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The human polycomb group complex associates with pericentromeric heterochromatin to form a novel nuclear domain.

Saurin AJ, Shiels C, Williamson J, Satijn DP, Otte AP, Sheer D, Freemont PS - J. Cell Biol. (1998)

Bottom Line: The Polycomb group (PcG) complex is a chromatin-associated multiprotein complex, involved in the stable repression of homeotic gene activity in Drosophila.Furthermore, these heterochromatin-bound PcG complexes remain stably associated throughout mitosis, thereby allowing the potential inheritance of the PcG complex through successive cell divisions.We discuss these results in terms of the known function of the PcG complex as a transcriptional repression complex.

View Article: PubMed Central - PubMed

Affiliation: Molecular Structure and Function Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom.

ABSTRACT
The Polycomb group (PcG) complex is a chromatin-associated multiprotein complex, involved in the stable repression of homeotic gene activity in Drosophila. Recently, a mammalian PcG complex has been identified with several PcG proteins implicated in the regulation of Hox gene expression. Although the mammalian PcG complex appears analogous to the complex in Drosophila, the molecular mechanisms and functions for the mammalian PcG complex remain unknown. Here we describe a detailed characterization of the human PcG complex in terms of cellular localization and chromosomal association. By using antibodies that specifically recognize three human PcG proteins- RING1, BMI1, and hPc2-we demonstrate in a number of human cell lines that the PcG complex forms a unique discrete nuclear structure that we term PcG bodies. PcG bodies are prominent novel nuclear structures with the larger PcG foci generally localized near the centromeres, as visualized with a kinetochore antibody marker. In both normal fetal and adult fibroblasts, PcG bodies are not randomly dispersed, but appear clustered into defined areas within the nucleus. We show in three different human cell lines that the PcG complex can tightly associate with large pericentromeric heterochromatin regions (1q12) on chromosome 1, and with related pericentromeric sequences on different chromosomes, providing evidence for a mammalian PcG-heterochromatin association. Furthermore, these heterochromatin-bound PcG complexes remain stably associated throughout mitosis, thereby allowing the potential inheritance of the PcG complex through successive cell divisions. We discuss these results in terms of the known function of the PcG complex as a transcriptional repression complex.

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The mammalian  PcG complex forms a novel  nuclear structure. The mammalian PcG complex was immunolocalized in human  cells using the RING1-specific polyclonal antibody  ASA3, and was stained using an FITC-conjugated secondary antibody. By double  immunofluorescence microscopy, the staining pattern obtained for RING1 in 2C4 human fibrosarcoma cells was  compared with that of known  nuclear structures. The green  signal shows labeled RING1,  and the red signal given by  Texas red–conjugated secondary antibodies shows immunolocalization of the following: (A) PML nuclear  bodies from the anti-PML antibody 5E10 (Stuurman et al., 1992); (B) speckled domains recognized by the anti-SC35 antibody (Fu and  Maniatis, 1990); (C) the snRNP family of proteins, recognized by the anti-Sm antigen antibody Y12 (Lerner et al., 1981); (D) coiled bodies recognized by the anti-p80 coilin antibody p-δ; (E) gems recognized by the anti-SMN antibody 2B1 (Liu and Dreyfuss, 1996); (F)  PTB protein (Huang et al., 1997); (G) DNA replication foci detected using antibodies to PCNA; (H) sites of RNA synthesis localized  with antibodies to in vivo–incorporated bromo-UTP nucleotides (note this panel is shown for U-2 OS cells). All images are a digital  overlay of the two optical channels obtained from a single optical confocal section.
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Figure 2: The mammalian PcG complex forms a novel nuclear structure. The mammalian PcG complex was immunolocalized in human cells using the RING1-specific polyclonal antibody ASA3, and was stained using an FITC-conjugated secondary antibody. By double immunofluorescence microscopy, the staining pattern obtained for RING1 in 2C4 human fibrosarcoma cells was compared with that of known nuclear structures. The green signal shows labeled RING1, and the red signal given by Texas red–conjugated secondary antibodies shows immunolocalization of the following: (A) PML nuclear bodies from the anti-PML antibody 5E10 (Stuurman et al., 1992); (B) speckled domains recognized by the anti-SC35 antibody (Fu and Maniatis, 1990); (C) the snRNP family of proteins, recognized by the anti-Sm antigen antibody Y12 (Lerner et al., 1981); (D) coiled bodies recognized by the anti-p80 coilin antibody p-δ; (E) gems recognized by the anti-SMN antibody 2B1 (Liu and Dreyfuss, 1996); (F) PTB protein (Huang et al., 1997); (G) DNA replication foci detected using antibodies to PCNA; (H) sites of RNA synthesis localized with antibodies to in vivo–incorporated bromo-UTP nucleotides (note this panel is shown for U-2 OS cells). All images are a digital overlay of the two optical channels obtained from a single optical confocal section.

Mentions: To test whether PcG bodies are associated with any of the other macromolecular nuclear domains (for review see Lamond and Earnshaw, 1998), we performed rigorous double-immunofluorescent labeling with antibodies against other known and previously characterized nuclear antigens. All double labeling was performed on 2C4 and U-2 OS cells since both contain differing numbers of PcG bodies (see above). For brevity, the data for 2C4 cells is shown in Fig. 2, although both cell lines gave consistent results.


The human polycomb group complex associates with pericentromeric heterochromatin to form a novel nuclear domain.

Saurin AJ, Shiels C, Williamson J, Satijn DP, Otte AP, Sheer D, Freemont PS - J. Cell Biol. (1998)

The mammalian  PcG complex forms a novel  nuclear structure. The mammalian PcG complex was immunolocalized in human  cells using the RING1-specific polyclonal antibody  ASA3, and was stained using an FITC-conjugated secondary antibody. By double  immunofluorescence microscopy, the staining pattern obtained for RING1 in 2C4 human fibrosarcoma cells was  compared with that of known  nuclear structures. The green  signal shows labeled RING1,  and the red signal given by  Texas red–conjugated secondary antibodies shows immunolocalization of the following: (A) PML nuclear  bodies from the anti-PML antibody 5E10 (Stuurman et al., 1992); (B) speckled domains recognized by the anti-SC35 antibody (Fu and  Maniatis, 1990); (C) the snRNP family of proteins, recognized by the anti-Sm antigen antibody Y12 (Lerner et al., 1981); (D) coiled bodies recognized by the anti-p80 coilin antibody p-δ; (E) gems recognized by the anti-SMN antibody 2B1 (Liu and Dreyfuss, 1996); (F)  PTB protein (Huang et al., 1997); (G) DNA replication foci detected using antibodies to PCNA; (H) sites of RNA synthesis localized  with antibodies to in vivo–incorporated bromo-UTP nucleotides (note this panel is shown for U-2 OS cells). All images are a digital  overlay of the two optical channels obtained from a single optical confocal section.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132874&req=5

Figure 2: The mammalian PcG complex forms a novel nuclear structure. The mammalian PcG complex was immunolocalized in human cells using the RING1-specific polyclonal antibody ASA3, and was stained using an FITC-conjugated secondary antibody. By double immunofluorescence microscopy, the staining pattern obtained for RING1 in 2C4 human fibrosarcoma cells was compared with that of known nuclear structures. The green signal shows labeled RING1, and the red signal given by Texas red–conjugated secondary antibodies shows immunolocalization of the following: (A) PML nuclear bodies from the anti-PML antibody 5E10 (Stuurman et al., 1992); (B) speckled domains recognized by the anti-SC35 antibody (Fu and Maniatis, 1990); (C) the snRNP family of proteins, recognized by the anti-Sm antigen antibody Y12 (Lerner et al., 1981); (D) coiled bodies recognized by the anti-p80 coilin antibody p-δ; (E) gems recognized by the anti-SMN antibody 2B1 (Liu and Dreyfuss, 1996); (F) PTB protein (Huang et al., 1997); (G) DNA replication foci detected using antibodies to PCNA; (H) sites of RNA synthesis localized with antibodies to in vivo–incorporated bromo-UTP nucleotides (note this panel is shown for U-2 OS cells). All images are a digital overlay of the two optical channels obtained from a single optical confocal section.
Mentions: To test whether PcG bodies are associated with any of the other macromolecular nuclear domains (for review see Lamond and Earnshaw, 1998), we performed rigorous double-immunofluorescent labeling with antibodies against other known and previously characterized nuclear antigens. All double labeling was performed on 2C4 and U-2 OS cells since both contain differing numbers of PcG bodies (see above). For brevity, the data for 2C4 cells is shown in Fig. 2, although both cell lines gave consistent results.

Bottom Line: The Polycomb group (PcG) complex is a chromatin-associated multiprotein complex, involved in the stable repression of homeotic gene activity in Drosophila.Furthermore, these heterochromatin-bound PcG complexes remain stably associated throughout mitosis, thereby allowing the potential inheritance of the PcG complex through successive cell divisions.We discuss these results in terms of the known function of the PcG complex as a transcriptional repression complex.

View Article: PubMed Central - PubMed

Affiliation: Molecular Structure and Function Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom.

ABSTRACT
The Polycomb group (PcG) complex is a chromatin-associated multiprotein complex, involved in the stable repression of homeotic gene activity in Drosophila. Recently, a mammalian PcG complex has been identified with several PcG proteins implicated in the regulation of Hox gene expression. Although the mammalian PcG complex appears analogous to the complex in Drosophila, the molecular mechanisms and functions for the mammalian PcG complex remain unknown. Here we describe a detailed characterization of the human PcG complex in terms of cellular localization and chromosomal association. By using antibodies that specifically recognize three human PcG proteins- RING1, BMI1, and hPc2-we demonstrate in a number of human cell lines that the PcG complex forms a unique discrete nuclear structure that we term PcG bodies. PcG bodies are prominent novel nuclear structures with the larger PcG foci generally localized near the centromeres, as visualized with a kinetochore antibody marker. In both normal fetal and adult fibroblasts, PcG bodies are not randomly dispersed, but appear clustered into defined areas within the nucleus. We show in three different human cell lines that the PcG complex can tightly associate with large pericentromeric heterochromatin regions (1q12) on chromosome 1, and with related pericentromeric sequences on different chromosomes, providing evidence for a mammalian PcG-heterochromatin association. Furthermore, these heterochromatin-bound PcG complexes remain stably associated throughout mitosis, thereby allowing the potential inheritance of the PcG complex through successive cell divisions. We discuss these results in terms of the known function of the PcG complex as a transcriptional repression complex.

Show MeSH
Related in: MedlinePlus