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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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PcG bodies remain  chromosomally associated  throughout mitosis. Mitotic  chromosomes were visualized by propidium iodide  staining of formaldehyde-fixed cells, and are shown in  red. The various stages of mitosis were distinguished according to criteria used by  Chaly et al. (1984). PcG bodies were visualized by immunofluorescent labeling of  (A) 2C4 cells with antibodies  against either RING1 (top;  green channel), hPc2 (middle; green channel) or BMI1  (bottom; green channel) and  (B) U-2 OS cells with antibodies against RING1 (green  channel). A digital overlay of  the two-color channels shows  the relation of PcG bodies  with the condensed chromosomes during the given  stages of mitosis. (i) Enlargement of BMI1 during  prometaphase in 2C4 cells in  the main figure shows direct  chromosome association  with concentration of the  protein on the sister chromatids (arrowheads). (ii) Enlargement of hPc2 during  anaphase in 2C4 cells in the  main figure shows direct  chromosome association of  hPc2 to a pericentromeric region on chromosome 1 (arrowheads).
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Figure 6: PcG bodies remain chromosomally associated throughout mitosis. Mitotic chromosomes were visualized by propidium iodide staining of formaldehyde-fixed cells, and are shown in red. The various stages of mitosis were distinguished according to criteria used by Chaly et al. (1984). PcG bodies were visualized by immunofluorescent labeling of (A) 2C4 cells with antibodies against either RING1 (top; green channel), hPc2 (middle; green channel) or BMI1 (bottom; green channel) and (B) U-2 OS cells with antibodies against RING1 (green channel). A digital overlay of the two-color channels shows the relation of PcG bodies with the condensed chromosomes during the given stages of mitosis. (i) Enlargement of BMI1 during prometaphase in 2C4 cells in the main figure shows direct chromosome association with concentration of the protein on the sister chromatids (arrowheads). (ii) Enlargement of hPc2 during anaphase in 2C4 cells in the main figure shows direct chromosome association of hPc2 to a pericentromeric region on chromosome 1 (arrowheads).

Mentions: To study the dynamics and stability of PcG bodies during mitosis and cell division, we fixed and labeled cells with propidium iodide to identify mitotic cells. It was previously observed by others that the large nuclear mammalian PcG protein–containing complexes were not antibody-labeled during cell division until the latter stages of mitosis. However, we attribute this finding to the possible destruction of protein antigenicity by fixation (see Materials and Methods). Using a milder fixation protocol while maintaining attachment of mitotic cells to the coverslip, we are able to use double immunofluorescent labeling with propidium iodide and FITC-labeled PcG components (RING1, BMI1, and hPc2) to study PcG bodies through all stages of mitosis in 2C4 cells (Fig. 6 A) and RING1 in U-2 OS cells (Fig. 6 B). Upon chromosome condensation at prophase, PcG bodies become much reduced in size and less conspicuous than those in interphase cells (Fig. 6). However, upon disassembly of the nuclear envelope and matrix at prometaphase, PcG bodies are once again, large concentrated foci seen by staining with either the anti-hPc2 or anti-BMI1 antibodies (Fig. 6 A). Interestingly, in the majority of cells at this stage, PcG bodies have doubled in number (see Fig. 6 A). This is most apparent in one particular 2C4 cell, which contains four PcG bodies, probably due to the known small number of tetraploid cells present in this cell line (J. Williamson, unpublished observations). Here we are able to see eight PcG-stained regions that associate on both pairs of chromatids concentrated into smaller foci (Fig. 6 I; arrowheads). This is similar to the pattern observed in the chromatin-released cell with RING1 associating to 1q12 in discrete smaller foci (see Fig. 5 A [ii]; arrowheads). We are unable to locate RING1 in 2C4 cells at prometaphase (Fig. 6 A), although we can see RING1 at a similar stage in U2-OS cells (Fig. 6 B). This is surprising, since we observe RING1 when the chromosomes align at the metaphase plate during metaphase (Fig. 6). BMI1 and hPc2 can also be clearly seen as part of the duplicated PcG bodies during metaphase (Fig. 6 A). We attribute this observation either to loss of RING1 from PcG bodies, or more likely to an inaccessibility of RING1 to antibody labeling.


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)

PcG bodies remain  chromosomally associated  throughout mitosis. Mitotic  chromosomes were visualized by propidium iodide  staining of formaldehyde-fixed cells, and are shown in  red. The various stages of mitosis were distinguished according to criteria used by  Chaly et al. (1984). PcG bodies were visualized by immunofluorescent labeling of  (A) 2C4 cells with antibodies  against either RING1 (top;  green channel), hPc2 (middle; green channel) or BMI1  (bottom; green channel) and  (B) U-2 OS cells with antibodies against RING1 (green  channel). A digital overlay of  the two-color channels shows  the relation of PcG bodies  with the condensed chromosomes during the given  stages of mitosis. (i) Enlargement of BMI1 during  prometaphase in 2C4 cells in  the main figure shows direct  chromosome association  with concentration of the  protein on the sister chromatids (arrowheads). (ii) Enlargement of hPc2 during  anaphase in 2C4 cells in the  main figure shows direct  chromosome association of  hPc2 to a pericentromeric region on chromosome 1 (arrowheads).
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Figure 6: PcG bodies remain chromosomally associated throughout mitosis. Mitotic chromosomes were visualized by propidium iodide staining of formaldehyde-fixed cells, and are shown in red. The various stages of mitosis were distinguished according to criteria used by Chaly et al. (1984). PcG bodies were visualized by immunofluorescent labeling of (A) 2C4 cells with antibodies against either RING1 (top; green channel), hPc2 (middle; green channel) or BMI1 (bottom; green channel) and (B) U-2 OS cells with antibodies against RING1 (green channel). A digital overlay of the two-color channels shows the relation of PcG bodies with the condensed chromosomes during the given stages of mitosis. (i) Enlargement of BMI1 during prometaphase in 2C4 cells in the main figure shows direct chromosome association with concentration of the protein on the sister chromatids (arrowheads). (ii) Enlargement of hPc2 during anaphase in 2C4 cells in the main figure shows direct chromosome association of hPc2 to a pericentromeric region on chromosome 1 (arrowheads).
Mentions: To study the dynamics and stability of PcG bodies during mitosis and cell division, we fixed and labeled cells with propidium iodide to identify mitotic cells. It was previously observed by others that the large nuclear mammalian PcG protein–containing complexes were not antibody-labeled during cell division until the latter stages of mitosis. However, we attribute this finding to the possible destruction of protein antigenicity by fixation (see Materials and Methods). Using a milder fixation protocol while maintaining attachment of mitotic cells to the coverslip, we are able to use double immunofluorescent labeling with propidium iodide and FITC-labeled PcG components (RING1, BMI1, and hPc2) to study PcG bodies through all stages of mitosis in 2C4 cells (Fig. 6 A) and RING1 in U-2 OS cells (Fig. 6 B). Upon chromosome condensation at prophase, PcG bodies become much reduced in size and less conspicuous than those in interphase cells (Fig. 6). However, upon disassembly of the nuclear envelope and matrix at prometaphase, PcG bodies are once again, large concentrated foci seen by staining with either the anti-hPc2 or anti-BMI1 antibodies (Fig. 6 A). Interestingly, in the majority of cells at this stage, PcG bodies have doubled in number (see Fig. 6 A). This is most apparent in one particular 2C4 cell, which contains four PcG bodies, probably due to the known small number of tetraploid cells present in this cell line (J. Williamson, unpublished observations). Here we are able to see eight PcG-stained regions that associate on both pairs of chromatids concentrated into smaller foci (Fig. 6 I; arrowheads). This is similar to the pattern observed in the chromatin-released cell with RING1 associating to 1q12 in discrete smaller foci (see Fig. 5 A [ii]; arrowheads). We are unable to locate RING1 in 2C4 cells at prometaphase (Fig. 6 A), although we can see RING1 at a similar stage in U2-OS cells (Fig. 6 B). This is surprising, since we observe RING1 when the chromosomes align at the metaphase plate during metaphase (Fig. 6). BMI1 and hPc2 can also be clearly seen as part of the duplicated PcG bodies during metaphase (Fig. 6 A). We attribute this observation either to loss of RING1 from PcG bodies, or more likely to an inaccessibility of RING1 to antibody labeling.

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