Limits...
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.

Show MeSH

Related in: MedlinePlus

Oxidative stress leads to down-regulation of Zfp277.A. H2O2 treatment of MEFs. The whole-cell extract from wild-type MEFs treated with 100 mM H2O2 for 6 hrs was subjected to a Western blot analysis for the proteins indicated on the left. B. N-acetyl cysteine (NAC) treatment of MEFs. Wild-type and Zfp277−/− MEFs (P-2) were cultured in the presence of 10 mM NAC. MEFs were harvested at passage 6 and the levels of Zfp277 and PcG proteins were determined by Western blotting. Tubulin was used as a loading control. C. Quantitative RT-PCR analysis of p19Arf and p16Ink4a mRNA in wild-type and Zfp277−/− MEFs at passage 6 cultured in the presence of 10 mM NAC. mRNA levels were normalized to Hprt1 expression. Expression levels relative to those in wild-type MEFs without NAC treatment are shown as the mean ± SD for three independent experiments. D. NAC treatment rescues the growth of Zfp277−/− MEFs. Wild-type and Zfp277−/− MEFs were cultured with or without 10 µM NAC 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. E. Photomicrographs of wild-type or Zfp277−/− MEFs with or without NAC observed under an inverted microscope. Scale bar represents 200 µm. F. Expression of p19Arf in Zfp277−/− MEFs. The protein levels of p19Arf in wild-type MEFs at the indicated passages were determined by Western blot analyses (left panel). Tubulin was used as a loading control. Quantitative RT-PCR analysis of p19Arf mRNA in wild-type MEFs at passages 2 and 6 (right panel). mRNA levels were normalized to Hprt1 expression. Relative expression levels are shown as the mean ± SD for three independent experiments. G. MG132 treatment of MEFs. MEFs at passage 4 were treated with 10 µM MG132 for 6 hrs and Zfp277 protein levels were determined by Western blotting. Tubulin was used as a loading control. Statistical significance was determined with Student's t-test; **p<0.01.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2927437&req=5

pone-0012373-g003: Oxidative stress leads to down-regulation of Zfp277.A. H2O2 treatment of MEFs. The whole-cell extract from wild-type MEFs treated with 100 mM H2O2 for 6 hrs was subjected to a Western blot analysis for the proteins indicated on the left. B. N-acetyl cysteine (NAC) treatment of MEFs. Wild-type and Zfp277−/− MEFs (P-2) were cultured in the presence of 10 mM NAC. MEFs were harvested at passage 6 and the levels of Zfp277 and PcG proteins were determined by Western blotting. Tubulin was used as a loading control. C. Quantitative RT-PCR analysis of p19Arf and p16Ink4a mRNA in wild-type and Zfp277−/− MEFs at passage 6 cultured in the presence of 10 mM NAC. mRNA levels were normalized to Hprt1 expression. Expression levels relative to those in wild-type MEFs without NAC treatment are shown as the mean ± SD for three independent experiments. D. NAC treatment rescues the growth of Zfp277−/− MEFs. Wild-type and Zfp277−/− MEFs were cultured with or without 10 µM NAC 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. E. Photomicrographs of wild-type or Zfp277−/− MEFs with or without NAC observed under an inverted microscope. Scale bar represents 200 µm. F. Expression of p19Arf in Zfp277−/− MEFs. The protein levels of p19Arf in wild-type MEFs at the indicated passages were determined by Western blot analyses (left panel). Tubulin was used as a loading control. Quantitative RT-PCR analysis of p19Arf mRNA in wild-type MEFs at passages 2 and 6 (right panel). mRNA levels were normalized to Hprt1 expression. Relative expression levels are shown as the mean ± SD for three independent experiments. G. MG132 treatment of MEFs. MEFs at passage 4 were treated with 10 µM MG132 for 6 hrs and Zfp277 protein levels were determined by Western blotting. Tubulin was used as a loading control. Statistical significance was determined with Student's t-test; **p<0.01.

Mentions: In general, culture-induced stress promotes the generation of reactive oxygen species (ROS), the main causative factor for oxidative stress. The balance between the generation and scavenging of ROS is critical for cellular senescence and organismal aging [10], [34]. To assess whether oxidative stress affects the levels of Zfp277 and PcG proteins, we treated MEFs at an early passage (P-2) with hydrogen peroxide (H2O2) which induces the generation of ROS [35]. At 6 hrs after the treatment with H2O2 (100 µM), phosphorylation of Jnk, an indicator of activated oxidative stress pathways, was detected (Figure 3A). This treatment led to a significant reduction in Zfp277 and PcG protein (Bmi1, Mel18, Ring1B and Ezh2) levels. The level of H3K27 trimethylation also decreased. In contrast, expression of a histone H3K27 demethylase Jmjd3 was up-regulated (Figure 3A), consistent with previous results showing Jmjd3 expression to be induced in response to stress and oncogenic signaling [31]. Because Zfp277−/− MEFs undergo premature senescence, we next checked their levels of PcG proteins. As demonstrated in Figure 3B, they were decreased in Zfp277−/− MEFs compared to the passage-matched wild-type MEFs. As expected, the level of reactive oxygen species (ROS) was significantly higher in Zfp277−/− MEFs than the wild-type MEFs at the same passage (Figure S3), suggesting that the loss of Zfp277 leads to enhanced oxidative stress which could promote senescence.


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)

Oxidative stress leads to down-regulation of Zfp277.A. H2O2 treatment of MEFs. The whole-cell extract from wild-type MEFs treated with 100 mM H2O2 for 6 hrs was subjected to a Western blot analysis for the proteins indicated on the left. B. N-acetyl cysteine (NAC) treatment of MEFs. Wild-type and Zfp277−/− MEFs (P-2) were cultured in the presence of 10 mM NAC. MEFs were harvested at passage 6 and the levels of Zfp277 and PcG proteins were determined by Western blotting. Tubulin was used as a loading control. C. Quantitative RT-PCR analysis of p19Arf and p16Ink4a mRNA in wild-type and Zfp277−/− MEFs at passage 6 cultured in the presence of 10 mM NAC. mRNA levels were normalized to Hprt1 expression. Expression levels relative to those in wild-type MEFs without NAC treatment are shown as the mean ± SD for three independent experiments. D. NAC treatment rescues the growth of Zfp277−/− MEFs. Wild-type and Zfp277−/− MEFs were cultured with or without 10 µM NAC 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. E. Photomicrographs of wild-type or Zfp277−/− MEFs with or without NAC observed under an inverted microscope. Scale bar represents 200 µm. F. Expression of p19Arf in Zfp277−/− MEFs. The protein levels of p19Arf in wild-type MEFs at the indicated passages were determined by Western blot analyses (left panel). Tubulin was used as a loading control. Quantitative RT-PCR analysis of p19Arf mRNA in wild-type MEFs at passages 2 and 6 (right panel). mRNA levels were normalized to Hprt1 expression. Relative expression levels are shown as the mean ± SD for three independent experiments. G. MG132 treatment of MEFs. MEFs at passage 4 were treated with 10 µM MG132 for 6 hrs and Zfp277 protein levels were determined by Western blotting. Tubulin was used as a loading control. Statistical significance was determined with Student's t-test; **p<0.01.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2927437&req=5

pone-0012373-g003: Oxidative stress leads to down-regulation of Zfp277.A. H2O2 treatment of MEFs. The whole-cell extract from wild-type MEFs treated with 100 mM H2O2 for 6 hrs was subjected to a Western blot analysis for the proteins indicated on the left. B. N-acetyl cysteine (NAC) treatment of MEFs. Wild-type and Zfp277−/− MEFs (P-2) were cultured in the presence of 10 mM NAC. MEFs were harvested at passage 6 and the levels of Zfp277 and PcG proteins were determined by Western blotting. Tubulin was used as a loading control. C. Quantitative RT-PCR analysis of p19Arf and p16Ink4a mRNA in wild-type and Zfp277−/− MEFs at passage 6 cultured in the presence of 10 mM NAC. mRNA levels were normalized to Hprt1 expression. Expression levels relative to those in wild-type MEFs without NAC treatment are shown as the mean ± SD for three independent experiments. D. NAC treatment rescues the growth of Zfp277−/− MEFs. Wild-type and Zfp277−/− MEFs were cultured with or without 10 µM NAC 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. E. Photomicrographs of wild-type or Zfp277−/− MEFs with or without NAC observed under an inverted microscope. Scale bar represents 200 µm. F. Expression of p19Arf in Zfp277−/− MEFs. The protein levels of p19Arf in wild-type MEFs at the indicated passages were determined by Western blot analyses (left panel). Tubulin was used as a loading control. Quantitative RT-PCR analysis of p19Arf mRNA in wild-type MEFs at passages 2 and 6 (right panel). mRNA levels were normalized to Hprt1 expression. Relative expression levels are shown as the mean ± SD for three independent experiments. G. MG132 treatment of MEFs. MEFs at passage 4 were treated with 10 µM MG132 for 6 hrs and Zfp277 protein levels were determined by Western blotting. Tubulin was used as a loading control. Statistical significance was determined with Student's t-test; **p<0.01.
Mentions: In general, culture-induced stress promotes the generation of reactive oxygen species (ROS), the main causative factor for oxidative stress. The balance between the generation and scavenging of ROS is critical for cellular senescence and organismal aging [10], [34]. To assess whether oxidative stress affects the levels of Zfp277 and PcG proteins, we treated MEFs at an early passage (P-2) with hydrogen peroxide (H2O2) which induces the generation of ROS [35]. At 6 hrs after the treatment with H2O2 (100 µM), phosphorylation of Jnk, an indicator of activated oxidative stress pathways, was detected (Figure 3A). This treatment led to a significant reduction in Zfp277 and PcG protein (Bmi1, Mel18, Ring1B and Ezh2) levels. The level of H3K27 trimethylation also decreased. In contrast, expression of a histone H3K27 demethylase Jmjd3 was up-regulated (Figure 3A), consistent with previous results showing Jmjd3 expression to be induced in response to stress and oncogenic signaling [31]. Because Zfp277−/− MEFs undergo premature senescence, we next checked their levels of PcG proteins. As demonstrated in Figure 3B, they were decreased in Zfp277−/− MEFs compared to the passage-matched wild-type MEFs. As expected, the level of reactive oxygen species (ROS) was significantly higher in Zfp277−/− MEFs than the wild-type MEFs at the same passage (Figure S3), suggesting that the loss of Zfp277 leads to enhanced oxidative stress which could promote senescence.

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