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A novel zinc finger protein Zfp277 mediates transcriptional repression of the Ink4a/arf locus through polycomb repressive complex 1.

Negishi M, Saraya A, Mochizuki S, Helin K, Koseki H, Iwama A - PLoS ONE (2010)

Bottom Line: Zfp277 binds to the Ink4a/Arf locus in a Bmi1-independent manner and interacts with polycomb repressor complex (PRC) 1 through direct interaction with Bmi1.Our findings implicate Zfp277 in the transcriptional regulation of the Ink4a/Arf locus and suggest that the interaction of Zfp277 with Bmi1 is essential for the recruitment of PRC1 to the Ink4a/Arf locus.Our findings also highlight dynamic regulation of both Zfp277 and PcG proteins by the oxidative stress pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan.

ABSTRACT

Background: Polycomb group (PcG) proteins play a crucial role in cellular senescence as key transcriptional regulators of the Ink4a/Arf tumor suppressor gene locus. However, how PcG complexes target and contribute to stable gene silencing of the Ink4a/Arf locus remains little understood.

Methodology/principal findings: We examined the function of Zinc finger domain-containing protein 277 (Zfp277), a novel zinc finger protein that interacts with the PcG protein Bmi1. Zfp277 binds to the Ink4a/Arf locus in a Bmi1-independent manner and interacts with polycomb repressor complex (PRC) 1 through direct interaction with Bmi1. Loss of Zfp277 in mouse embryonic fibroblasts (MEFs) caused dissociation of PcG proteins from the Ink4a/Arf locus, resulting in premature senescence associated with derepressed p16(Ink4a) and p19(Arf) expression. Levels of both Zfp277 and PcG proteins inversely correlated with those of reactive oxygen species (ROS) in senescing MEFs, but the treatment of Zfp277(-/-) MEFs with an antioxidant restored the binding of PRC2 but not PRC1 to the Ink4a/Arf locus. Notably, forced expression of Bmi1 in Zfp277(-/-) MEFs did not restore the binding of Bmi1 to the Ink4a/Arf locus and failed to bypass cellular senescence. A Zfp277 mutant that could not bind Bmi1 did not rescue Zfp277(-/-) MEFs from premature senescence.

Conclusions/significance: Our findings implicate Zfp277 in the transcriptional regulation of the Ink4a/Arf locus and suggest that the interaction of Zfp277 with Bmi1 is essential for the recruitment of PRC1 to the Ink4a/Arf locus. Our findings also highlight dynamic regulation of both Zfp277 and PcG proteins by the oxidative stress pathways.

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Forced expression of Bmi1 does not repress the Ink4a-Arf genes in Zfp277−/− MEFs.A. Bmi1 does not rescue Zfp277−/− MEFs from premature senescence. Wild-type and Zfp277−/− MEFs were transduced with either a control or a Bmi1 retrovirus and cell growth was monitored every three days by replating at 1×105 cells/plate. Cumulative cell numbers are shown as the mean ± SD for three independent triplicate experiments. B. Bmi1 does not repress the Ink4a-Arf genes in Zfp277−/− MEFs. Wild-type and Zfp277−/− MEFs were transduced with either a control or a Bmi1 retrovirus and the expression of p19Arf was detected by Western blot analysis. Tubulin was used as a loading control. C. ChIP analysis of Bmi1 in Bmi1-transduced Zfp277−/− MEFs. Zfp277−/− MEFs transduced with either a control or a Bmi1 retrovirus were subjected to ChIP analyses using an anti-Bmi1 antibody and control IgG. The ChIP analysis of Bmi1 in wild-type MEFs at passage 2 is shown as a control. Percentages of input DNA are shown as the mean ± S.D. for three independent experiments. Statistical significance was determined with Student's t-test; **p<0.01, ***p<0.001.
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pone-0012373-g005: Forced expression of Bmi1 does not repress the Ink4a-Arf genes in Zfp277−/− MEFs.A. Bmi1 does not rescue Zfp277−/− MEFs from premature senescence. Wild-type and Zfp277−/− MEFs were transduced with either a control or a Bmi1 retrovirus and cell growth was monitored every three days by replating at 1×105 cells/plate. Cumulative cell numbers are shown as the mean ± SD for three independent triplicate experiments. B. Bmi1 does not repress the Ink4a-Arf genes in Zfp277−/− MEFs. Wild-type and Zfp277−/− MEFs were transduced with either a control or a Bmi1 retrovirus and the expression of p19Arf was detected by Western blot analysis. Tubulin was used as a loading control. C. ChIP analysis of Bmi1 in Bmi1-transduced Zfp277−/− MEFs. Zfp277−/− MEFs transduced with either a control or a Bmi1 retrovirus were subjected to ChIP analyses using an anti-Bmi1 antibody and control IgG. The ChIP analysis of Bmi1 in wild-type MEFs at passage 2 is shown as a control. Percentages of input DNA are shown as the mean ± S.D. for three independent experiments. Statistical significance was determined with Student's t-test; **p<0.01, ***p<0.001.

Mentions: Forced expression of Bmi1 in MEFs extends their life span through tight gene silencing of the Ink4a/Arf locus [16], [38]. Therefore, we asked whether Bmi1 could extend the life span of Zfp277−/− MEFs. Forced expression of Bmi1 did not abrogate the premature senescence of Zfp277−/− MEFs at early passages (Figure 5A). Western blot analysis revealed that exogenous Bmi1 failed to repress the expression of p19Arf (Figure 5B). Furthermore, ChIP analysis of Bmi1 demonstrated that the forced expression of Bmi1 only minimally promoted the binding of Bmi1 to the Ink4a/Arf locus in Zfp277−/− MEFs (P-2) (Figure 5C). These findings suggest that Zfp277 is required for recruitment of Bmi1 to or its stable accumulation at the Ink4a/Arf locus in MEFs.


A novel zinc finger protein Zfp277 mediates transcriptional repression of the Ink4a/arf locus through polycomb repressive complex 1.

Negishi M, Saraya A, Mochizuki S, Helin K, Koseki H, Iwama A - PLoS ONE (2010)

Forced expression of Bmi1 does not repress the Ink4a-Arf genes in Zfp277−/− MEFs.A. Bmi1 does not rescue Zfp277−/− MEFs from premature senescence. Wild-type and Zfp277−/− MEFs were transduced with either a control or a Bmi1 retrovirus and cell growth was monitored every three days by replating at 1×105 cells/plate. Cumulative cell numbers are shown as the mean ± SD for three independent triplicate experiments. B. Bmi1 does not repress the Ink4a-Arf genes in Zfp277−/− MEFs. Wild-type and Zfp277−/− MEFs were transduced with either a control or a Bmi1 retrovirus and the expression of p19Arf was detected by Western blot analysis. Tubulin was used as a loading control. C. ChIP analysis of Bmi1 in Bmi1-transduced Zfp277−/− MEFs. Zfp277−/− MEFs transduced with either a control or a Bmi1 retrovirus were subjected to ChIP analyses using an anti-Bmi1 antibody and control IgG. The ChIP analysis of Bmi1 in wild-type MEFs at passage 2 is shown as a control. Percentages of input DNA are shown as the mean ± S.D. for three independent experiments. Statistical significance was determined with Student's t-test; **p<0.01, ***p<0.001.
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Related In: Results  -  Collection

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pone-0012373-g005: Forced expression of Bmi1 does not repress the Ink4a-Arf genes in Zfp277−/− MEFs.A. Bmi1 does not rescue Zfp277−/− MEFs from premature senescence. Wild-type and Zfp277−/− MEFs were transduced with either a control or a Bmi1 retrovirus and cell growth was monitored every three days by replating at 1×105 cells/plate. Cumulative cell numbers are shown as the mean ± SD for three independent triplicate experiments. B. Bmi1 does not repress the Ink4a-Arf genes in Zfp277−/− MEFs. Wild-type and Zfp277−/− MEFs were transduced with either a control or a Bmi1 retrovirus and the expression of p19Arf was detected by Western blot analysis. Tubulin was used as a loading control. C. ChIP analysis of Bmi1 in Bmi1-transduced Zfp277−/− MEFs. Zfp277−/− MEFs transduced with either a control or a Bmi1 retrovirus were subjected to ChIP analyses using an anti-Bmi1 antibody and control IgG. The ChIP analysis of Bmi1 in wild-type MEFs at passage 2 is shown as a control. Percentages of input DNA are shown as the mean ± S.D. for three independent experiments. Statistical significance was determined with Student's t-test; **p<0.01, ***p<0.001.
Mentions: Forced expression of Bmi1 in MEFs extends their life span through tight gene silencing of the Ink4a/Arf locus [16], [38]. Therefore, we asked whether Bmi1 could extend the life span of Zfp277−/− MEFs. Forced expression of Bmi1 did not abrogate the premature senescence of Zfp277−/− MEFs at early passages (Figure 5A). Western blot analysis revealed that exogenous Bmi1 failed to repress the expression of p19Arf (Figure 5B). Furthermore, ChIP analysis of Bmi1 demonstrated that the forced expression of Bmi1 only minimally promoted the binding of Bmi1 to the Ink4a/Arf locus in Zfp277−/− MEFs (P-2) (Figure 5C). These findings suggest that Zfp277 is required for recruitment of Bmi1 to or its stable accumulation at the Ink4a/Arf locus in MEFs.

Bottom Line: Zfp277 binds to the Ink4a/Arf locus in a Bmi1-independent manner and interacts with polycomb repressor complex (PRC) 1 through direct interaction with Bmi1.Our findings implicate Zfp277 in the transcriptional regulation of the Ink4a/Arf locus and suggest that the interaction of Zfp277 with Bmi1 is essential for the recruitment of PRC1 to the Ink4a/Arf locus.Our findings also highlight dynamic regulation of both Zfp277 and PcG proteins by the oxidative stress pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan.

ABSTRACT

Background: Polycomb group (PcG) proteins play a crucial role in cellular senescence as key transcriptional regulators of the Ink4a/Arf tumor suppressor gene locus. However, how PcG complexes target and contribute to stable gene silencing of the Ink4a/Arf locus remains little understood.

Methodology/principal findings: We examined the function of Zinc finger domain-containing protein 277 (Zfp277), a novel zinc finger protein that interacts with the PcG protein Bmi1. Zfp277 binds to the Ink4a/Arf locus in a Bmi1-independent manner and interacts with polycomb repressor complex (PRC) 1 through direct interaction with Bmi1. Loss of Zfp277 in mouse embryonic fibroblasts (MEFs) caused dissociation of PcG proteins from the Ink4a/Arf locus, resulting in premature senescence associated with derepressed p16(Ink4a) and p19(Arf) expression. Levels of both Zfp277 and PcG proteins inversely correlated with those of reactive oxygen species (ROS) in senescing MEFs, but the treatment of Zfp277(-/-) MEFs with an antioxidant restored the binding of PRC2 but not PRC1 to the Ink4a/Arf locus. Notably, forced expression of Bmi1 in Zfp277(-/-) MEFs did not restore the binding of Bmi1 to the Ink4a/Arf locus and failed to bypass cellular senescence. A Zfp277 mutant that could not bind Bmi1 did not rescue Zfp277(-/-) MEFs from premature senescence.

Conclusions/significance: Our findings implicate Zfp277 in the transcriptional regulation of the Ink4a/Arf locus and suggest that the interaction of Zfp277 with Bmi1 is essential for the recruitment of PRC1 to the Ink4a/Arf locus. Our findings also highlight dynamic regulation of both Zfp277 and PcG proteins by the oxidative stress pathways.

Show MeSH
Related in: MedlinePlus