Polycomb group protein complexes exchange rapidly in livingDrosophila
10.1242/dev.01950
Crossref journal-article
The Company of Biologists
Development (237)
Abstract

Fluorescence recovery after photobleaching (FRAP) microscopy was used to determine the kinetic properties of Polycomb group (PcG) proteins in whole living Drosophila organisms (embryos) and tissues (wing imaginal discs and salivary glands).PcG genes are essential genes in higher eukaryotes responsible for the maintenance of the spatially distinct repression of developmentally important regulators such as the homeotic genes. Their absence, as well as overexpression, causes transformations in the axial organization of the body. Although protein complexes have been isolated in vitro, little is known about their stability or exact mechanism of repression in vivo.We determined the translational diffusion constants of PcG proteins,dissociation constants and residence times for complexes in vivo at different developmental stages. In polytene nuclei, the rate constants suggest heterogeneity of the complexes. Computer simulations with new models for spatially distributed protein complexes were performed in systems showing both diffusion and binding equilibria, and the results compared with our experimental data. We were able to determine forward and reverse rate constants for complex formation. Complexes exchanged within a period of 1-10 minutes, more than an order of magnitude faster than the cell cycle time,ruling out models of repression in which access of transcription activators to the chromatin is limited and demonstrating that long-term repression primarily reflects mass-action chemical equilibria.

Bibliography

Ficz, G., Heintzmann, R., & Arndt-Jovin, D. J. (2005). Polycomb group protein complexes exchange rapidly in livingDrosophila. Development, 132(17), 3963–3976.

Authors 3
  1. Gabriella Ficz (first)
  2. Rainer Heintzmann (additional)
  3. Donna J. Arndt-Jovin (additional)
References 73 Referenced 106
  1. Baxter, J., Sauer, S., Peters, A., John, R., Williams, R.,Caparros, M. L., Arney, K., Otte, A., Jenuwein, T., Merkenschlager, M. and Fisher, A. G. (2004). Histone hypomethylation is an indicator of epogenetic plasticity in quiescent lymphocytes. EMBO J.23,4462-4472. (10.1038/sj.emboj.7600414)
  2. Beuchle, D., Struhl, G. and Muller, J. (2001). Polycomb group proteins and heritable silencing of Drosophila Hox genes. Development128,993-1004. (10.1242/dev.128.6.993)
  3. Bienz, M. and Muller, J. (1995). Transcriptional silencing of homeotic genes in Drosophila.BioEssays17,775-784. (10.1002/bies.950170907)
  4. Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development118,401-415. (10.1242/dev.118.2.401)
  5. Breiling, A., Turner, B., Bianchi, M. and Orlando, V.(2001). General transcription factors bind promoters repressed by Polycomb group proteins. Nature412,651-655. (10.1038/35088090)
  6. Breiling, A., Bonte, E., Ferrari, S., Becker, P. B. and Paro,R. (1999). The Drosophila Polycomb protein interacts with nucleosomal core particles in vitro via its repression domain. Mol. Cell. Biol.19,8451-8460. (10.1128/MCB.19.12.8451)
  7. Breiling, A., O'Neill, L. P., D'Eliseo, D., Turner, B. M. and Orlando, V. (2004). Epigenome changes in active and inactive polycomb-group-controlled regions. EMBO Rep.5, 976-982. (10.1038/sj.embor.7400260)
  8. Buchenau, P., Hodgson, J., Strutt, H. and Arndt-Jovin, D. J.(1998). The distribution of polycomb-group proteins during cell division and development in Drosophila embryos: impact on models for silencing. J. Cell Biol.141,469-481. (10.1083/jcb.141.2.469)
  9. Busturia, A. and Morata, G. (1988). Ectopic expression of homeotic genes caused by the elimination of the Polycomb gene in Drosophila imaginal epidermis. Development104,713-720. (10.1242/dev.104.4.713)
  10. Busturia, A., Wightman, C. D. and Sakonju, S.(1997). A silencer is required for maintenance of transcriptional repression throughout Drosophila development. Development124,4343-4350. (10.1242/dev.124.21.4343)
  11. Cao, R., Wang, L., Wang, H., Xia, L., Erdjument-Bromage, H.,Tempst, P., Jones, R. S. and Zhang, Y. (2002). Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science298,1039-1043. (10.1126/science.1076997)
  12. Capdevila, J. and Guerrero, I. (1994). Targeted expression of the signaling molecule Decapentaplegic Induces pattern duplications and growth alterations in Drosophila wings. EMBO J.13,4459-4468. (10.1002/j.1460-2075.1994.tb06768.x)
  13. Carrero, G., McDonald, D., Crawford, E., de Vries, G. and Hendzel, M. J. (2003). Using FRAP and mathematical modeling to determine the in vivo kinetics of nuclear proteins. Methods29,14-28. (10.1016/S1046-2023(02)00288-8)
  14. Catez, F., Yang, H., Tracey, K. J., Reeves, R., Misteli, T. and Bustin, M. (2004). Network of dynamic interactions between histone H1 and high-mobility-group proteins in chromatin. Mol. Cell. Biol.24,4321-4328. (10.1128/MCB.24.10.4321-4328.2004)
  15. Chan, C. S., Rastelli, L. and Pirrotta, V.(1994). A Polycomb response element in the Ubx gene that determines an epigenetically inherited state of repression. EMBO J.13,2553-2564. (10.1002/j.1460-2075.1994.tb06545.x)
  16. Cheutin, T., McNairn, A. J., Jenuwein, T., Gilbert, D. M.,Sihgh, P. B. and Misteli, T. (2003). Maintanance of stable heterochromatin domains by dynamic HP1 binding. Science299,721-725. (10.1126/science.1078572)
  17. Chiang, A., O'Connor, M. B., Paro, R., Simon, J. and Bender,W. (1995). Discrete Polycomb-binding sites in each parasegmental domain of the bithorax complex. Development121,1681-1689. (10.1242/dev.121.6.1681)
  18. Czermin, B., Melfi, R., McCabe, D., Seitz, V., Imhof, A. and Pirrotta, V. (2002). Drosophila Enhancer of Zeste/ESC complexes have a histone H3 methyltransferase activity that marks chromosomal polycomb sites. Cell111,185-196. (10.1016/S0092-8674(02)00975-3)
  19. Dellino, G. I., Schwartz, Y. B., Farkas, G., McCabe, D., Elgin,S. C. R. and Pirrotta, V. (2004). Polycomb silencing blocks transcription initiation. Mol. Cell13,887-893. (10.1016/S1097-2765(04)00128-5)
  20. Dietzel, S., Niemann, H., Brückner, B., Maurange, C. and Paro, R. (1999). The nuclear distribution of Polycomb during Drosophila melanogaster development shown with a GFP fusion protein. Chromosoma108,83-94. (10.1007/s004120050355)
  21. Dou, Y., Bowen, J., Liu, Y. and Gorovsky, M. A.(2002). Phosophorylation and an ATP-dependent process increase the dynamic exchange of H1 in chromatin. J. Cell Biol.158,1161-1170. (10.1083/jcb.200202131)
  22. Dundr, M., Hoffmann-Rohrer, U., Hu, Q., Grummt, I., Rothblum, L. I., Phair, R. D. and Misteli, T. (2002). A kinetic framework for a mammalian RNA polymerase in vivo. Science298,1623-1626. (10.1126/science.1076164)
  23. Dundr, M., Herbert, M. D., Karpova, T. S., Stanek, D., Xu, H.,Shpargel, K. B., Meier, U. T., Neugebauer, K. M., Matera, A. G. and Misteli,T. (2004). In vivo kinetics of Cajal body components. J. Cell Biol.164,831-842. (10.1083/jcb.200311121)
  24. Fauvarque, M. O., Zuber, V. and Dura, J. M.(1995). Regulation of polyhomeotic transcription may involve local changes in chromatin activity in Drosophila. Mech. Dev.52,343-355. (10.1016/0925-4773(95)00412-T)
  25. Festenstein, R., Pagakis, S. N., Hiragami, K., Lyon, D.,Verreault, A., Sekkali, B. and Kioussis, D. (2003). Modulation of heterochromatin protein 1 dynamics in primary mammalian cells. Science229,719-721. (10.1126/science.1078694)
  26. Fischle, W., Wang, Y., Jacobs, S. A., Kim, Y., Allis, C. D. and Khorasanizadeh, S. (2003). Molecular basis for the discrimination of repressive methyl-lysine marks in histone H3 by Polycomb and HP1 chromodomains. Genes Dev.17,1870-1881. (10.1101/gad.1110503)
  27. Fitzgerald, D. P. and Bender, W. (2001). Polycomb group repression reduces DNA accessibility. Mol. Cell. Biol.21,6585-6597. (10.1128/MCB.21.19.6585-6597.2001)
  28. Francis, N. J. and Kingston, R. E. (2001). Mechanisms of transcriptional memory. Nat. Rev. Mol. Cell Biol.2,409-421. (10.1038/35073039)
  29. Francis, N. J., Kingston, R. E. and Woodcock, C.(2004). Chromatin compaction by a Polycomb Group protein complex. Science306,1574-1577. (10.1126/science.1100576)
  30. Franke, A., DeCamillis, M., Zink, D., Cheng, N., Brock, H. W. and Paro, R. (1992). Polycomb and polyhomeotic are constituents of a multimeric protein complex in chromatin of Drosophila melanogaster.EMBO J.11,2941-2950. (10.1002/j.1460-2075.1992.tb05364.x)
  31. Garcia-Bellido, A., PRipoll, P. and Morata, G.(1976). Developmental compartmentalization in the dorsal mesothoracic disc of Drosophila. Dev. Biol.48,132-147. (10.1016/0012-1606(76)90052-X)
  32. Gaul, U. and Jäckle, H. (1989). Analysis of maternal effect mutant combinations elucidates regulation and function of the overlap of hunchback and Krüppel gene expression in the Drosophila blastoderm embryo. Development107,651-662.
  33. Grewal, S. I. and Moazed, D. (2003). Heterochromatin and epigenetic control of gene expression. Science301,798-802. (10.1126/science.1086887)
  34. Hernández, G., Lalioti, V., Vandekerckhove, J., Sierra,J. M. and Santarén, J. F. (2004). Identification and characterization of the expression of the translation initiation factor 4A(eIF4A) from Drosophila melanogaster. Proteomics4, 316-326. (10.1002/pmic.200300555)
  35. Houtsmuller, A. B. and Vermeulen, W. (2001). Macromolecular dynamics in living cell nuclei revealed by fluorescence redistribution after photobleaching. Histochem. Cell Biol.115,13-21. (10.1007/s004180000234)
  36. Houtsmuller, A. B., Rademakers, S., Nigg, A. L., Hoogstraten,D., Hoeijmakers, J. H. J. and Vermeulen, W. (1999). Action of DNA repair endonuclease ERCC1/XPF in living cells. Science284,958-961. (10.1126/science.284.5416.958)
  37. Kuzmichev, A., Nishioka, K., Erdjument-Bromage, H., Tempst, P. and Reinberg, D. (2002). Histone methyltransferase activity associated with a human multiprotein complex containing the Enhancer of Zeste protein. Genes Dev.16,2893-2905. (10.1101/gad.1035902)
  38. Levine, S. S., Weiss, A., Erdjument-Bromage, H., Shao, Z.,Tempst, P. and Kingston, R. E. (2002). The core of the polycomb repressive complex is compositionally and functionally conserved in flies and humans. Mol. Cell. Biol.22,6070-6078. (10.1128/MCB.22.17.6070-6078.2002)
  39. Levine, S. S., King, I. F. G. and Kingston, R. E.(2004). Division of labor in Polycomb group repression. Trends Biochem. Sci.29,478-485. (10.1016/j.tibs.2004.07.007)
  40. Lewis, E. B. (1963). Genes and developmental pathways. Am. Zool.3,33-56. (10.1093/icb/3.1.33)
  41. Lewis, E. B. (1978). A gene complex controlling segmentation in Drosophila. Nature276,565-570. (10.1038/276565a0)
  42. McNally, J. G., Müller, W. G., Walker, D., Wolford, R. and Hager, G. L. (2000). The glucocorticoid receptor: rapid exchange with regulatory sites in living cells. Science287,1262-1265. (10.1126/science.287.5456.1262)
  43. Milan, M., Diaz-Benjumea, F. L. and Cohen, S. M.(1998). Beadex encodes an LMO protein that regulates Apterous LIM-homeodomain activity in Drosophila wing development: a model for LMO oncogene function. Genes Dev.12,2912-2920. (10.1101/gad.12.18.2912)
  44. Min, J., Zhang, Y. and Xu, R. M. (2003). Structural basis for specific binding of Polycomb chromodomain to histone H3 methylated at Lys 27. Genes Dev.17,1823-1828. (10.1101/gad.269603)
  45. Misteli, T., Gunjan, A., Hock, R., Bustin, M. and Brown, D. T. (2000). Dynamic binding of histone H1 to chromatin in living cells. Nature408,877-881. (10.1038/35048610)
  46. Mulholland, N. M., King, I. F. G. and Kingston, R. E.(2003). Regulation of Polycomb roup complexes by the sequence-specific DNA binding proteins Zeste and GAGA. Genes Dev.17,2741-2746. (10.1101/gad.1143303)
  47. Müller, J., Hart, C. M., Francis, N. J., Vargas, M. L.,Sengupta, A., Wild, B., Miller, E. L., O'Connor, M. B., Kingston, R. E. and Simon, J. A. (2002). Histone methyltransferase activity of a Drosophila Polycomb group repressor complex. Cell111,197-208. (10.1016/S0092-8674(02)00976-5)
  48. Netter, S., Faucheux, M. and Theodore, L.(2001). Developmental dynamics of a polyhomeotic-EGFP fusion in vivo. DNA Cell Biol.20,483-492. (10.1089/104454901316976118)
  49. Ng, J., Hart, C. M., Morgan, K. and Simon, J. A.(2000). A Drosophila ESC-E(Z) protein complex is distinct from other polycomb group complexes and contains covalently modified ESC. Mol. Cell. Biol.20,3069-3078. (10.1128/MCB.20.9.3069-3078.2000)
  50. Orlando, V. (2003). Polycomb, epigenomes, and control of cell identity. Cell112,599-606. (10.1016/S0092-8674(03)00157-0)
  51. Orlando, V., Jane, E. P., Chinwalla, V., Harte, P. J. and Paro,R. (1998). Binding of trithorax and Polycomb proteins to the bithorax complex: dynamic changes during early Drosophilaembryogenesis. EMBO J.17,5141-5150. (10.1093/emboj/17.17.5141)
  52. Paro, R. and Hogness, D. S. (1991). The Polycomb protein shares a homologous domain with a heterochromatin-associated protein of Drosophila. Proc. Natl. Acad. Sci. USA88,263-267. (10.1073/pnas.88.1.263)
  53. Phair, R. D. and Misteli, T. (2001). Kinetic modeling approaches to in vivo imaging. Nat. Rev. Mol. Cell Biol.2,898-907. (10.1038/35103000)
  54. Phair, R. D., Scaffidi, P., Elbi, C., Vacerova, J., Dey, A.,Ozato, K., Brown, D. T., Hager, G., Bustin, M. and Misteli, T.(2004). Global nature of dynamic protein-chromatin interactions in vivo: three dimensional genome scanning and dynamic interaction networks of chromatin proteins. Mol. Cell. Biol.24,6393-6402. (10.1128/MCB.24.14.6393-6402.2004)
  55. Post, J. N., Lidke, K. A., Rieger, B. and Arndt-Jovin, D. J.(2005). One- and two-photon photoactivation of a paGFP-fusion protein in live Drosophila embryos. FEBS Lett.579,325-330. (10.1016/j.febslet.2004.11.092)
  56. Rastelli, L., Chan, C. S. and Pirrotta, V.(1993). Related chromosome binding sites for zeste,suppressors of zeste and Polycomb group proteins in Drosophila and their dependence on Enhancer of zestefunction. EMBO J.12,1513-1522. (10.1002/j.1460-2075.1993.tb05795.x)
  57. Ringrose, L., Rehmsmeier, M., Dura, J. M. and Paro, R.(2003). Genome-wide prediction of Polycomb/Trithorax response elements in Drosophila melanogaster. Dev. Cell5, 759-771. (10.1016/S1534-5807(03)00337-X)
  58. Ringrose, L., Ehret, H. and Paro, R. (2004). Distinct contributions of histone H3 Lysine 9 and 27 Methylation to locus-specific stability of polycomb complexes. Mol. Cell16,641. (10.1016/j.molcel.2004.10.015)
  59. Roy, G., Miron, M., Khaleghpour, K., Lasko, P. and Sonenberg,N. (2004). The Drosophila Poly(A) binding protein-interacting protein, dPaip2, is a novel effector of cell growth. Mol. Cell. Biol.24,1143-1154. (10.1128/MCB.24.3.1143-1154.2004)
  60. Saurin, A. J., Shao, Z., Erdjument-Bromage, H., Tempst, P. and Kingston, R. E. (2001). A Drosophila Polycomb group complex includes ZESTE and dTAFII proteins. Nature412,655-660. (10.1038/35088096)
  61. Sengel, P. and Mandaron, P. (1969). Aspects morphologiques du développement in vitro des disques imaginaux de la Drosophila. Arch. Dev. Biol.174,303-311.
  62. Shao, Z. H., Raible, F., Mollaaghababa, R., Guyon, J. R., Wu, C. T., Bender, W. and Kingston, R. E. (1999). Stabilization of chromatin structure by PRC1, a Polycomb complex. Cell98, 37-46. (10.1016/S0092-8674(00)80604-2)
  63. Simon, J. A. and Tamkun, J. W. (2002). Programming off and on states in chromatin: mechanism of Polycomb and trithorax group complexes. Curr. Opin. Genet. Dev.12,210-218. (10.1016/S0959-437X(02)00288-5)
  64. Spradling, A. C. and Rubin, G. M. (1982). Transposition of cloned P elements into Drosophila germ line chromosomes. Science218,341-347. (10.1126/science.6289435)
  65. Sprague, B. L., Pego, R. L., Stavreva, D. A. and McNally, J. G. (2004). Analysis of binding reactions by fluorescence recovery after photobleaching. Biophys. J.86,3473-3495. (10.1529/biophysj.103.026765)
  66. Strutt, H. and Paro, R. (1997). The Polycomb group protein complex of Drosophila melanogaster has different compositions at different target genes. Mol. Cell. Biol.17,6773-6783. (10.1128/MCB.17.12.6773)
  67. Strutt, H., Cavalli, G. and Paro, R. (1997). Co-localization of Polycomb protein and GAGA factor on regulatory elements responsible for the maintenance of homeotic gene expression. EMBO J.16,3621-3632. (10.1093/emboj/16.12.3621)
  68. Tautz, D. (1988). Regulation of the Drosophila segmentation gene hunchback by two maternal morphogenetic centres. Nature332,281-284. (10.1038/332281a0)
  69. Verkman, A. S. (2002). Solute and macromolecule diffusion in cellular aqueous compartments. Trends Biochem. Sci.27,27-33. (10.1016/S0968-0004(01)02003-5)
  70. Vermaak, D., Ahmad, K. and Henikoff, S. (2003). Maintenance of chromatin states:an open-and-shut case. Curr. Opin. Cell Biol.15,266-274. (10.1016/S0955-0674(03)00043-7)
  71. Wang, L., Brown, J. L., Cao, R., Zhang, Y., Kassis, J. A. and Jones, R. S. (2004). Hierarchical recruitment of Polycomb group silencing complexes. Mol. Cell14,637-646. (10.1016/j.molcel.2004.05.009)
  72. White, J. and Stelzer, E. (1999). Photobleaching GFP reveals protein dynamics inside live cells. Trends Cell Biol.9,61-65. (10.1016/S0962-8924(98)01433-0)
  73. Yamamoto, K., Sonoda, M., Inokuchi, J., Shirasawa, S. and Sasazuki, T. (2004). Polycomb group suppressor of zeste 12 links heterochromatin protein 1α and enhancer of zeste 2. J. Biol. Chem.279,401-406. (10.1074/jbc.M307344200)
Dates
Type When
Created 20 years ago (Aug. 3, 2005, 8:13 p.m.)
Deposited 2 years, 3 months ago (May 3, 2023, 8:32 p.m.)
Indexed 3 weeks, 3 days ago (Aug. 6, 2025, 9:23 a.m.)
Issued 19 years, 11 months ago (Sept. 1, 2005)
Published 19 years, 11 months ago (Sept. 1, 2005)
Published Print 19 years, 11 months ago (Sept. 1, 2005)
Funders 0

None

@article{Ficz_2005, title={Polycomb group protein complexes exchange rapidly in livingDrosophila}, volume={132}, ISSN={0950-1991}, url={http://dx.doi.org/10.1242/dev.01950}, DOI={10.1242/dev.01950}, number={17}, journal={Development}, publisher={The Company of Biologists}, author={Ficz, Gabriella and Heintzmann, Rainer and Arndt-Jovin, Donna J.}, year={2005}, month=sep, pages={3963–3976} }