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. 2006 Dec 18;175(6):869-80.
doi: 10.1083/jcb.200604005. Epub 2006 Dec 11.

Loss of linker histone H1 in cellular senescence

Ryo Funayama  1 , Motoki Saito, Hiroko Tanobe, Fuyuki Ishikawa
Affiliations

Loss of linker histone H1 in cellular senescence

Ryo Funayama et al. J Cell Biol. .

Abstract

Cellular senescence is a tumor-suppressing mechanism that is accompanied by characteristic chromatin condensation called senescence-associated heterochromatic foci (SAHFs). We found that individual SAHFs originate from individual chromosomes. SAHFs do not show alterations of posttranslational modifications of core histones that mark condensed chromatin in mitotic chromosomes, apoptotic chromatin, or transcriptionally inactive heterochromatin. Remarkably, SAHF-positive senescent cells lose linker histone H1 and exhibit increased levels of chromatin-bound high mobility group A2 (HMGA2). The expression of N-terminally enhanced green fluorescent protein (EGFP)-tagged histone H1 induces premature senescence phenotypes, including increased levels of phosphorylated p53, p21, and hypophosphorylated Rb, and a decrease in the chromatin-bound endogenous histone H1 level but not in p16 level accumulation or SAHF formation. However, the simultaneous ectopic expression of hemagglutinin-tagged HMGA2 and N-terminally EGFP-tagged histone H1 leads to significant SAHF formation (P < 0.001). It is known that histone H1 and HMG proteins compete for a common binding site, the linker DNA. These results suggest that SAHFs are a novel type of chromatin condensation involving alterations in linker DNA-binding proteins.

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Figures

Figure 1.
Figure 1.
A single SAHF originates from a single chromosome. (A) Similar sizes of SAHFs and inactive X chromosomes. The top two figures show a single mock-transfected WI-38 nucleus stained with DAPI (left) or anti-macroH2A antibody (right). The inactive X chromosomes are indicated by arrowheads. Two separate RasG12V-transfected WI-38 SAHF nuclei stained with DAPI are shown at the bottom. (B) Mock- or RasG12V-transfected WI-38 cells were stained with anti–CENP-B antibody and DAPI. Schematic drawings illustrating the localization of SAHFs and CENP-B foci are shown (right). (C) Percentages of CENP-B foci that colocalized with (+) or excluded (−) SAHF signals (n = 418 derived from 16 senescent cells). Error bars show the SD from the mean of three independent experiments. (D) Percentages of SAHFs that contained zero, one, or more than one CENP-B foci (n = 665 derived from 24 senescent cells). The mean number of SAHFs per optical section was 27.7 ± 5.8 (mean ± SD). (E) RasG12V-transfected senescent WI-38 cells were hybridized with a probe specific for chromosome 12 (Chr12) and stained with DAPI. The probe stained two equal-sized metaphase chromosomes in WI-38 cell metaphase spreads, showing its specificity (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200604005/DC1). Enlarged images of regions A and B are optical sections at 0.4-μm intervals (right). Arrowheads indicate the overlapping of a single SAHF and chromosome 12. The results are representative of observations of five independent cells. Bars, 10 μm.
Figure 2.
Figure 2.
SAHFs are surrounded by the ICD. (A) Mock- or RasG12V-transfected WI-38 cells were stained with anti-RNA polymerase II antibody and DAPI. (B) Percentages of SAHFs that overlapped with (+) or excluded (−) anti-RNA polymerase II signals (n = 341 derived from 13 senescent cells). Error bars represent SD. (C) Senescent cells were incubated with bromo-labeled UTP (BrU) and stained with anti-BrdU antibody. Enlarged images of the boxed areas are shown in bottom panels. No signals were observed when unlabeled UTP was used instead of BrU, when cells were treated with 1 μg/ml α-amanitin (the concentration that inhibits RNA polymerase II), or when they were treated with RNase A (Fig. S1 B, available at http://www.jcb.org/cgi/content/full/jcb.200604005/DC1). Bars, 10 μm.
Figure 3.
Figure 3.
SAHFs do not show histone modifications that characterize three types of chromatin condensation. (A) Mock- or RasG12V-transfected WI-38 cells as well as HeLa cells irradiated with 100 Jm−2 UV rays (HeLa-UV) were stained with antihistone antibodies and DAPI. Mitotic WI-38 nuclei and apoptotic HeLa nuclei are shown by arrowheads and arrows, respectively. (B) WI-38 cells were mock- or RasG12V-transfected and harvested at the indicated time points after drug selection. HeLa cells were untreated (non-treat), treated with 100 ng/ml colcemid, or irradiated with 100 Jm−2 UV rays (UV). Histones were prepared by acid extraction and analyzed by immunoblotting. Percentages of SAHF-positive cells are shown (bottom; n = 300). Bars, 10 μm.
Figure 4.
Figure 4.
Histone H1 is lost from the senescent cell. (A) Scheme showing the fractionation of WI-38 cells to isolate chromatin fractions. Hoechst 33342 staining of the chromatin-rich fraction (fraction N2) showed that SAHFs are retained in the nuclei present in this fraction (not depicted). (B) Coomassie staining of fractions C, N1, and N2 derived from mock- and RasG12V-transfected WI-38 cells. The 37-kD protein band from which peptide sequences of human histone H1 were obtained (right) is indicated with an arrow. (C) Chromatin-rich fractions (N2) were prepared as shown in A and analyzed by Coomassie staining and immunoblotting. AE fractions prepared from mock-transfected cells were used for comparison. The amount of loaded proteins was standardized with core histones (vertical bold line). Arrowheads indicate the positions of histone H1. The band of which signal intensity appeared to increase in RasG12V-transfected cells (lane 9, open circle) was detected in the absence of the first antibody, indicating that it is unrelated to histone H1 (Fig. S2 A, available at http://www.jcb.org/cgi/content/full/jcb.200604005/DC1). (D) Histones were prepared from mock- or RasG12V-transfected WI-38 cells by acid extraction and analyzed by Coomassie staining and immunoblotting. HeLa cells with or without colcemid treatment were used for comparison. (E) Quiescent cells were prepared by culturing mock-transfected cells in 0.1% FBS for 1 wk. WCEs were prepared from mock-transfected, RasG12V-transfected, or serum-starved quiescent cells and analyzed by immunoblotting. AE fractions obtained from mock-transfected cells were also examined. Two bands that showed slightly slower and faster migration than the 37-kD band were detected in RasG12V-transfected cells (lane 3; open and filled circles, respectively). However, these bands were also detected in the absence of the first antibody, indicating that they are unrelated to histone H1 (Fig. S2 B). (F) WCEs were prepared from mock- or RasG12V-transfected cells at the indicated time points after drug selection. Samples were analyzed by immunoblotting.
Figure 5.
Figure 5.
Histone H1 is lost from various types of senescent cells. (A) Mock- or RasG12V-transfected cells (WI-38, MRC-5, IMR-90, and BJ) were fractionated as shown in Fig. 4 A at day 5 after drug selection. AE and cytoplasmic fractions (C) were analyzed by immunoblotting. The amounts of loaded proteins were calibrated by anti-H2B or antiactin signals, respectively. Transfected cells were scored for the percentages of BrdU- and SAHF-positive cells at harvest time (bottom; n = 200). (B) WI-38 cells were induced to senesce by the retroviral expression of RasG12V or a constitutive active form of MKK6 (MKK6EE). Replicative senescent cells were analyzed at 57 population doubling levels, at which cells could not reach confluence for 3 wk. Quiescent cells were prepared by culturing the mock-transfected cells in 0.1% FBS for 1 wk. WCEs were analyzed by immunoblotting. The percentages of BrdU- and SAHF-positive cells are indicated (bottom; n = 200).
Figure 6.
Figure 6.
Loss of histone H1 from senescent cell chromatin is posttranslationally regulated. (A) Mock- and RasG12V-transfected WI-38 cells in 10% FBS and quiescent cells prepared by culturing noninfected WI-38 cells in 0.1% FBS for 1 wk were analyzed for mRNA expressions of HIST1H1D (encoding histone H1.3) and GAPDH by RT-PCR from total RNA in the presence (lanes 1–3) or absence (lanes 4–6) of reverse transcriptase (RT). (B) Cells were stained with DAPI (left) or propidium iodide and were analyzed for DNA contents with a laser-scanning cytometer (right). Although RasG12V-transfected cells completely stopped dividing, the laser-scanning cytometer analysis showed a large percentage of the cells in S phase. It is not clear whether this reflects cell cycle arrest in S phase or whether the laser-scanning cytometer did not accurately measure DNA contents in senescent cells, presumably as a result of the highly irregular patterns of DNA staining. (C) AE fractions were prepared from cells described in A and were analyzed by Coomassie staining (left) and immunoblotting (right). Bars, 10 μm.
Figure 7.
Figure 7.
Ectopic expression of histone H1 does not prevent RasG12V-induced senescence. WI-38 cells were first infected with retrovirus (retro) or lentivirus (lenti) expressing histone H1 under the control of the indicated promoters and were infected with RasG12V-expressing retrovirus. Cell lysates were prepared from transfected cells at day 7 after drug selection and analyzed by immunoblotting. Transfected cells were scored for the percentages of BrdU- and SAHF-positive cells at day 3 after drug selection (bottom; n = 200). CMV, cytomegalovirus; LTR, long terminal repeat.
Figure 8.
Figure 8.
Ectopic expression of N-terminally EGFP-tagged histone H1 induces premature senescence. (A) Nuclear localization of histone H1 fusion proteins. Schematic structures of histone H1 (histone H1.3) fusion proteins are indicated (top). Transfected WI-38 cells were examined for EGFP and stained with anti-HA antibody (HA) and DAPI (DNA) at day 2 after drug selection. Images are projections of whole nuclei. (B) Cell lysates were prepared from transfected cells at day 3 after drug selection and analyzed by immunoblotting. The amounts of loaded proteins were calibrated by anti-H2B signals. Endogenous histone H1 (H1) and H1 fusion proteins were analyzed using anti-H1 sheep antibody. (C) The number of transfected cells was scored at the indicated time points after drug selection. Error bars represent SD. (D) Transfected cells were scored for the percentages of BrdU- (top) and SA–β-gal–positive cells (bottom) at days 3 and 5 after drug selection, respectively (n = 200). (E) Cell lysates were prepared from transfected cells at day 3 after drug selection and analyzed by immunoblotting. The amounts of loaded proteins were calibrated by antiactin signals. (F) Cell lysates were analyzed as described in B and E. The percentages of BrdU- and SAHF-positive cells are indicated (bottom; n = 200). Bars, 10 μm.
Figure 9.
Figure 9.
Coexpression of HA-tagged HMGA2 and N-fusion histone H1 induces SAHF formation. (A) AE fractions were prepared from mock- and RasG12V-transfected WI-38 cells and analyzed by Coomassie staining and immunoblotting. A protein band having an apparent molecular mass of 22 kD from which peptide sequences of human HMGA2 were obtained is indicated with an arrowhead. (B) AE fractions were prepared from mock- and RasG12V-transfected WI-38 cells at the indicated time points after drug selection and were analyzed by immunoblotting. Histone H1 was analyzed using anti-H1 rabbit antibody. (C) Cell lysates were prepared from transfected WI-38 cells at day 7 after drug selection and were analyzed by immunoblotting as described in Fig. 8 B. CMV, cytomegalovirus; LTR, long terminal repeat. (D) Transfected WI-38 cells were analyzed as described in B. Histone H1 was analyzed using anti-H1 sheep antibody. The percentages of SAHF-positive cells are indicated (bottom; n = 200). (E) Transfected WI-38 cells were scored for the percentage of SAHF-positive cells at day 3 after drug selection (top; n = 200). *, P < 0.001 by t test. Error bars represent SD. Transfected WI-38 cells were analyzed as described in Fig. 8 B (bottom). (F) Representative images of SAHF-positive cells. Transfected WI-38 cells were stained with DAPI at day 3 after drug selection. Arrowheads indicate SAHF-positive nuclei. (G) A model of the role of histone H1 dissociation and HMGA2 accumulation in inducing growth arrest and SAHF formation. p53-p21-pRb–dependent growth arrest is mediated by either histone H1–free chromatin itself or signaling elicited by unbound histone H1. SAHFs are formed when histone H1 is replaced with HMGA2, which occurs only when RasG12V is expressed or N-fusion H1 and HMGA2 are coexpressed. Bars, 10 μm.
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