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Quantitative assessment of higher‐order chromatin structure of the INK4/ARF locus in human senescent cells

Quantitative assessment of higher‐order chromatin structure of the INK4/ARF locus in human... Somatic fibroblasts can be reprogrammed to senescent cells by oncogenic activation ( Serrano , 1997 ) or to induced pluripotent stem (iPS) cells by the expression of transcription factors associated with pluripotency ( Takahashi , 2007 ). The INK4/ARF locus, which encodes p15 INK4b , p16 INK4a , and ARF genes ( Gil & Peters, 2006 ; Kim & Sharpless, 2006 ), is an inducer of senescence ( Collado , 2007 ) and a barrier for reprogramming into iPS cells ( Banito , 2009 ; Li , 2009 ). In fact, the conversion to iPS cells is strongly impaired by senescent program. The CCCTC‐binding factor CTCF is known to possess essential functions including transcriptional control, chromatin insulation, and chromosomal interactions within the three‐dimensional context of the nucleus ( Phillips & Corces, 2009 ; Ohlsson , 2010 ). Here, we analyzed the role of CTCF in well‐established human cell lines: 201B7 cells, induced from fibroblasts and recognized as the iPS cell standard, human diploid fibroblasts (IMR90), and the same cells undergoing oncogene‐induced senescence after Ras activation (oncogene‐induced senescent, OIS) (Fig. S1, Supporting information). To avoid the variability between the cells, we used early passage IMR90 cells ( Narita , 2003 ) and repeated experiments in other cell lines such as 253G1 cells for iPS cells. Interestingly, CTCF mRNA was remarkably up‐regulated in iPS cells and reversely down‐regulated in OIS cells, compared with that of IMR90 cells ( Fig. 1A ). We also found increased CTCF protein in iPS cells, but decreased levels of expression in senescent cells (Fig. S2, Supporting information). Using the same samples, p15 INK4b , ARF , and p16 INK4a were significantly silenced in iPS cells and IMR90 cells ( Fig. 1A and Fig. S2 , Supporting information). In contrast, as previously shown, p15 INK4b and p16 INK4a were up‐regulated in OIS cells (day 6), with no change in the expression of ARF . 1 CTCF is required for chromosomal conformation at the INK4/ARF locus. (A) Unique expression of CTCF and INK4/ARF genes. (B) Presence of CTCF at IC sites in human diploid fibroblasts (IMR90), Ras‐induced senescent IMR90 cells (oncogene‐induced senescent (OIS) cells), and 201B7 cells derived from human fibroblasts (iPS). CTCF‐enriched sites (IC1, IC2 and IC3) are shown in Fig. S3A (Supporting information). (C–E) The relative interacting frequency with the references IC1/ p15 INK4b (C), IC2/ ARF (D), and IC3 (E) in control and CTCF knockdown IMR90 cells. In chromosome conformation capture (3C) assay, interaction frequencies between the reference and its physically close site were normalized to 1 [IC1‐fragment 6 (C), IC2‐fragment 8 (D) and IC3‐fragment 10 (E)]. Samples without ligation after Eco RI digestion are shown as ligation minus. Primer in each fragment is indicated by small arrow. In the right panel, the radar chart shows the average relative interacting frequencies between the reference (central yellow circle) and each fragment. Values are the means and standard deviations from more than three independent experiments. * P < 0.05, ** P < 0.01. Using genome‐wide CTCF‐binding profiles available at the websites and our published data ( Wendt , 2008 ; Mishiro , 2009 ), there were at least three CTCF‐enriched sites in this locus, named IC1, IC2, and IC3 (Fig. S3, Supporting information). IC1 and IC2 were downstream of the p15 INK4b and ARF transcription start sites, respectively, while IC3 was downstream of p16 INK4a exon 3. Chromatin immunoprecipitation showed that CTCF bound to IC1, IC2, and IC3 sites in all three cell lines ( Fig. 1B ), but did not bind to p16 INK4a exon 1 as a negative control (data not shown). Compared with IMR90 cells, the amount of CTCF decreased at IC1 and IC3 sites in OIS cells. In contrast, CTCF binding was significantly high in iPS cells. Using a chromosome conformation capture (3C) assay, we measured the interaction frequencies of the reference IC1/ p15 INK4b with nine distinct Eco RI fragments in the locus in IMR90 cells ( Fig. 1C ). The IC1 site was colocalized with IC2/ ARF , the p16 INK4a promoter, and IC3 (red line). The IC2/ ARF reference strongly interacted with IC1/ p15 INK4b ( Fig. 1D ). Further, interaction frequencies of the IC3 reference increased at IC1/ p15 INK4b ( Fig. 1E ). These data indicate that IC1/ p15 INK4b , IC2/ ARF , the p16 INK4 promoter, and IC3 are closely localized in nuclei, leading to possible formation of chromatin loops in the INK4/ARF locus (as modeled in Fig. 2D ). Importantly, CTCF knockdown decreased their colocalization (purple line), resulting in increased expression of p15 INK4b and p16 INK4a (Fig. S4A–D, Supporting information). The depletion of the cofactor cohesin RAD21 ( Wendt , 2008 ), which coexisted with CTCF at the IC sites, also induced the INK4/ARF genes (Fig. S4E–G, Supporting information). These data suggest that CTCF complex is involved in the compact chromatin formation at the INK4/ARF locus. 2 Higher‐order chromatin structure of the INK4/ARF locus in reprogrammed cells. (A–C) As described in Fig. 1 , 3C analysis was performed in iPS, IMR90, and oncogene‐induced senescent cells. (D) Somatic fibroblasts show compact chromatin loops for coordinate repression of INK4/ARF genes (repressive compaction), while senescent cells reduce CTCF binding and lose higher‐order chromatin structure leading to activation of p15 INK4b and p16 INK4a (active decompaction). In iPS cells, the silenced INK4/ARF locus has moderately compact chromatin (intermediate compaction). We then performed a 3C assay in IMR90, OIS, and iPS cells ( Fig. 2 ). Using three reference sites (yellow bars), the IC sites and the p16 INK4a promoter closely associated to form compact chromatin in IMR90 cells (red line). However, in OIS cells (green line), interaction frequencies of these references with other sites were remarkably reduced, paralleling CTCF binding decreases at IC1 and IC3 sites, compared with those in IMR90 cells. This suggests that the loose chromatin structure of the INK4/ARF locus occurred in OIS cells with INK4 up‐regulation. In iPS cells (blue line), interactions between IC sites also decreased to some extent. The interactions of the IC2/ ARF with p16 INK4a promoter and IC3 were retained in iPS cells, suggesting that the INK4/ARF locus in iPS cells has an intermediate state of chromatin compaction. We demonstrated that CTCF is crucial for higher‐order chromatin organization in the INK4/ARF locus in reprogrammed cells. As shown in Fig. 2D , we proposed the CTCF‐mediated chromatin conformation model of the INK4/ARF locus: repressive compaction in growing fibroblasts, active decompaction in OIS cells, and intermediate compaction in iPS cells. Especially, OIS cells show a unique chromatin decompaction in the INK4/ARF locus, with marked induction of the INK 4 genes and senescence‐associated nuclear changes (Figs S5 and S6, Supporting information), which may be a barrier for reprogramming to iPS cells. Our study uncovers that the higher‐order chromatin signature provides the identity of senescent cells. Acknowledgments We would like to thank Dr. Hiroyuki Aburatani (University of Tokyo) for previous collaboration. This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, from the Japan Science and Technology Agency (CREST), and from the Naito Foundation (M.N.). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Aging Cell Wiley

Quantitative assessment of higher‐order chromatin structure of the INK4/ARF locus in human senescent cells

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References (12)

Publisher
Wiley
Copyright
© 2012 The Authors. Aging Cell © 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland
ISSN
1474-9718
eISSN
1474-9726
DOI
10.1111/j.1474-9726.2012.00809.x
pmid
22340434
Publisher site
See Article on Publisher Site

Abstract

Somatic fibroblasts can be reprogrammed to senescent cells by oncogenic activation ( Serrano , 1997 ) or to induced pluripotent stem (iPS) cells by the expression of transcription factors associated with pluripotency ( Takahashi , 2007 ). The INK4/ARF locus, which encodes p15 INK4b , p16 INK4a , and ARF genes ( Gil & Peters, 2006 ; Kim & Sharpless, 2006 ), is an inducer of senescence ( Collado , 2007 ) and a barrier for reprogramming into iPS cells ( Banito , 2009 ; Li , 2009 ). In fact, the conversion to iPS cells is strongly impaired by senescent program. The CCCTC‐binding factor CTCF is known to possess essential functions including transcriptional control, chromatin insulation, and chromosomal interactions within the three‐dimensional context of the nucleus ( Phillips & Corces, 2009 ; Ohlsson , 2010 ). Here, we analyzed the role of CTCF in well‐established human cell lines: 201B7 cells, induced from fibroblasts and recognized as the iPS cell standard, human diploid fibroblasts (IMR90), and the same cells undergoing oncogene‐induced senescence after Ras activation (oncogene‐induced senescent, OIS) (Fig. S1, Supporting information). To avoid the variability between the cells, we used early passage IMR90 cells ( Narita , 2003 ) and repeated experiments in other cell lines such as 253G1 cells for iPS cells. Interestingly, CTCF mRNA was remarkably up‐regulated in iPS cells and reversely down‐regulated in OIS cells, compared with that of IMR90 cells ( Fig. 1A ). We also found increased CTCF protein in iPS cells, but decreased levels of expression in senescent cells (Fig. S2, Supporting information). Using the same samples, p15 INK4b , ARF , and p16 INK4a were significantly silenced in iPS cells and IMR90 cells ( Fig. 1A and Fig. S2 , Supporting information). In contrast, as previously shown, p15 INK4b and p16 INK4a were up‐regulated in OIS cells (day 6), with no change in the expression of ARF . 1 CTCF is required for chromosomal conformation at the INK4/ARF locus. (A) Unique expression of CTCF and INK4/ARF genes. (B) Presence of CTCF at IC sites in human diploid fibroblasts (IMR90), Ras‐induced senescent IMR90 cells (oncogene‐induced senescent (OIS) cells), and 201B7 cells derived from human fibroblasts (iPS). CTCF‐enriched sites (IC1, IC2 and IC3) are shown in Fig. S3A (Supporting information). (C–E) The relative interacting frequency with the references IC1/ p15 INK4b (C), IC2/ ARF (D), and IC3 (E) in control and CTCF knockdown IMR90 cells. In chromosome conformation capture (3C) assay, interaction frequencies between the reference and its physically close site were normalized to 1 [IC1‐fragment 6 (C), IC2‐fragment 8 (D) and IC3‐fragment 10 (E)]. Samples without ligation after Eco RI digestion are shown as ligation minus. Primer in each fragment is indicated by small arrow. In the right panel, the radar chart shows the average relative interacting frequencies between the reference (central yellow circle) and each fragment. Values are the means and standard deviations from more than three independent experiments. * P < 0.05, ** P < 0.01. Using genome‐wide CTCF‐binding profiles available at the websites and our published data ( Wendt , 2008 ; Mishiro , 2009 ), there were at least three CTCF‐enriched sites in this locus, named IC1, IC2, and IC3 (Fig. S3, Supporting information). IC1 and IC2 were downstream of the p15 INK4b and ARF transcription start sites, respectively, while IC3 was downstream of p16 INK4a exon 3. Chromatin immunoprecipitation showed that CTCF bound to IC1, IC2, and IC3 sites in all three cell lines ( Fig. 1B ), but did not bind to p16 INK4a exon 1 as a negative control (data not shown). Compared with IMR90 cells, the amount of CTCF decreased at IC1 and IC3 sites in OIS cells. In contrast, CTCF binding was significantly high in iPS cells. Using a chromosome conformation capture (3C) assay, we measured the interaction frequencies of the reference IC1/ p15 INK4b with nine distinct Eco RI fragments in the locus in IMR90 cells ( Fig. 1C ). The IC1 site was colocalized with IC2/ ARF , the p16 INK4a promoter, and IC3 (red line). The IC2/ ARF reference strongly interacted with IC1/ p15 INK4b ( Fig. 1D ). Further, interaction frequencies of the IC3 reference increased at IC1/ p15 INK4b ( Fig. 1E ). These data indicate that IC1/ p15 INK4b , IC2/ ARF , the p16 INK4 promoter, and IC3 are closely localized in nuclei, leading to possible formation of chromatin loops in the INK4/ARF locus (as modeled in Fig. 2D ). Importantly, CTCF knockdown decreased their colocalization (purple line), resulting in increased expression of p15 INK4b and p16 INK4a (Fig. S4A–D, Supporting information). The depletion of the cofactor cohesin RAD21 ( Wendt , 2008 ), which coexisted with CTCF at the IC sites, also induced the INK4/ARF genes (Fig. S4E–G, Supporting information). These data suggest that CTCF complex is involved in the compact chromatin formation at the INK4/ARF locus. 2 Higher‐order chromatin structure of the INK4/ARF locus in reprogrammed cells. (A–C) As described in Fig. 1 , 3C analysis was performed in iPS, IMR90, and oncogene‐induced senescent cells. (D) Somatic fibroblasts show compact chromatin loops for coordinate repression of INK4/ARF genes (repressive compaction), while senescent cells reduce CTCF binding and lose higher‐order chromatin structure leading to activation of p15 INK4b and p16 INK4a (active decompaction). In iPS cells, the silenced INK4/ARF locus has moderately compact chromatin (intermediate compaction). We then performed a 3C assay in IMR90, OIS, and iPS cells ( Fig. 2 ). Using three reference sites (yellow bars), the IC sites and the p16 INK4a promoter closely associated to form compact chromatin in IMR90 cells (red line). However, in OIS cells (green line), interaction frequencies of these references with other sites were remarkably reduced, paralleling CTCF binding decreases at IC1 and IC3 sites, compared with those in IMR90 cells. This suggests that the loose chromatin structure of the INK4/ARF locus occurred in OIS cells with INK4 up‐regulation. In iPS cells (blue line), interactions between IC sites also decreased to some extent. The interactions of the IC2/ ARF with p16 INK4a promoter and IC3 were retained in iPS cells, suggesting that the INK4/ARF locus in iPS cells has an intermediate state of chromatin compaction. We demonstrated that CTCF is crucial for higher‐order chromatin organization in the INK4/ARF locus in reprogrammed cells. As shown in Fig. 2D , we proposed the CTCF‐mediated chromatin conformation model of the INK4/ARF locus: repressive compaction in growing fibroblasts, active decompaction in OIS cells, and intermediate compaction in iPS cells. Especially, OIS cells show a unique chromatin decompaction in the INK4/ARF locus, with marked induction of the INK 4 genes and senescence‐associated nuclear changes (Figs S5 and S6, Supporting information), which may be a barrier for reprogramming to iPS cells. Our study uncovers that the higher‐order chromatin signature provides the identity of senescent cells. Acknowledgments We would like to thank Dr. Hiroyuki Aburatani (University of Tokyo) for previous collaboration. This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, from the Japan Science and Technology Agency (CREST), and from the Naito Foundation (M.N.).

Journal

Aging CellWiley

Published: Jun 1, 2012

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