCP2 function during neural development and are there clues as to how it may regulate gene expression? The MeCP2 protein has a domain responsible for recognizing a single methylated CpG dinucleotide, as well as a transcriptional repression domain (TRD) that interacts with a corepressor complex containing Sin3A and histone deacetylase (HDAC) activity, arguing that MeCP2 binding to methylated promoter regions
causes changes in histone acetylation and chromatin structure (Wade, 2001). But does MeCP2 confer “welltimed silence,” or is it a more constitutive repressor of gene expression in the nervous system? In addition, are there critical genes during nervous system development that are regulated by MeCP2? In the August issue of Molecular Cell, Meehan and colleagues show that reduction of MeCP2 expression in Xenopus laevis inhibits primary neurogenesis, providing an experimental model for determining how xMeCP2 regulates nervous system development (Stancheva et al., 2003). They examined the expression of genes known to be essential for primary neurogenesis and showed that xMeCP2 is involved in inhibiting expression of xHairy2a, a known target of the Notch/Delta signaling pathway (Davis et al., 2001). Meehan and colleagues show that xHairy2a expression is increased by depletion of xMeCP2, and that xHairy2a functions as a repressor to inhibit primary neurogenesis. During development, Notch target genes, such as xHairy2a, are repressed in the absence of Notch signaling by Suppressor of Hairless [Su(H)] or its mammalian homolog C promoter binding factor 1 (CBF1), which bind to the promoter regions of Notch-regulated genes and recruit a corepressor complex that includes Sin3A, HDAC, and SMRT (Silencing Mediator of Retinoic Acid and Thyroid Receptors; Davis and Turner, 2001; Kao et al., 1998). Upon Notch activation by a ligand such as Delta, the intracellular domain of Notch is cleaved, translocates to the nucleus, and displaces the corepressor complex from Su(H)/ CBF1, converting it from a repressor to a transcriptional activator. They elegantly showed that xMeCP2 interacts with the SMRT corepressor complex, including CBF1, through the TRD and that this interaction depends upon Sin3A (Stancheva et al., 2003). Using selective inhibitors, Meehan and colleagues provide evidence that methylation and HDAC activity act synergistically to repress xHairy2a expression. Furthermore, chromatin immunoprecipitation (ChIP) analysis revealed that binding of xMeCP2 to CpG-rich regions of the xHairy2a promoter could be displaced by expression of the activated Notch intracellular domain (NICD). Interestingly, this is also dependent upon Sin3A, because in Sin3A-depleted embryos, expression of NICD could no longer displace xMeCP2 from the xHairy2a promoter but could still interfere with binding of the SMRT complex. This argues that normally there is Sin3A-mediated coupling between xMeCP2 and the SMRT complex that allows xMeCP2 binding to be regulated by Notch. The finding that xMeCP2 binding to the xHairy2a promoter can be modulated by the Notch signaling pathway is particularly significant because it provides compelling evidence that methyl-CpG binding proteins may indeed be able to confer “well-timed silence” by responding to developmentally relevant signals. A beautiful aspect of this work is that Meehan and colleagues used the Xenopus assay system to explore the functional significance of two common mutations in human Rett syndrome: R168X, which truncates MeCP2
Developmental Cell 360
after the methyl-CpG binding domain (MBD) and eliminates the TRD; and R306C, which is a missense mutation in the TRD. R168X lacks the TRD and does not interact with components of the SMRT complex, while R306C shows weaker interaction. R168X retains binding to CpG-rich regions of the xHairy2a promoter, but Meehan and colleagues show that it was unable to be displaced by expression of NICD. This further argues that xMeCP2 must be associated with the SMRT complex in order for its binding activity to be modulated by Notch signaling. Although modulation of xMeCP2 binding by Notch signaling depends upon interaction with Sin3A and the SMRT corepressor complex, it remains to be determined whether all aspects of MeCP2 function will depend upon this interaction. This work advances our understanding of MeCP2 as a regulator of gene expression during neural development but raises many intriguing questions. During Xenopus primary neurogenesis, xMeCP2 clearly modulates the expression of xHairy2a, which has significant effects on neurogenesis. An obvious question is whether hairyrelated genes are misregulated in Rett syndrome or in mouse models of the disease, and whether this explains all or part of the mutant phenotype. In humans, the Rett syndrome phenotype becomes apparent postnatally beginning at 6–18 months of age, arguing that early aspects of nervous system development may be less dependent upon MeCP2 activity than later aspects (Shahbazian and Zoghbi, 2002). This would suggest that other gene targets for xMeCP2 may exist that play a significant role in postmitotic neurons. The relationship between Notch signaling and xMeCP2 binding to the xHairy2a promoter is particularly striking because it shows that methyl-CpG binding proteins can be regulated by extrinsic signaling during development. It will be interesting to determine whether other Notch target genes are also coregulated by
MeCP2 together with the SMRT corepressor complex. The observation that other Notch target genes, such as the Enhancer of Split-related (ESR) genes, were not affected by depletion of xMeCP2 argues that repression by MeCP2 is not a uniform feature of Notch-responsive promoters (Stancheva et al., 2003). However, Notch can regulate aspects of neuronal maturation, including dendritic branching, which is reduced in neurons affected by Rett syndrome, leaving open the possibility that subtle alterations in Notch signaling may contribute to the Rett syndrome phenotype. Clearly much remains to be learned about this tragic, debilitating disease.
A Cytokine in the Drosophila Stress Response
Drosophila (Ekengren et al., 2001; Ekengren and Hultmark, 2001). Under severe stress, flies secrete a family of eight related peptides of uncertain function, the Turandot (Tot) peptides, which accumulate in their circulating body fluid (the hemolymph). At least one of them, TotA, is produced in the fat body (Ekengren et al., 2001), a tissue with a function similar to that of the mammalian liver. This humoral stress response differs in many ways from the classical heat shock and antimicrobial responses. It can be triggered by many cues, including infection and heat shock as well as tissue damage, dehydration, and other severe treatments of the animal (Ekengren et al., 2001; Ekengren and Hultmark, 2001). Agaisse et al. (2003) have now found that this response is mediated by a cytokine produced in the blood cells (the hemocytes). In a microarray screen for genes that are regulated by the JAK/STAT pathway in response to bacterial infection, Agaisse et al. (2003) found that the TotA gene is upregulated in hopTum (also called TumL) mutant flies, which carry a constitutively active form of the single Drosophila JAK homolog, Hopscotch. Conversely, the induction of TotA by a septic injury is abolished in hop
The fruit fly, Drosophila melanogaster, has become a popular tool for studying immediate reactions to environmental hazards, such as the heat shock and innate immune responses. In mammals, protective responses to infections and other insults are coordinated by a complex network of cytokines that mediate cell-to-cell signaling. By contrast, the corresponding heat shock and innate immune responses in Drosophila have usually been regarded as cell-autonomous processes. However, in this issue of Developmental Cell, Agaisse et al. (2003) show that cytokines do play a role in mediating an acute phase response in this organism. Agaisse et al. (2003) have studied the induction of a novel kind of stress reaction, which was recently discovered in
Monica L. Vetter Department of Neurobiology and Anatomy University of Utah Salt Lake City, Utah 84132 Selected Reading Amir, R.E., Van den Veyver, I.B., Wan, M., Tran, C.Q., Francke, U., and Zoghbi, H.Y. (1999). Nat. Genet. 23, 185–188. Chen, R.Z., Akbarian, S., Tudor, M., and Jaenisch, R. (2001). Nat. Genet. 27, 327–331. Davis, R.L., and Turner, D.L. (2001). Oncogene 20, 8342–8357. Davis, R.L., Turner, D.L., Evans, L.M., and Kirschner, M.W. (2001). Dev. Cell 1, 553–565. Guy, J., Hendrich, B., Holmes, M., Martin, J.E., and Bird, A. (2001). Nat. Genet. 27, 322–326. Kao, H.Y., Ordentlich, P., Koyano-Nakagawa, N., Tang, Z., Downes, M., Kintner, C.R., Evans, R.M., and Kadesch, T. (1998). Genes Dev. 12, 2269–2277. Meehan, R.R. (2003). Semin. Cell Dev. Biol. 14, 53–65. Shahbazian, M.D., and Zoghbi, H.Y. (2002). Am. J. Hum. Genet. 71, 1259–1272. Stancheva, I., Collins, A.L., Van den Veyver, I.B., Zoghbi, H., and Meehan, R.R. (2003). Mol. Cell 12, 425–435. Wade, P.A. (2001). Bioessays 23, 1131–1137.
Previews
Methylation Gets SMRT: Functional Insights into Rett Syndrome Rett syndrome, a neurodevelopmental disorder, is caused by mutations in the methyl-CpG binding protein MeCP2. A recent report demonstrates that MeCP2 cooperates with the SMRT corepressor complex to inhibit expression of a hairy-related repressor during primary neurogenesis in Xenopus, and that this can be modulated by Notch signaling. Rett syndrome mutations that disrupt interaction with the SMRT corepressor complex also prevent regulation of MeCP2 by activated Notch. “Well-timed silence hath more eloquence than speech.” —Martin Farquhar Tupper (1810–1889) During animal development, well-timed repression of gene expression can be as important as activation. DNA methylation is one mechanism for maintaining genes in a transcriptionally silent state (Meehan, 2003). DNA methylation occurs at cytosine residues within the context of a CpG dinucleotide site, allowing these sites to be recognized by methyl-CpG binding proteins, which function as transcriptional repressors (Wade, 2001). MeCP2 was the first methyl-CpG binding protein to be purified and sequenced, and this subsequently led to the identification of other family members. It is tempting to think of methyl-CpG binding proteins as global repressors of transcription at all methylated sites. This idea was first called into question when it was determined that mutations in the human MeCP2 gene result in Rett syndrome, which is an X-linked neurological disorder characterized by reduced brain growth, regressive effects on motor, speech, and cognitive development, and stereotyped hand-wringing behavior affecting 1/10,000–1/15,000 female children (Amir et al., 1999). Disruption of the MeCP2 gene in mouse produces a Rett syndrome-like phenotype including the hallmark neurological and behavioral features (Chen et al., 2001; Guy et al., 2001). Most strikingly, an identical phenotype is obtained when MeCP2 is selectively deleted embryonically in the CNS, arguing that MeCP2 is primarily required in the nervous system (Chen et al., 2001). In fact, MeCP2 is expressed at high levels in neurons but not glia, and may play a role in regulating neuronal maturation and dendrite development (Shahbazian and Zoghbi, 2002). How does MeCP2 function during neural development and are there clues as to how it may regulate gene expression? The MeCP2 protein has a domain responsible for recognizing a single methylated CpG dinucleotide, as well as a transcriptional repression domain (TRD) that interacts with a corepressor complex containing Sin3A and histone deacetylase (HDAC) activity, arguing that MeCP2 binding to methylated promoter regions
causes changes in histone acetylation and chromatin structure (Wade, 2001). But does MeCP2 confer “welltimed silence,” or is it a more constitutive repressor of gene expression in the nervous system? In addition, are there critical genes during nervous system development that are regulated by MeCP2? In the August issue of Molecular Cell, Meehan and colleagues show that reduction of MeCP2 expression in Xenopus laevis inhibits primary neurogenesis, providing an experimental model for determining how xMeCP2 regulates nervous system development (Stancheva et al., 2003). They examined the expression of genes known to be essential for primary neurogenesis and showed that xMeCP2 is involved in inhibiting expression of xHairy2a, a known target of the Notch/Delta signaling pathway (Davis et al., 2001). Meehan and colleagues show that xHairy2a expression is increased by depletion of xMeCP2, and that xHairy2a functions as a repressor to inhibit primary neurogenesis. During development, Notch target genes, such as xHairy2a, are repressed in the absence of Notch signaling by Suppressor of Hairless [Su(H)] or its mammalian homolog C promoter binding factor 1 (CBF1), which bind to the promoter regions of Notch-regulated genes and recruit a corepressor complex that includes Sin3A, HDAC, and SMRT (Silencing Mediator of Retinoic Acid and Thyroid Receptors; Davis and Turner, 2001; Kao et al., 1998). Upon Notch activation by a ligand such as Delta, the intracellular domain of Notch is cleaved, translocates to the nucleus, and displaces the corepressor complex from Su(H)/ CBF1, converting it from a repressor to a transcriptional activator. They elegantly showed that xMeCP2 interacts with the SMRT corepressor complex, including CBF1, through the TRD and that this interaction depends upon Sin3A (Stancheva et al., 2003). Using selective inhibitors, Meehan and colleagues provide evidence that methylation and HDAC activity act synergistically to repress xHairy2a expression. Furthermore, chromatin immunoprecipitation (ChIP) analysis revealed that binding of xMeCP2 to CpG-rich regions of the xHairy2a promoter could be displaced by expression of the activated Notch intracellular domain (NICD). Interestingly, this is also dependent upon Sin3A, because in Sin3A-depleted embryos, expression of NICD could no longer displace xMeCP2 from the xHairy2a promoter but could still interfere with binding of the SMRT complex. This argues that normally there is Sin3A-mediated coupling between xMeCP2 and the SMRT complex that allows xMeCP2 binding to be regulated by Notch. The finding that xMeCP2 binding to the xHairy2a promoter can be modulated by the Notch signaling pathway is particularly significant because it provides compelling evidence that methyl-CpG binding proteins may indeed be able to confer “well-timed silence” by responding to developmentally relevant signals. A beautiful aspect of this work is that Meehan and colleagues used the Xenopus assay system to explore the functional significance of two common mutations in human Rett syndrome: R168X, which truncates MeCP2
Developmental Cell 360
after the methyl-CpG binding domain (MBD) and eliminates the TRD; and R306C, which is a missense mutation in the TRD. R168X lacks the TRD and does not interact with components of the SMRT complex, while R306C shows weaker interaction. R168X retains binding to CpG-rich regions of the xHairy2a promoter, but Meehan and colleagues show that it was unable to be displaced by expression of NICD. This further argues that xMeCP2 must be associated with the SMRT complex in order for its binding activity to be modulated by Notch signaling. Although modulation of xMeCP2 binding by Notch signaling depends upon interaction with Sin3A and the SMRT corepressor complex, it remains to be determined whether all aspects of MeCP2 function will depend upon this interaction. This work advances our understanding of MeCP2 as a regulator of gene expression during neural development but raises many intriguing questions. During Xenopus primary neurogenesis, xMeCP2 clearly modulates the expression of xHairy2a, which has significant effects on neurogenesis. An obvious question is whether hairyrelated genes are misregulated in Rett syndrome or in mouse models of the disease, and whether this explains all or part of the mutant phenotype. In humans, the Rett syndrome phenotype becomes apparent postnatally beginning at 6–18 months of age, arguing that early aspects of nervous system development may be less dependent upon MeCP2 activity than later aspects (Shahbazian and Zoghbi, 2002). This would suggest that other gene targets for xMeCP2 may exist that play a significant role in postmitotic neurons. The relationship between Notch signaling and xMeCP2 binding to the xHairy2a promoter is particularly striking because it shows that methyl-CpG binding proteins can be regulated by extrinsic signaling during development. It will be interesting to determine whether other Notch target genes are also coregulated by
MeCP2 together with the SMRT corepressor complex. The observation that other Notch target genes, such as the Enhancer of Split-related (ESR) genes, were not affected by depletion of xMeCP2 argues that repression by MeCP2 is not a uniform feature of Notch-responsive promoters (Stancheva et al., 2003). However, Notch can regulate aspects of neuronal maturation, including dendritic branching, which is reduced in neurons affected by Rett syndrome, leaving open the possibility that subtle alterations in Notch signaling may contribute to the Rett syndrome phenotype. Clearly much remains to be learned about this tragic, debilitating disease.
A Cytokine in the Drosophila Stress Response
Drosophila (Ekengren et al., 2001; Ekengren and Hultmark, 2001). Under severe stress, flies secrete a family of eight related peptides of uncertain function, the Turandot (Tot) peptides, which accumulate in their circulating body fluid (the hemolymph). At least one of them, TotA, is produced in the fat body (Ekengren et al., 2001), a tissue with a function similar to that of the mammalian liver. This humoral stress response differs in many ways from the classical heat shock and antimicrobial responses. It can be triggered by many cues, including infection and heat shock as well as tissue damage, dehydration, and other severe treatments of the animal (Ekengren et al., 2001; Ekengren and Hultmark, 2001). Agaisse et al. (2003) have now found that this response is mediated by a cytokine produced in the blood cells (the hemocytes). In a microarray screen for genes that are regulated by the JAK/STAT pathway in response to bacterial infection, Agaisse et al. (2003) found that the TotA gene is upregulated in hopTum (also called TumL) mutant flies, which carry a constitutively active form of the single Drosophila JAK homolog, Hopscotch. Conversely, the induction of TotA by a septic injury is abolished in hop
The fruit fly, Drosophila melanogaster, has become a popular tool for studying immediate reactions to environmental hazards, such as the heat shock and innate immune responses. In mammals, protective responses to infections and other insults are coordinated by a complex network of cytokines that mediate cell-to-cell signaling. By contrast, the corresponding heat shock and innate immune responses in Drosophila have usually been regarded as cell-autonomous processes. However, in this issue of Developmental Cell, Agaisse et al. (2003) show that cytokines do play a role in mediating an acute phase response in this organism. Agaisse et al. (2003) have studied the induction of a novel kind of stress reaction, which was recently discovered in
Monica L. Vetter Department of Neurobiology and Anatomy University of Utah Salt Lake City, Utah 84132 Selected Reading Amir, R.E., Van den Veyver, I.B., Wan, M., Tran, C.Q., Francke, U., and Zoghbi, H.Y. (1999). Nat. Genet. 23, 185–188. Chen, R.Z., Akbarian, S., Tudor, M., and Jaenisch, R. (2001). Nat. Genet. 27, 327–331. Davis, R.L., and Turner, D.L. (2001). Oncogene 20, 8342–8357. Davis, R.L., Turner, D.L., Evans, L.M., and Kirschner, M.W. (2001). Dev. Cell 1, 553–565. Guy, J., Hendrich, B., Holmes, M., Martin, J.E., and Bird, A. (2001). Nat. Genet. 27, 322–326. Kao, H.Y., Ordentlich, P., Koyano-Nakagawa, N., Tang, Z., Downes, M., Kintner, C.R., Evans, R.M., and Kadesch, T. (1998). Genes Dev. 12, 2269–2277. Meehan, R.R. (2003). Semin. Cell Dev. Biol. 14, 53–65. Shahbazian, M.D., and Zoghbi, H.Y. (2002). Am. J. Hum. Genet. 71, 1259–1272. Stancheva, I., Collins, A.L., Van den Veyver, I.B., Zoghbi, H., and Meehan, R.R. (2003). Mol. Cell 12, 425–435. Wade, P.A. (2001). Bioessays 23, 1131–1137.