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The RITS Complex—A Direct Link between Small RNA and Heterochromatin
Molecular Cell 304
Selected Reading
Shen, H., Kan, J.L.C., and Green, M.R. (2004). Mol. Cell 13, this issue, 367–376.
Cartegni, L., and Krainer, A.R. (2003). Nat. Struct. Biol. 10, 120–125. Graveley, B.R. (2000). RNA 6, 1197–1211.
Valca´rcel, J., Gaur, R.K., Singh, R., and Green, M.R. (1996). Science 273, 1706–1709.
Hertel, K.J., and Maniatis, T. (1999). Proc. Natl. Acad. Sci. USA 96, 2651–2655.
Wu, J.Y., and Maniatis, T. (1993). Cell 75, 1061–1070.
Jurica, M.S., and Moore, M.J. (2003). Mol. Cell 12, 5–14.
Zuo, P., and Maniatis, T. (1996). Genes Dev. 10, 1356–1368.
Wang, Z., Hoffmann, H.M., and Grabowski, P.J. (1995). RNA 1, 21–35.
Philipps, D., Celotto, A.M., Wang, Q.Q., Tarng, R.S., and Graveley, B.R. (2003). Nucleic Acids Res. 31, 6502–6508.
The RITS Complex—A Direct Link between Small RNA and Heterochromatin
In a recent report, Moazed, Grewal, and colleagues (Verdel et al., 2004) characterize the RITS (RNAinduced initiation of transcriptional silencing) protein complex in fission yeast. They provide a sought-for link between the small RNA produced by the RNA interference machinery and heterochromatin components, suggesting a mechanism for how heterochromatin formation can be targeted in trans to specific chromosomal regions. RNA interference (RNAi) was initially thought of as a purely posttranscriptional process in which small (21–23 nucleotide) inhibitory RNA molecules (siRNA) trigger degradation of homologous mRNA by the RNase IIIlike enzyme, Dicer, with the help of the RNA induced silencing complex (RISC) containing Argonaute (Fire et al., 1998; Zamore et al., 2000; Bernstein et al., 2001). However, in 2002 it was demonstrated that centromeric silencing in fission yeast (Schizosaccharomyces pombe) requires components in the RNAi pathway and therefore in this case RNAi also acts at the transcriptional level to silence genes (Volpe et al., 2002). Subsequently, similar links between RNAi and chromatin modifications have also been established in plants (Arabidopsis)where silencing of genes and retrotransposons depend on RNAi (Zilberman et al., 2003), in Drosophila (Pal-Bhadra et al., 2002), and in the ciliate (Tetrahymena) where RNAi was shown to direct chromatin modifications and DNA elimination (Mochizuki et al., 2002). Hence it seems as though RNAi coupled to transcriptional regulation by chromatin modification could be quite widespread in eukaryotes. In fission yeast, Dicer (Dcr1), the RISC component Argonaute (Ago1), an RNA-dependent polymerase (Rdp1), and additional uncharacterized Csp gene products (centromere suppressor of position effect) (Ekwall et al., 1999) have been shown to be required for heterochromatin formation and silencing of centromeres (Volpe et al., 2002, 2003). In these mutants, long unprocessed transcripts from both strands of the centromere dg and dh sequences accumulate. The long transcripts are normally short lived since the RNAi pathway rapidly processes them to produce small siRNA. The siRNA produced by Dicer then somehow leads to formation of
heterochromatin, i.e., methylation of histone H3 at K9 by the methyl-transferase Clr4 and subsequent binding of the heterochromatin protein Swi6 over the dg and dh repeats. Conversely, in RNAi pathway mutants both H3 K9 methylation and Swi6 binding are lost. Since the role of Swi6 in centromere function is to mediate cohesin binding, the RNAi pathway mutants display a typical chromosome segregation defect, i.e., lagging chromosomes in anaphase and sensitivity to microtubule inhibitors (Provost et al., 2002; Volpe et al., 2003). Another role of RNAi in fission yeast is to direct localized repressive chromatin formation to genes in euchromatin. This was elegantly shown by expressing an inverted repeat RNA, which can form short hairpin RNA (shRNA) capable of targeting heterochromatin formation and cohesin binding in trans (Schramke and Allshire, 2003). The gene becomes methylated at H3 K9 and Swi6 protein binds locally, thus silencing the gene homologous to the shRNA. This type of RNAi-mediated gene repression is dependent on Ago1, Dcr1, Rdp1, Clr4, and Swi6 and is normally used to silence meiotic genes in mitotic fission yeast cells via nearby retrotransposable long terminal repeats (LTR) elements (Schramke and Allshire, 2003). One of the biggest challenges in the heterochromatin/ RNAi field is to understand how the siRNA produced by Dicer is able to direct heterochromatin formation in trans. In a recent issue of Science, Moazed and Grewal’s groups address this issue by purifying and characterizing a new protein complex called RITS (RNA-induced initiation of transcriptional silencing) (Verdel et al., 2004). RITS directly links the siRNA produced by Dicer to heterochromatin because it contains both a previously known chromodomain protein Chp1 that binds centromeres (Partridge et al., 2002) and the S. pombe Argonaute homolog, Ago1. The RITS also contains a previously uncharacterized protein (Tas3). In posttranscriptional RNAi silencing, Argonaute has an important role in the RISC complex, whereby it binds siRNA, thus mediating the contact with complementary mRNA required for its destruction by Dicer. The authors propose that RITS could play an analogous role by mediating targeting of specific regions of chromatin for heterochromatin formation. They showed that the siRNA molecules copurified with the RITS complex are homologous to centromeric dg and dh sequences. Furthermore, in Dicer mutants these siRNA molecules are absent from an inactive form of the RITS complex. In addition, they showed by chromatin immunoprecipitation that two RITS proteins, Ago1 and Chp1, bind centromeres, and that all three RITS proteins are required for methylation
Previews 305
Figure 1. A Schematic Representation of the Proposed Model for Initiation of Heterochromatin Assembly by RITS From Verdel et al. (2004). See text for details.
of H3 K9 and Swi6 binding to centromeric chromatin. Thus a model is suggested in which RITS mediates heterochromatin formation by RNA-RNA interaction with nascent RNA being transcribed from the dg-dh region of the centromere or by RNA-DNA interactions with dg and dh DNA (see Figure 1). It is currently unknown which of these targeting mechanisms is used. Binding of Chp1 chromodomain to methylated H3 K9 (Partridge et al., 2002) may further stabilize the interactions of RITS with chromatin. Whether this mechanism, involving RITS, is also required for repression of LTR-mediated silencing of meiotic genes remains to be tested. The siRNA of RITS complexes did hybridize to dg-dh but not to LTR sequences in a Southern blot, but as pointed out by the authors this could be due to relatively low abundance of siRNA homologous to LTR in comparison to dg-dh repeats (Verdel et al., 2004). Since Ago1 is required for LTR-mediated silencing (Schramke and Allshire, 2003), it seems plausible that RITS would be required for LTR silencing. It will also be important to elucidate exactly how methylation of H3K9 via Clr4 methyltransferase is directed and whether the function of RITS complexes is conserved in other organisms. Perhaps the biggest paradox to be explained is how dg-dh promoters escape silencing from the repressive chromatin structures that their transcripts help to create. One attractive idea is that dg-dh transcription is feedback regulated and only occurs at stages during the cell cycle when heterochromatin structure is temporally compromised during DNA replication or in other situations where heterochromatin integrity is damaged. Karl Ekwall Karolinska Institutet Department of Biosciences University College Sodertorn Department of Natural Sciences S-141 89, Huddinge Sweden
Selected Reading Bernstein, E., Caudy, A.A., Hammond, S.M., and Hannon, G.J. (2001). Nature 409, 363–366. Ekwall, K., Cranston, G., and Allshire, R.C. (1999). Genetics 153, 1153–1169. Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, C.C. (1998). Nature 391, 806–811. Mochizuki, K., Fine, N.A., Fujisawa, T., and Gorovsky, M.A. (2002). Cell 110, 689–699. Pal-Bhadra, M., Bhadra, U., and Birchler, J.A. (2002). Mol. Cell 9, 315–327. Partridge, J.F., Scott, K.S., Bannister, A.J., Kouzarides, T., and Allshire, R.C. (2002). Curr. Biol. 12, 1652–1660. Provost, P., Silverstein, R.A., Dishart, D., Walfridsson, J., Djupedal, I., Kniola, B., Wright, A., Samuelsson, B., Radmark, O., and Ekwall, K. (2002). Proc. Natl. Acad. Sci. USA 99, 16648–16653. Schramke, V., and Allshire, R. (2003). Science 301, 1069–1074. Verdel, A., Jia, S., Gerber, S., Sugiyama, T., Gygi, S., Grewal, S.I., and Moazed, D. (2004). Science. published online 2 January (Sciencexpress). Volpe, T.A., Kidner, C., Hall, I.M., Teng, G., Grewal, S.I., and Martienssen, R.A. (2002). Science 297, 1833–1837. Volpe, T., Schramke, V., Hamilton, G.L., White, S.A., Teng, G., Martienssen, R.A., and Allshire, R.C. (2003). Chromosome Res. 11, 137–146. Zamore, P.D., Tuschl, T., Sharp, P.A., and Bartel, D.P. (2000). Cell 101, 25–33. Zilberman, D., Cao, X., and Jacobsen, S.E. (2003). Science 299, 716–719.
Selected Reading
Shen, H., Kan, J.L.C., and Green, M.R. (2004). Mol. Cell 13, this issue, 367–376.
Cartegni, L., and Krainer, A.R. (2003). Nat. Struct. Biol. 10, 120–125. Graveley, B.R. (2000). RNA 6, 1197–1211.
Valca´rcel, J., Gaur, R.K., Singh, R., and Green, M.R. (1996). Science 273, 1706–1709.
Hertel, K.J., and Maniatis, T. (1999). Proc. Natl. Acad. Sci. USA 96, 2651–2655.
Wu, J.Y., and Maniatis, T. (1993). Cell 75, 1061–1070.
Jurica, M.S., and Moore, M.J. (2003). Mol. Cell 12, 5–14.
Zuo, P., and Maniatis, T. (1996). Genes Dev. 10, 1356–1368.
Wang, Z., Hoffmann, H.M., and Grabowski, P.J. (1995). RNA 1, 21–35.
Philipps, D., Celotto, A.M., Wang, Q.Q., Tarng, R.S., and Graveley, B.R. (2003). Nucleic Acids Res. 31, 6502–6508.
The RITS Complex—A Direct Link between Small RNA and Heterochromatin
In a recent report, Moazed, Grewal, and colleagues (Verdel et al., 2004) characterize the RITS (RNAinduced initiation of transcriptional silencing) protein complex in fission yeast. They provide a sought-for link between the small RNA produced by the RNA interference machinery and heterochromatin components, suggesting a mechanism for how heterochromatin formation can be targeted in trans to specific chromosomal regions. RNA interference (RNAi) was initially thought of as a purely posttranscriptional process in which small (21–23 nucleotide) inhibitory RNA molecules (siRNA) trigger degradation of homologous mRNA by the RNase IIIlike enzyme, Dicer, with the help of the RNA induced silencing complex (RISC) containing Argonaute (Fire et al., 1998; Zamore et al., 2000; Bernstein et al., 2001). However, in 2002 it was demonstrated that centromeric silencing in fission yeast (Schizosaccharomyces pombe) requires components in the RNAi pathway and therefore in this case RNAi also acts at the transcriptional level to silence genes (Volpe et al., 2002). Subsequently, similar links between RNAi and chromatin modifications have also been established in plants (Arabidopsis)where silencing of genes and retrotransposons depend on RNAi (Zilberman et al., 2003), in Drosophila (Pal-Bhadra et al., 2002), and in the ciliate (Tetrahymena) where RNAi was shown to direct chromatin modifications and DNA elimination (Mochizuki et al., 2002). Hence it seems as though RNAi coupled to transcriptional regulation by chromatin modification could be quite widespread in eukaryotes. In fission yeast, Dicer (Dcr1), the RISC component Argonaute (Ago1), an RNA-dependent polymerase (Rdp1), and additional uncharacterized Csp gene products (centromere suppressor of position effect) (Ekwall et al., 1999) have been shown to be required for heterochromatin formation and silencing of centromeres (Volpe et al., 2002, 2003). In these mutants, long unprocessed transcripts from both strands of the centromere dg and dh sequences accumulate. The long transcripts are normally short lived since the RNAi pathway rapidly processes them to produce small siRNA. The siRNA produced by Dicer then somehow leads to formation of
heterochromatin, i.e., methylation of histone H3 at K9 by the methyl-transferase Clr4 and subsequent binding of the heterochromatin protein Swi6 over the dg and dh repeats. Conversely, in RNAi pathway mutants both H3 K9 methylation and Swi6 binding are lost. Since the role of Swi6 in centromere function is to mediate cohesin binding, the RNAi pathway mutants display a typical chromosome segregation defect, i.e., lagging chromosomes in anaphase and sensitivity to microtubule inhibitors (Provost et al., 2002; Volpe et al., 2003). Another role of RNAi in fission yeast is to direct localized repressive chromatin formation to genes in euchromatin. This was elegantly shown by expressing an inverted repeat RNA, which can form short hairpin RNA (shRNA) capable of targeting heterochromatin formation and cohesin binding in trans (Schramke and Allshire, 2003). The gene becomes methylated at H3 K9 and Swi6 protein binds locally, thus silencing the gene homologous to the shRNA. This type of RNAi-mediated gene repression is dependent on Ago1, Dcr1, Rdp1, Clr4, and Swi6 and is normally used to silence meiotic genes in mitotic fission yeast cells via nearby retrotransposable long terminal repeats (LTR) elements (Schramke and Allshire, 2003). One of the biggest challenges in the heterochromatin/ RNAi field is to understand how the siRNA produced by Dicer is able to direct heterochromatin formation in trans. In a recent issue of Science, Moazed and Grewal’s groups address this issue by purifying and characterizing a new protein complex called RITS (RNA-induced initiation of transcriptional silencing) (Verdel et al., 2004). RITS directly links the siRNA produced by Dicer to heterochromatin because it contains both a previously known chromodomain protein Chp1 that binds centromeres (Partridge et al., 2002) and the S. pombe Argonaute homolog, Ago1. The RITS also contains a previously uncharacterized protein (Tas3). In posttranscriptional RNAi silencing, Argonaute has an important role in the RISC complex, whereby it binds siRNA, thus mediating the contact with complementary mRNA required for its destruction by Dicer. The authors propose that RITS could play an analogous role by mediating targeting of specific regions of chromatin for heterochromatin formation. They showed that the siRNA molecules copurified with the RITS complex are homologous to centromeric dg and dh sequences. Furthermore, in Dicer mutants these siRNA molecules are absent from an inactive form of the RITS complex. In addition, they showed by chromatin immunoprecipitation that two RITS proteins, Ago1 and Chp1, bind centromeres, and that all three RITS proteins are required for methylation
Previews 305
Figure 1. A Schematic Representation of the Proposed Model for Initiation of Heterochromatin Assembly by RITS From Verdel et al. (2004). See text for details.
of H3 K9 and Swi6 binding to centromeric chromatin. Thus a model is suggested in which RITS mediates heterochromatin formation by RNA-RNA interaction with nascent RNA being transcribed from the dg-dh region of the centromere or by RNA-DNA interactions with dg and dh DNA (see Figure 1). It is currently unknown which of these targeting mechanisms is used. Binding of Chp1 chromodomain to methylated H3 K9 (Partridge et al., 2002) may further stabilize the interactions of RITS with chromatin. Whether this mechanism, involving RITS, is also required for repression of LTR-mediated silencing of meiotic genes remains to be tested. The siRNA of RITS complexes did hybridize to dg-dh but not to LTR sequences in a Southern blot, but as pointed out by the authors this could be due to relatively low abundance of siRNA homologous to LTR in comparison to dg-dh repeats (Verdel et al., 2004). Since Ago1 is required for LTR-mediated silencing (Schramke and Allshire, 2003), it seems plausible that RITS would be required for LTR silencing. It will also be important to elucidate exactly how methylation of H3K9 via Clr4 methyltransferase is directed and whether the function of RITS complexes is conserved in other organisms. Perhaps the biggest paradox to be explained is how dg-dh promoters escape silencing from the repressive chromatin structures that their transcripts help to create. One attractive idea is that dg-dh transcription is feedback regulated and only occurs at stages during the cell cycle when heterochromatin structure is temporally compromised during DNA replication or in other situations where heterochromatin integrity is damaged. Karl Ekwall Karolinska Institutet Department of Biosciences University College Sodertorn Department of Natural Sciences S-141 89, Huddinge Sweden
Selected Reading Bernstein, E., Caudy, A.A., Hammond, S.M., and Hannon, G.J. (2001). Nature 409, 363–366. Ekwall, K., Cranston, G., and Allshire, R.C. (1999). Genetics 153, 1153–1169. Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, C.C. (1998). Nature 391, 806–811. Mochizuki, K., Fine, N.A., Fujisawa, T., and Gorovsky, M.A. (2002). Cell 110, 689–699. Pal-Bhadra, M., Bhadra, U., and Birchler, J.A. (2002). Mol. Cell 9, 315–327. Partridge, J.F., Scott, K.S., Bannister, A.J., Kouzarides, T., and Allshire, R.C. (2002). Curr. Biol. 12, 1652–1660. Provost, P., Silverstein, R.A., Dishart, D., Walfridsson, J., Djupedal, I., Kniola, B., Wright, A., Samuelsson, B., Radmark, O., and Ekwall, K. (2002). Proc. Natl. Acad. Sci. USA 99, 16648–16653. Schramke, V., and Allshire, R. (2003). Science 301, 1069–1074. Verdel, A., Jia, S., Gerber, S., Sugiyama, T., Gygi, S., Grewal, S.I., and Moazed, D. (2004). Science. published online 2 January (Sciencexpress). Volpe, T.A., Kidner, C., Hall, I.M., Teng, G., Grewal, S.I., and Martienssen, R.A. (2002). Science 297, 1833–1837. Volpe, T., Schramke, V., Hamilton, G.L., White, S.A., Teng, G., Martienssen, R.A., and Allshire, R.C. (2003). Chromosome Res. 11, 137–146. Zamore, P.D., Tuschl, T., Sharp, P.A., and Bartel, D.P. (2000). Cell 101, 25–33. Zilberman, D., Cao, X., and Jacobsen, S.E. (2003). Science 299, 716–719.