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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-Niedenf├╝hr 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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Related in: MedlinePlus

CBX7 and CBX8 bind simultaneously to the same INK4a allele.A and B. Sequential ChIP using HDFs that co-express FLAG-tagged mCbx7 and HA-tagged mCbx8. In A, the first ChIP was performed with the FLAG antibody, followed by either anti-HA or irrelevant IgG. In B, the first ChIP was performed with the HA antibody, followed by either anti-FLAG or IgG. The precipitated DNA was analyzed by qPCR with the indicated primer sets. The histograms represent the average and SD values of biological replicates. C and D, Sequential ChIP of endogenous CBX8 and CBX7 in FDF cells. Chromatin was first precipitated with affinity purified anti-CBX8 antibody and enrichment for INK4a primer set 7 (PS7) was confirmed by q-PCR. The chromatin was divided into four equal fractions and re-precipitated with either rabbit IgG as a negative control, anti-H3K27me3 or anti-CBX8 as positive controls, or anti-CBX7. The enrichment is calculated relative to the original input chromatin. The figure shows the results of a single representative experiment.
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pone-0006380-g005: CBX7 and CBX8 bind simultaneously to the same INK4a allele.A and B. Sequential ChIP using HDFs that co-express FLAG-tagged mCbx7 and HA-tagged mCbx8. In A, the first ChIP was performed with the FLAG antibody, followed by either anti-HA or irrelevant IgG. In B, the first ChIP was performed with the HA antibody, followed by either anti-FLAG or IgG. The precipitated DNA was analyzed by qPCR with the indicated primer sets. The histograms represent the average and SD values of biological replicates. C and D, Sequential ChIP of endogenous CBX8 and CBX7 in FDF cells. Chromatin was first precipitated with affinity purified anti-CBX8 antibody and enrichment for INK4a primer set 7 (PS7) was confirmed by q-PCR. The chromatin was divided into four equal fractions and re-precipitated with either rabbit IgG as a negative control, anti-H3K27me3 or anti-CBX8 as positive controls, or anti-CBX7. The enrichment is calculated relative to the original input chromatin. The figure shows the results of a single representative experiment.

Mentions: As we work with pools of cells, the ability of several PRC1 components to modulate INK4a expression is open to different interpretations. For example, it is conceivable that different PRC1 complexes regulate the locus in different cells or that several PRC1 complexes congregate at the locus in every cell. To try to distinguish between these possibilities, we set out to determine whether CBX7 and CBX8 can be detected on the same piece of DNA, using a ChIP-reChIP strategy. HDFs were co-infected with retroviruses encoding FLAG-tagged mCbx7 and HA-tagged mCbx8 to generate pools of drug-resistant cells expressing both proteins. An initial ChIP was performed using the FLAG antibody and the chromatin was eluted from the beads with the FLAG peptide. The recovered chromatin was then immunoprecipitated with the HA antibody or with an irrelevant IgG and subjected to qPCR analysis with a subset of the primer sets described in Figure 3A. The signal was quantified relative to the input material used in the first ChIP. As illustrated in Figure 5A, mCbx8 was substantially enriched at the INK4a locus following a mCbx7 ChIP. In the reciprocal experiment, the chromatin was first precipitated with the HA antibody and eluted with HA peptide. Again, it was clear that mCbx7 was enriched in the chromatin precipitated through mCbx8 (Fig. 5B), implying that mCbx7 and mCbx8 bind simultaneously to the same fragment of chromatin.


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-Niedenf├╝hr M, Gil J, Peters G - PLoS ONE (2009)

CBX7 and CBX8 bind simultaneously to the same INK4a allele.A and B. Sequential ChIP using HDFs that co-express FLAG-tagged mCbx7 and HA-tagged mCbx8. In A, the first ChIP was performed with the FLAG antibody, followed by either anti-HA or irrelevant IgG. In B, the first ChIP was performed with the HA antibody, followed by either anti-FLAG or IgG. The precipitated DNA was analyzed by qPCR with the indicated primer sets. The histograms represent the average and SD values of biological replicates. C and D, Sequential ChIP of endogenous CBX8 and CBX7 in FDF cells. Chromatin was first precipitated with affinity purified anti-CBX8 antibody and enrichment for INK4a primer set 7 (PS7) was confirmed by q-PCR. The chromatin was divided into four equal fractions and re-precipitated with either rabbit IgG as a negative control, anti-H3K27me3 or anti-CBX8 as positive controls, or anti-CBX7. The enrichment is calculated relative to the original input chromatin. The figure shows the results of a single representative experiment.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2713427&req=5

pone-0006380-g005: CBX7 and CBX8 bind simultaneously to the same INK4a allele.A and B. Sequential ChIP using HDFs that co-express FLAG-tagged mCbx7 and HA-tagged mCbx8. In A, the first ChIP was performed with the FLAG antibody, followed by either anti-HA or irrelevant IgG. In B, the first ChIP was performed with the HA antibody, followed by either anti-FLAG or IgG. The precipitated DNA was analyzed by qPCR with the indicated primer sets. The histograms represent the average and SD values of biological replicates. C and D, Sequential ChIP of endogenous CBX8 and CBX7 in FDF cells. Chromatin was first precipitated with affinity purified anti-CBX8 antibody and enrichment for INK4a primer set 7 (PS7) was confirmed by q-PCR. The chromatin was divided into four equal fractions and re-precipitated with either rabbit IgG as a negative control, anti-H3K27me3 or anti-CBX8 as positive controls, or anti-CBX7. The enrichment is calculated relative to the original input chromatin. The figure shows the results of a single representative experiment.
Mentions: As we work with pools of cells, the ability of several PRC1 components to modulate INK4a expression is open to different interpretations. For example, it is conceivable that different PRC1 complexes regulate the locus in different cells or that several PRC1 complexes congregate at the locus in every cell. To try to distinguish between these possibilities, we set out to determine whether CBX7 and CBX8 can be detected on the same piece of DNA, using a ChIP-reChIP strategy. HDFs were co-infected with retroviruses encoding FLAG-tagged mCbx7 and HA-tagged mCbx8 to generate pools of drug-resistant cells expressing both proteins. An initial ChIP was performed using the FLAG antibody and the chromatin was eluted from the beads with the FLAG peptide. The recovered chromatin was then immunoprecipitated with the HA antibody or with an irrelevant IgG and subjected to qPCR analysis with a subset of the primer sets described in Figure 3A. The signal was quantified relative to the input material used in the first ChIP. As illustrated in Figure 5A, mCbx8 was substantially enriched at the INK4a locus following a mCbx7 ChIP. In the reciprocal experiment, the chromatin was first precipitated with the HA antibody and eluted with HA peptide. Again, it was clear that mCbx7 was enriched in the chromatin precipitated through mCbx8 (Fig. 5B), implying that mCbx7 and mCbx8 bind simultaneously to the same fragment of chromatin.

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