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Several distinct polycomb complexes regulate and co-localize on the INK4a tumor suppressor locus.

Maertens GN, El Messaoudi-Aubert S, Racek T, Stock JK, Nicholls J, Rodriguez-Niedenführ M, Gil J, Peters G - PLoS ONE (2009)

Bottom Line: In primary human fibroblasts, CBX7, CBX8, MEL18 and BMI1 are present at the INK4a locus and shRNA-mediated knockdown of any one of these components results in de-repression of INK4a and proliferative arrest.Sequential chromatin immunoprecipitation (ChIP) reveals that CBX7 and CBX8 bind simultaneously to the same region of chromatin and knockdown of one of the Pc or Psc proteins results in release of the other, suggesting that the binding of PRC1 complexes is interdependent.Our findings provide the first evidence that a single gene can be regulated by several distinct PRC1 complexes and raise important questions about their configuration and relative functions.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK, London Research Institute, London, United Kingdom.

ABSTRACT
Misexpression of Polycomb repressive complex 1 (PRC1) components in human cells profoundly influences the onset of cellular senescence by modulating transcription of the INK4a tumor suppressor gene. Using tandem affinity purification, we find that CBX7 and CBX8, two Polycomb (Pc) homologs that repress INK4a, both participate in PRC1-like complexes with at least two Posterior sex combs (Psc) proteins, MEL18 and BMI1. Each complex contains a single representative of the Pc and Psc families. In primary human fibroblasts, CBX7, CBX8, MEL18 and BMI1 are present at the INK4a locus and shRNA-mediated knockdown of any one of these components results in de-repression of INK4a and proliferative arrest. Sequential chromatin immunoprecipitation (ChIP) reveals that CBX7 and CBX8 bind simultaneously to the same region of chromatin and knockdown of one of the Pc or Psc proteins results in release of the other, suggesting that the binding of PRC1 complexes is interdependent. Our findings provide the first evidence that a single gene can be regulated by several distinct PRC1 complexes and raise important questions about their configuration and relative functions.

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Involvement of BMI1 and MEL18 in mCbx7-mediated repression of INK4a.A. Immunoblot analyses of FDF cells infected with retroviruses encoding either mCbx7 or GFP (negative control) showing down-regulation of p16INK4a. B. qRT-PCR analyses of INK4a and ARF RNAs in cells described in panel A. C. ChIP analyses of CBX7 (mouse+human) and H3K27me3 levels across the INK4a-ARF locus in cells expressing GFP or mCbx7, using the indicated primer sets (as in Fig. 3). D. The mCbx7 expressing cells were infected with lentivirus-based shRNA vectors targeting BMI1, MEL18, or an irrelevant control (C). Effects on the respective target proteins and on p16INK4a were assessed by immunoblotting with the indicated antibodies. GAPDH was used as a loading control. E. qRT-PCR analyses of INK4a and ARF RNAs in cells described in panel D. F. ChIP analyses of CBX7 (mouse+human) and H3K27me3 in cells expressing shRNAs against BMI1 or MEL18. Anti-rabbit IgG was used as a negative control. The analyses were confined to primers corresponding to the proximal part of INK4a exon 1α (PS7) and ARF exon 1β (PS2).
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pone-0006380-g004: Involvement of BMI1 and MEL18 in mCbx7-mediated repression of INK4a.A. Immunoblot analyses of FDF cells infected with retroviruses encoding either mCbx7 or GFP (negative control) showing down-regulation of p16INK4a. B. qRT-PCR analyses of INK4a and ARF RNAs in cells described in panel A. C. ChIP analyses of CBX7 (mouse+human) and H3K27me3 levels across the INK4a-ARF locus in cells expressing GFP or mCbx7, using the indicated primer sets (as in Fig. 3). D. The mCbx7 expressing cells were infected with lentivirus-based shRNA vectors targeting BMI1, MEL18, or an irrelevant control (C). Effects on the respective target proteins and on p16INK4a were assessed by immunoblotting with the indicated antibodies. GAPDH was used as a loading control. E. qRT-PCR analyses of INK4a and ARF RNAs in cells described in panel D. F. ChIP analyses of CBX7 (mouse+human) and H3K27me3 in cells expressing shRNAs against BMI1 or MEL18. Anti-rabbit IgG was used as a negative control. The analyses were confined to primers corresponding to the proximal part of INK4a exon 1α (PS7) and ARF exon 1β (PS2).

Mentions: As a more cogent test of the functional relevance of each component, we next asked whether the ability of CBX7 to downregulate p16INK4a was dependent on either BMI1 or MEL18. HDFs that had been stably transduced with mCbx7 were superinfected with lentiviral vectors expressing shRNAs against BMI1 and MEL18. Relative to the GFP control, mCbx7 caused a substantial reduction in p16INK4a RNA and protein levels, as expected, whereas p14ARF RNA levels were essentially unchanged (Fig. 4A and 4B). ChIP analyses confirmed that ectopic mCbx7 caused a substantial increase in CBX7/mCbx7 binding around the first exon of INK4a, detected using an antibody that recognizes both the mouse and human versions of the protein, with little if any change at the ARF promoter (Fig. 4C). This was accompanied by an increase in the H3K27me3 modification, particularly at positions coincident with CBX7 (Fig. 4C). Importantly, shRNA-mediated knockdown of either BMI1 or MEL18 restored p16INK4a levels in the mCbx7-expressing cells, again without affecting ARF (Fig. 4D and 4E). Moreover, ChIP analyses revealed that the depletion of BMI1 and MEL18 led to a decrease in CBX7/mCbx7 binding and H3K27me3 at the INK4a locus (Fig. 4F). These findings suggest that BMI1 and MEL18 are rate limiting for repression of INK4a by mCbx7.


Several distinct polycomb complexes regulate and co-localize on the INK4a tumor suppressor locus.

Maertens GN, El Messaoudi-Aubert S, Racek T, Stock JK, Nicholls J, Rodriguez-Niedenführ M, Gil J, Peters G - PLoS ONE (2009)

Involvement of BMI1 and MEL18 in mCbx7-mediated repression of INK4a.A. Immunoblot analyses of FDF cells infected with retroviruses encoding either mCbx7 or GFP (negative control) showing down-regulation of p16INK4a. B. qRT-PCR analyses of INK4a and ARF RNAs in cells described in panel A. C. ChIP analyses of CBX7 (mouse+human) and H3K27me3 levels across the INK4a-ARF locus in cells expressing GFP or mCbx7, using the indicated primer sets (as in Fig. 3). D. The mCbx7 expressing cells were infected with lentivirus-based shRNA vectors targeting BMI1, MEL18, or an irrelevant control (C). Effects on the respective target proteins and on p16INK4a were assessed by immunoblotting with the indicated antibodies. GAPDH was used as a loading control. E. qRT-PCR analyses of INK4a and ARF RNAs in cells described in panel D. F. ChIP analyses of CBX7 (mouse+human) and H3K27me3 in cells expressing shRNAs against BMI1 or MEL18. Anti-rabbit IgG was used as a negative control. The analyses were confined to primers corresponding to the proximal part of INK4a exon 1α (PS7) and ARF exon 1β (PS2).
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pone-0006380-g004: Involvement of BMI1 and MEL18 in mCbx7-mediated repression of INK4a.A. Immunoblot analyses of FDF cells infected with retroviruses encoding either mCbx7 or GFP (negative control) showing down-regulation of p16INK4a. B. qRT-PCR analyses of INK4a and ARF RNAs in cells described in panel A. C. ChIP analyses of CBX7 (mouse+human) and H3K27me3 levels across the INK4a-ARF locus in cells expressing GFP or mCbx7, using the indicated primer sets (as in Fig. 3). D. The mCbx7 expressing cells were infected with lentivirus-based shRNA vectors targeting BMI1, MEL18, or an irrelevant control (C). Effects on the respective target proteins and on p16INK4a were assessed by immunoblotting with the indicated antibodies. GAPDH was used as a loading control. E. qRT-PCR analyses of INK4a and ARF RNAs in cells described in panel D. F. ChIP analyses of CBX7 (mouse+human) and H3K27me3 in cells expressing shRNAs against BMI1 or MEL18. Anti-rabbit IgG was used as a negative control. The analyses were confined to primers corresponding to the proximal part of INK4a exon 1α (PS7) and ARF exon 1β (PS2).
Mentions: As a more cogent test of the functional relevance of each component, we next asked whether the ability of CBX7 to downregulate p16INK4a was dependent on either BMI1 or MEL18. HDFs that had been stably transduced with mCbx7 were superinfected with lentiviral vectors expressing shRNAs against BMI1 and MEL18. Relative to the GFP control, mCbx7 caused a substantial reduction in p16INK4a RNA and protein levels, as expected, whereas p14ARF RNA levels were essentially unchanged (Fig. 4A and 4B). ChIP analyses confirmed that ectopic mCbx7 caused a substantial increase in CBX7/mCbx7 binding around the first exon of INK4a, detected using an antibody that recognizes both the mouse and human versions of the protein, with little if any change at the ARF promoter (Fig. 4C). This was accompanied by an increase in the H3K27me3 modification, particularly at positions coincident with CBX7 (Fig. 4C). Importantly, shRNA-mediated knockdown of either BMI1 or MEL18 restored p16INK4a levels in the mCbx7-expressing cells, again without affecting ARF (Fig. 4D and 4E). Moreover, ChIP analyses revealed that the depletion of BMI1 and MEL18 led to a decrease in CBX7/mCbx7 binding and H3K27me3 at the INK4a locus (Fig. 4F). These findings suggest that BMI1 and MEL18 are rate limiting for repression of INK4a by mCbx7.

Bottom Line: In primary human fibroblasts, CBX7, CBX8, MEL18 and BMI1 are present at the INK4a locus and shRNA-mediated knockdown of any one of these components results in de-repression of INK4a and proliferative arrest.Sequential chromatin immunoprecipitation (ChIP) reveals that CBX7 and CBX8 bind simultaneously to the same region of chromatin and knockdown of one of the Pc or Psc proteins results in release of the other, suggesting that the binding of PRC1 complexes is interdependent.Our findings provide the first evidence that a single gene can be regulated by several distinct PRC1 complexes and raise important questions about their configuration and relative functions.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK, London Research Institute, London, United Kingdom.

ABSTRACT
Misexpression of Polycomb repressive complex 1 (PRC1) components in human cells profoundly influences the onset of cellular senescence by modulating transcription of the INK4a tumor suppressor gene. Using tandem affinity purification, we find that CBX7 and CBX8, two Polycomb (Pc) homologs that repress INK4a, both participate in PRC1-like complexes with at least two Posterior sex combs (Psc) proteins, MEL18 and BMI1. Each complex contains a single representative of the Pc and Psc families. In primary human fibroblasts, CBX7, CBX8, MEL18 and BMI1 are present at the INK4a locus and shRNA-mediated knockdown of any one of these components results in de-repression of INK4a and proliferative arrest. Sequential chromatin immunoprecipitation (ChIP) reveals that CBX7 and CBX8 bind simultaneously to the same region of chromatin and knockdown of one of the Pc or Psc proteins results in release of the other, suggesting that the binding of PRC1 complexes is interdependent. Our findings provide the first evidence that a single gene can be regulated by several distinct PRC1 complexes and raise important questions about their configuration and relative functions.

Show MeSH
Related in: MedlinePlus