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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 localize specifically  to the pericentromeric region of chromosome 1 in a number of cell lines. Digitally  merged fluorescent-channel images are  shown of interphase cell nuclei showing  immunofluorescent localization of the in  situ–hybridized pUC1.77 probe, specific  for the q12 pericentromeric region of  chromosome 1 (red) and immunofluorescent localization of PcG bodies using anti-RING1 antibodies (green). Nuclei were  counterstained with DAPI (blue channel)  showing the nuclear volume. The separate  color channels are shown under the main  merged images. (A) Interphase 2C4 cell  nuclei showing (i) normal interphase nuclei and (ii) interphase chromatin from a  released nucleus by a cytospin where the  nuclear architecture and territories are  compromised, thereby releasing the chromosome domains throughout the length of  the nucleus. (B) U-2 OS cells labeled after  (i) the normal high-stringency immuno-FISH protocol, and (ii) lower stringency  immuno-FISH protocol showing additional regions of pUC1.77 hybridization  that colocalize with smaller PcG bodies  (arrowheads); (iii) low-stringency hybridizations of the pUC1.77 probe were localized on metaphase chromosome spreads  counterstained with DAPI (blue channel)  showing the three large 1q12-specific hybridizing regions (white arrowheads) that  are due to breakage of the q12 region in  one chromosome 1 (double white arrowheads), together with the additional sites  of hybridization during low-stringency  conditions (yellow arrowheads). (C)  SAOS-2 cells labeled after hybrdization of  the pUC1.77 probe (i) and hybridization  at lower stringency showing an additional  two sites of hybridization (ii; arrowheads);  (iii) low-stringency hybridizations of the  pUC1.77 probe were localized on metaphase chromosome spreads counterstained with DAPI (blue channel), showing the multiple rearrangements of the  1q12 region.
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Figure 5: PcG bodies localize specifically to the pericentromeric region of chromosome 1 in a number of cell lines. Digitally merged fluorescent-channel images are shown of interphase cell nuclei showing immunofluorescent localization of the in situ–hybridized pUC1.77 probe, specific for the q12 pericentromeric region of chromosome 1 (red) and immunofluorescent localization of PcG bodies using anti-RING1 antibodies (green). Nuclei were counterstained with DAPI (blue channel) showing the nuclear volume. The separate color channels are shown under the main merged images. (A) Interphase 2C4 cell nuclei showing (i) normal interphase nuclei and (ii) interphase chromatin from a released nucleus by a cytospin where the nuclear architecture and territories are compromised, thereby releasing the chromosome domains throughout the length of the nucleus. (B) U-2 OS cells labeled after (i) the normal high-stringency immuno-FISH protocol, and (ii) lower stringency immuno-FISH protocol showing additional regions of pUC1.77 hybridization that colocalize with smaller PcG bodies (arrowheads); (iii) low-stringency hybridizations of the pUC1.77 probe were localized on metaphase chromosome spreads counterstained with DAPI (blue channel) showing the three large 1q12-specific hybridizing regions (white arrowheads) that are due to breakage of the q12 region in one chromosome 1 (double white arrowheads), together with the additional sites of hybridization during low-stringency conditions (yellow arrowheads). (C) SAOS-2 cells labeled after hybrdization of the pUC1.77 probe (i) and hybridization at lower stringency showing an additional two sites of hybridization (ii; arrowheads); (iii) low-stringency hybridizations of the pUC1.77 probe were localized on metaphase chromosome spreads counterstained with DAPI (blue channel), showing the multiple rearrangements of the 1q12 region.

Mentions: Using the protein–chromatin immuno-FISH labeling method described above, but reducing the size of the hybridizing probe, we can specifically localize PcG bodies to areas of constitutive heterochromatin in the pericentromeric region of chromosome 1. The DNA probe pUC1.77, which is specific for the pericentromeric region q12 of chromosome 1 (Cooke, 1979), gives total immunofluorescent colocalization with PcG bodies identified using the anti-RING1 antibody (Fig. 5 A [i]). This colocalization is identical for both pairs of chromosomes in all interphase cells showing the specificity of PcG bodies for this region of chromosome 1 in 2C4 cells. Furthermore, discrete areas of hybridization of the pUC1.77 probe overlap with PcG body staining in chromatin released from a nucleus using a cytospin (Fig. 5 A [ii]). This overlap suggests that RING1, as observed in the large foci in 2C4 interphase cells, is concentrated into smaller foci spanning almost the entire q12-hybridizing region of chromosome 1 (see Fig. 5 A [ii]; arrowheads). Given the demonstrated biochemical associations among RING1, BMI1, and hPc2 (Satijn et al., 1997b), it would seem likely that all three components are present in these smaller RING1 foci.


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 localize specifically  to the pericentromeric region of chromosome 1 in a number of cell lines. Digitally  merged fluorescent-channel images are  shown of interphase cell nuclei showing  immunofluorescent localization of the in  situ–hybridized pUC1.77 probe, specific  for the q12 pericentromeric region of  chromosome 1 (red) and immunofluorescent localization of PcG bodies using anti-RING1 antibodies (green). Nuclei were  counterstained with DAPI (blue channel)  showing the nuclear volume. The separate  color channels are shown under the main  merged images. (A) Interphase 2C4 cell  nuclei showing (i) normal interphase nuclei and (ii) interphase chromatin from a  released nucleus by a cytospin where the  nuclear architecture and territories are  compromised, thereby releasing the chromosome domains throughout the length of  the nucleus. (B) U-2 OS cells labeled after  (i) the normal high-stringency immuno-FISH protocol, and (ii) lower stringency  immuno-FISH protocol showing additional regions of pUC1.77 hybridization  that colocalize with smaller PcG bodies  (arrowheads); (iii) low-stringency hybridizations of the pUC1.77 probe were localized on metaphase chromosome spreads  counterstained with DAPI (blue channel)  showing the three large 1q12-specific hybridizing regions (white arrowheads) that  are due to breakage of the q12 region in  one chromosome 1 (double white arrowheads), together with the additional sites  of hybridization during low-stringency  conditions (yellow arrowheads). (C)  SAOS-2 cells labeled after hybrdization of  the pUC1.77 probe (i) and hybridization  at lower stringency showing an additional  two sites of hybridization (ii; arrowheads);  (iii) low-stringency hybridizations of the  pUC1.77 probe were localized on metaphase chromosome spreads counterstained with DAPI (blue channel), showing the multiple rearrangements of the  1q12 region.
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Related In: Results  -  Collection

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Figure 5: PcG bodies localize specifically to the pericentromeric region of chromosome 1 in a number of cell lines. Digitally merged fluorescent-channel images are shown of interphase cell nuclei showing immunofluorescent localization of the in situ–hybridized pUC1.77 probe, specific for the q12 pericentromeric region of chromosome 1 (red) and immunofluorescent localization of PcG bodies using anti-RING1 antibodies (green). Nuclei were counterstained with DAPI (blue channel) showing the nuclear volume. The separate color channels are shown under the main merged images. (A) Interphase 2C4 cell nuclei showing (i) normal interphase nuclei and (ii) interphase chromatin from a released nucleus by a cytospin where the nuclear architecture and territories are compromised, thereby releasing the chromosome domains throughout the length of the nucleus. (B) U-2 OS cells labeled after (i) the normal high-stringency immuno-FISH protocol, and (ii) lower stringency immuno-FISH protocol showing additional regions of pUC1.77 hybridization that colocalize with smaller PcG bodies (arrowheads); (iii) low-stringency hybridizations of the pUC1.77 probe were localized on metaphase chromosome spreads counterstained with DAPI (blue channel) showing the three large 1q12-specific hybridizing regions (white arrowheads) that are due to breakage of the q12 region in one chromosome 1 (double white arrowheads), together with the additional sites of hybridization during low-stringency conditions (yellow arrowheads). (C) SAOS-2 cells labeled after hybrdization of the pUC1.77 probe (i) and hybridization at lower stringency showing an additional two sites of hybridization (ii; arrowheads); (iii) low-stringency hybridizations of the pUC1.77 probe were localized on metaphase chromosome spreads counterstained with DAPI (blue channel), showing the multiple rearrangements of the 1q12 region.
Mentions: Using the protein–chromatin immuno-FISH labeling method described above, but reducing the size of the hybridizing probe, we can specifically localize PcG bodies to areas of constitutive heterochromatin in the pericentromeric region of chromosome 1. The DNA probe pUC1.77, which is specific for the pericentromeric region q12 of chromosome 1 (Cooke, 1979), gives total immunofluorescent colocalization with PcG bodies identified using the anti-RING1 antibody (Fig. 5 A [i]). This colocalization is identical for both pairs of chromosomes in all interphase cells showing the specificity of PcG bodies for this region of chromosome 1 in 2C4 cells. Furthermore, discrete areas of hybridization of the pUC1.77 probe overlap with PcG body staining in chromatin released from a nucleus using a cytospin (Fig. 5 A [ii]). This overlap suggests that RING1, as observed in the large foci in 2C4 interphase cells, is concentrated into smaller foci spanning almost the entire q12-hybridizing region of chromosome 1 (see Fig. 5 A [ii]; arrowheads). Given the demonstrated biochemical associations among RING1, BMI1, and hPc2 (Satijn et al., 1997b), it would seem likely that all three components are present in these smaller RING1 foci.

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