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The retinoblastoma protein – a bridge to heterochromatin
trends in plant science Correspondence
The retinoblastoma protein – a bridge to heterochromatin The retinoblastoma protein (pRb) is a master regulator of the cell cycle, cell differentiation and apoptosis1. In humans, pRb and its related proteins, p107 and p130, are potent inhibitors
of zeste, possesses a putative Rb-binding motif (LICSD), which is embedded within an acidic region (Fig. 1). CLF encodes a SETdomain protein that negatively regulates the floral homeotic gene AGAMOUS (Ref. 8). Heterochromatin protein 1 (HP1), which contains a chromo domain, a motif found in a large superfamily of proteins involved in chromatin organization and gene regulation, was of particular interest6,7. Structural
AtClf
SVVGRRRIYYDQTGGEA L I C S D SEEEAIDDEEEKRD
HP1-chromo domain structure
MoMod1 MoMod2 Hs Gg Dm
(Fig. 3). Several previous findings provide further support for this proposition. (1) pRb can be found at the heterochromatin– euchromatin boundary12. (2) Drosophila HP1 is a modifier of heterochromatin-induced position–effect variegation (PEV) – a phenomenon whereby euchromatic genes become silenced when brought into close proximity with heterochromatin13. (3) The Rb-associated protein E2F is involved in PEV (Ref. 14).
β1
β2
β3
α
MGKKQNKKKVEEVLEEEEEEYVVEKVLDRRVVKGKVEYLLKWKGFSDEDNTWEPEEN MGKKQNGKSKKVEEAEPEEF VVEKVLDRRVVNGKVEYFLKWKGFTDADNTWEPEEN MGKKTKRTADSSSSEDEEEY VVEKVLDRRVVKGQVEYLLKWKGFSEEHNTWEPEKN MGKKQNGKGKKVEEAEPEEF VVEKVLDRRVVNGKVEYYLKWKGFTDADNTWEPEEN MGKKQNGKSKKVEEAEPEEF VVEKVLDRRVVNGKVEYFLKWKGFTDADNTWEPEEN
L L L L L
D D D D D
C C C C C
P P P P P
D E E E E
Rb-binding motif
LIAEF LIEDF LISEF LIEAF LIEAF
Trends in Plant Science
Fig. 1. Chromatin-associated proteins posess an Rb-binding motif. Putative Rb-binding motif in the Arabidopsis SET-domain CURLY LEAF protein (AtClf) and in the chromo-domain of HP1 proteins from a variety of eukaryotes. The location of the chromo-domain secondary structures9 is shown at the top. b1, b2 and b3 indicate the three antiparallel b-sheet strands, and a indicates the C-terminal a-helix. The putative Rb-binding domain is boxed. MoMod1, mouse modifier 1; MoMod2, mouse modifier 2; Hs, Homo sapiens; Gg, Gallus gallus; Dm, Drosophila melanogaster.
of gene transcription mediated by the E2F family of transcription factors2. Interaction of pRb with E2F results in the formation of a transcription–repressive complex that constrains expression of E2F-target genes and, consequently, the progression of cells into S phase. The pRb–E2F repressive complex can act by sequestering transcription activators, or by recruiting histone deacetylases3 or repressor proteins4. Here we present data that suggests the operation of a novel transcriptional repression mechanism involving the interaction of pRb with heterochromatin-associated proteins. The capability of pRb to affect higher-order chromatin structure is suggested by the finding that chromatin from Rb2/2 cells is more susceptible to micrococcal nuclease than chromatin from Rb1/1 cells5. To study how pRb might bring about a more ‘closed’ chromatin structure we have examined the potential of pRb to interact with chromatinassociated proteins. We first focused on the polycomb group (PcG) and chromo-domain proteins, which are implicated in gene silencing via stabilization of heterochromatin6,7. A search of the GenBank Database yielded several chromatin-associated proteins from a variety of eukaryotes, each carrying a putative Rb-binding motif. For example, the Arabidopsis polycomb gene CURLY LEAF (CLF), a homologue of the Drosophila gene Enhancer
resolution of the chromo domain by nuclear magnetic resonance (NMR) spectroscopy revealed that it consists of three-stranded antiparallel b-sheets, which fold against a Cterminal a-helix9. The HP1 group of proteins from a variety of eukaryotes was found to contain a conventional Rb-binding motif located at the loop between the b3 sheet and the a-helix structure (Fig. 1). This loop is a variable region among the different chromo domain proteins, which might not affect its three-dimensional structure9. The ability of pRb proteins to interact with HP1 and CLF proteins was then tested by examining the binding of these proteins to members of the Rb protein family, maize Rb (ZmRB; Ref. 10) and human pRb1. As demonstrated in vitro by the GST pull-down assay (data not shown) and by the yeast twohybrid system (Fig. 2), both plant and human Rb proteins bind to CLF and HP1g proteins. Binding is dependent on the so-called Rb ‘pocket’ region inasmuch as human Rb lacking the pocket B domain (HuRbD22; Ref. 11) failed to interact with CLF and HP1g proteins. Taken together, these results point to the capacity for pRb to mediate gene silencing by bringing euchromatic genes, such as E2Ftarget genes, into the proximity of condensed, transcriptionally inactive heterochromatin via interaction with chromatin-associated proteins
1360 - 1385/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.
ZmRB CLF ZmRB/CLF HuRB/CLF HURB∆22/CLF
HuRB HuRB∆22 ZmRB HP1g
ZmRB/HP1g HuRB/HP1g HuRB∆22/HP1g
HuRB HuRB∆22
Fig. 2. Plant and human retinoblastoma (Rb) proteins bind to CLF and HP1g proteins in yeast two-hybrid assays (MATCHMAKER, Clontech). The HP1Hs-gamma cDNA (referred to as HP1g; Accession no. U26312), isolated from a human cDNA library by PCR, and CURLY LEAF (CLF) were subcloned into both pGEX-2TK and yeast twohybrid vectors. Interaction in yeast is demonstrated by the activity of bglactosidase (blue colonies). HuRBD22, which lacks the ‘pocket’ B domain, failed to interact with CLF (HuRBD22/CLF) and HP1g (HuRBD22/HP1g).
June 2000, Vol. 5, No. 6
239
trends in plant science Correspondence
Heterochromatin
Heterochromatin 5
Euchromatin CLF Rb DP E2F
Rb
CLF
6
CLF
CLF
7
8
E2F-target gene 9 CLF
Chromatin-associated protein complex
Nucleosome unit 10 Trends in Plant Science
Fig. 3. The retinoblastoma protein (pRb) – a bridge to heterochromatin. E2F-target genes might be brought into the proximity of heterochromatin by pRb. The E2F–Rb pathway has been characterized in plants15,16, and this model is applicable to both plants and animals. The heterodimer DP–E2F anchors pRb into the promoter region. A direct interaction can be carried out between pRb and L-x-C-x-[E/D]-containing heterochromatin-associated proteins, such as the Arabidopsis CLF and HP1 proteins. This interaction results in the localization of a euchromatic E2F-target gene in close proximity to heterochromatin, which, in turn, induces its packaging into condensed, transcriptionally inactive chromatin.
11
12
13
Our suggested mode of action of pRb provides a link between pRb-mediated cellular differentiation–dedifferentiation and chromatin structure. Acknowledgements
We thank Yigal Avivi for critical reading and editing of the manuscript, Justin Goodrich for providing CLF cDNA clone, and Boaz Kaplan for his assistance with the yeast two-hybrid system. This work was supported by a grant from the Israel Science Foundation. Leor Williams and Gideon Grafi* Dept of Plant Sciences, The Weizmann Institute of Science, 76100 Rehovot, Israel
*Author for correspondence (tel 1972 8 934 3505; fax 1972 8 934 4181; e-mail [email protected]) References 1 Herwig, S. and Strauss, M. (1997) The retinoblastoma protein: a master regulator of cell cycle, differentiation and apoptosis. Eur. J. Biochem. 246, 581–601 2 Dyson, N. (1998) The regulation of E2F by pRBfamily proteins. Genes Dev. 12, 2245–2262 3 Brehm, A. and Kouzarides, T. (1999) Retinoblastoma protein meets chromatin. Trends Biochem. Sci. 24, 142–145 4 Meloni, A.R. et al. (1999) A mechanism for Rb/p130-mediated transcriptional repression
14
15
16
involving recruitment of the CtBP corepressor. Proc. Natl. Acad. Sci. U. S. A. 96, 9574–9579 Herrera, R.E. et al. (1996) Increased histone H1 phosphorylation and relaxed chromatin structure in Rb-deficient fibroblasts. Proc. Natl. Acad. Sci. U. S. A. 93, 11510–11515 Cavalli, G. and Paro, R. (1998) Chromo-domain proteins: linking chromatin structure to epigenetic regulation. Curr. Opin. Cell Biol. 10, 354–360 Jenuwein, T. et al. (1998) SET domain proteins modulate chromatin domains in eu- and heterochromatin. Cell. Mol. Life Sci. 54, 80–93 Goodrich, J. et al. (1997) A polycomb-group gene regulates homeotic gene expression in Arabidopsis. Nature 386, 44–51 Ball, L.J. et al. (1997) Structure of the chromatin binding (chromo) domain from mouse modifier protein 1. EMBO J. 16, 2473–2481 Grafi, G. et al. (1996) A maize cDNA encoding a member of the retinoblastoma protein family: involvement in endoreduplication. Proc. Natl. Acad. Sci. U. S. A. 93, 8962–8967 Mori, N. et al. (1990) Variable mutations of the Rb gene in small-cell lung-carcinoma. Oncogene 5, 1713–1717 Szekely, L. et al. (1991) Subcellular-localization of the retinoblastoma protein. Cell Growth Differ. 2, 287–295 Eissenberg, J.C. et al. (1990) Mutation in a heterochromatin-specific chromosomal protein is associated with suppression of positioneffect variegation in Drosophila melanogaster. Proc. Natl. Acad. Sci. U. S. A. 87, 9923–9927 Seum, C. et al. (1996) Position-effect variegation in Drosophila depends on the dose of the gene encoding the E2F transcriptional activator and cell cycle. Development 122, 1949–1956 Ramirez-Parra, E. et al. (1999) The cloning of plant E2F, a retinoblastoma-binding protein, reveals unique and conserved features with animal G1/S regulators. Nucleic Acids Res. 27, 3527–3533 Sekine, M. et al. (1999) Isolation and characterization of the E2F-like gene in plants. FEBS Lett. 460, 117–122
Letters to Trends in Plant Science Correspondence in Trends in Plant Science may address topics raised in very recent issues of the magazine, or other matters of general current interest to plant scientists. Letters should be no more than 700–800 words long with a maximum of 12 references and one small figure. Letters should be sent, together with an electronic copy on disc to the Editor (or e-mail your text to [email protected]). The decision to publish rests with the Editor, and the author(s) of any article highlighted in Correspondence will normally be invited to reply.
240
June 2000, Vol. 5, No. 6
The retinoblastoma protein – a bridge to heterochromatin The retinoblastoma protein (pRb) is a master regulator of the cell cycle, cell differentiation and apoptosis1. In humans, pRb and its related proteins, p107 and p130, are potent inhibitors
of zeste, possesses a putative Rb-binding motif (LICSD), which is embedded within an acidic region (Fig. 1). CLF encodes a SETdomain protein that negatively regulates the floral homeotic gene AGAMOUS (Ref. 8). Heterochromatin protein 1 (HP1), which contains a chromo domain, a motif found in a large superfamily of proteins involved in chromatin organization and gene regulation, was of particular interest6,7. Structural
AtClf
SVVGRRRIYYDQTGGEA L I C S D SEEEAIDDEEEKRD
HP1-chromo domain structure
MoMod1 MoMod2 Hs Gg Dm
(Fig. 3). Several previous findings provide further support for this proposition. (1) pRb can be found at the heterochromatin– euchromatin boundary12. (2) Drosophila HP1 is a modifier of heterochromatin-induced position–effect variegation (PEV) – a phenomenon whereby euchromatic genes become silenced when brought into close proximity with heterochromatin13. (3) The Rb-associated protein E2F is involved in PEV (Ref. 14).
β1
β2
β3
α
MGKKQNKKKVEEVLEEEEEEYVVEKVLDRRVVKGKVEYLLKWKGFSDEDNTWEPEEN MGKKQNGKSKKVEEAEPEEF VVEKVLDRRVVNGKVEYFLKWKGFTDADNTWEPEEN MGKKTKRTADSSSSEDEEEY VVEKVLDRRVVKGQVEYLLKWKGFSEEHNTWEPEKN MGKKQNGKGKKVEEAEPEEF VVEKVLDRRVVNGKVEYYLKWKGFTDADNTWEPEEN MGKKQNGKSKKVEEAEPEEF VVEKVLDRRVVNGKVEYFLKWKGFTDADNTWEPEEN
L L L L L
D D D D D
C C C C C
P P P P P
D E E E E
Rb-binding motif
LIAEF LIEDF LISEF LIEAF LIEAF
Trends in Plant Science
Fig. 1. Chromatin-associated proteins posess an Rb-binding motif. Putative Rb-binding motif in the Arabidopsis SET-domain CURLY LEAF protein (AtClf) and in the chromo-domain of HP1 proteins from a variety of eukaryotes. The location of the chromo-domain secondary structures9 is shown at the top. b1, b2 and b3 indicate the three antiparallel b-sheet strands, and a indicates the C-terminal a-helix. The putative Rb-binding domain is boxed. MoMod1, mouse modifier 1; MoMod2, mouse modifier 2; Hs, Homo sapiens; Gg, Gallus gallus; Dm, Drosophila melanogaster.
of gene transcription mediated by the E2F family of transcription factors2. Interaction of pRb with E2F results in the formation of a transcription–repressive complex that constrains expression of E2F-target genes and, consequently, the progression of cells into S phase. The pRb–E2F repressive complex can act by sequestering transcription activators, or by recruiting histone deacetylases3 or repressor proteins4. Here we present data that suggests the operation of a novel transcriptional repression mechanism involving the interaction of pRb with heterochromatin-associated proteins. The capability of pRb to affect higher-order chromatin structure is suggested by the finding that chromatin from Rb2/2 cells is more susceptible to micrococcal nuclease than chromatin from Rb1/1 cells5. To study how pRb might bring about a more ‘closed’ chromatin structure we have examined the potential of pRb to interact with chromatinassociated proteins. We first focused on the polycomb group (PcG) and chromo-domain proteins, which are implicated in gene silencing via stabilization of heterochromatin6,7. A search of the GenBank Database yielded several chromatin-associated proteins from a variety of eukaryotes, each carrying a putative Rb-binding motif. For example, the Arabidopsis polycomb gene CURLY LEAF (CLF), a homologue of the Drosophila gene Enhancer
resolution of the chromo domain by nuclear magnetic resonance (NMR) spectroscopy revealed that it consists of three-stranded antiparallel b-sheets, which fold against a Cterminal a-helix9. The HP1 group of proteins from a variety of eukaryotes was found to contain a conventional Rb-binding motif located at the loop between the b3 sheet and the a-helix structure (Fig. 1). This loop is a variable region among the different chromo domain proteins, which might not affect its three-dimensional structure9. The ability of pRb proteins to interact with HP1 and CLF proteins was then tested by examining the binding of these proteins to members of the Rb protein family, maize Rb (ZmRB; Ref. 10) and human pRb1. As demonstrated in vitro by the GST pull-down assay (data not shown) and by the yeast twohybrid system (Fig. 2), both plant and human Rb proteins bind to CLF and HP1g proteins. Binding is dependent on the so-called Rb ‘pocket’ region inasmuch as human Rb lacking the pocket B domain (HuRbD22; Ref. 11) failed to interact with CLF and HP1g proteins. Taken together, these results point to the capacity for pRb to mediate gene silencing by bringing euchromatic genes, such as E2Ftarget genes, into the proximity of condensed, transcriptionally inactive heterochromatin via interaction with chromatin-associated proteins
1360 - 1385/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.
ZmRB CLF ZmRB/CLF HuRB/CLF HURB∆22/CLF
HuRB HuRB∆22 ZmRB HP1g
ZmRB/HP1g HuRB/HP1g HuRB∆22/HP1g
HuRB HuRB∆22
Fig. 2. Plant and human retinoblastoma (Rb) proteins bind to CLF and HP1g proteins in yeast two-hybrid assays (MATCHMAKER, Clontech). The HP1Hs-gamma cDNA (referred to as HP1g; Accession no. U26312), isolated from a human cDNA library by PCR, and CURLY LEAF (CLF) were subcloned into both pGEX-2TK and yeast twohybrid vectors. Interaction in yeast is demonstrated by the activity of bglactosidase (blue colonies). HuRBD22, which lacks the ‘pocket’ B domain, failed to interact with CLF (HuRBD22/CLF) and HP1g (HuRBD22/HP1g).
June 2000, Vol. 5, No. 6
239
trends in plant science Correspondence
Heterochromatin
Heterochromatin 5
Euchromatin CLF Rb DP E2F
Rb
CLF
6
CLF
CLF
7
8
E2F-target gene 9 CLF
Chromatin-associated protein complex
Nucleosome unit 10 Trends in Plant Science
Fig. 3. The retinoblastoma protein (pRb) – a bridge to heterochromatin. E2F-target genes might be brought into the proximity of heterochromatin by pRb. The E2F–Rb pathway has been characterized in plants15,16, and this model is applicable to both plants and animals. The heterodimer DP–E2F anchors pRb into the promoter region. A direct interaction can be carried out between pRb and L-x-C-x-[E/D]-containing heterochromatin-associated proteins, such as the Arabidopsis CLF and HP1 proteins. This interaction results in the localization of a euchromatic E2F-target gene in close proximity to heterochromatin, which, in turn, induces its packaging into condensed, transcriptionally inactive chromatin.
11
12
13
Our suggested mode of action of pRb provides a link between pRb-mediated cellular differentiation–dedifferentiation and chromatin structure. Acknowledgements
We thank Yigal Avivi for critical reading and editing of the manuscript, Justin Goodrich for providing CLF cDNA clone, and Boaz Kaplan for his assistance with the yeast two-hybrid system. This work was supported by a grant from the Israel Science Foundation. Leor Williams and Gideon Grafi* Dept of Plant Sciences, The Weizmann Institute of Science, 76100 Rehovot, Israel
*Author for correspondence (tel 1972 8 934 3505; fax 1972 8 934 4181; e-mail [email protected]) References 1 Herwig, S. and Strauss, M. (1997) The retinoblastoma protein: a master regulator of cell cycle, differentiation and apoptosis. Eur. J. Biochem. 246, 581–601 2 Dyson, N. (1998) The regulation of E2F by pRBfamily proteins. Genes Dev. 12, 2245–2262 3 Brehm, A. and Kouzarides, T. (1999) Retinoblastoma protein meets chromatin. Trends Biochem. Sci. 24, 142–145 4 Meloni, A.R. et al. (1999) A mechanism for Rb/p130-mediated transcriptional repression
14
15
16
involving recruitment of the CtBP corepressor. Proc. Natl. Acad. Sci. U. S. A. 96, 9574–9579 Herrera, R.E. et al. (1996) Increased histone H1 phosphorylation and relaxed chromatin structure in Rb-deficient fibroblasts. Proc. Natl. Acad. Sci. U. S. A. 93, 11510–11515 Cavalli, G. and Paro, R. (1998) Chromo-domain proteins: linking chromatin structure to epigenetic regulation. Curr. Opin. Cell Biol. 10, 354–360 Jenuwein, T. et al. (1998) SET domain proteins modulate chromatin domains in eu- and heterochromatin. Cell. Mol. Life Sci. 54, 80–93 Goodrich, J. et al. (1997) A polycomb-group gene regulates homeotic gene expression in Arabidopsis. Nature 386, 44–51 Ball, L.J. et al. (1997) Structure of the chromatin binding (chromo) domain from mouse modifier protein 1. EMBO J. 16, 2473–2481 Grafi, G. et al. (1996) A maize cDNA encoding a member of the retinoblastoma protein family: involvement in endoreduplication. Proc. Natl. Acad. Sci. U. S. A. 93, 8962–8967 Mori, N. et al. (1990) Variable mutations of the Rb gene in small-cell lung-carcinoma. Oncogene 5, 1713–1717 Szekely, L. et al. (1991) Subcellular-localization of the retinoblastoma protein. Cell Growth Differ. 2, 287–295 Eissenberg, J.C. et al. (1990) Mutation in a heterochromatin-specific chromosomal protein is associated with suppression of positioneffect variegation in Drosophila melanogaster. Proc. Natl. Acad. Sci. U. S. A. 87, 9923–9927 Seum, C. et al. (1996) Position-effect variegation in Drosophila depends on the dose of the gene encoding the E2F transcriptional activator and cell cycle. Development 122, 1949–1956 Ramirez-Parra, E. et al. (1999) The cloning of plant E2F, a retinoblastoma-binding protein, reveals unique and conserved features with animal G1/S regulators. Nucleic Acids Res. 27, 3527–3533 Sekine, M. et al. (1999) Isolation and characterization of the E2F-like gene in plants. FEBS Lett. 460, 117–122
Letters to Trends in Plant Science Correspondence in Trends in Plant Science may address topics raised in very recent issues of the magazine, or other matters of general current interest to plant scientists. Letters should be no more than 700–800 words long with a maximum of 12 references and one small figure. Letters should be sent, together with an electronic copy on disc to the Editor (or e-mail your text to [email protected]). The decision to publish rests with the Editor, and the author(s) of any article highlighted in Correspondence will normally be invited to reply.
240
June 2000, Vol. 5, No. 6