Depletion of chromatin-bound HP1 proteins makes primary cells exquisitely sensitive to apoptotic killing by HDIs. (A) Phase-contrast images of control or HP1βΔN stably expressing WI38 cells treated with or without 0.5 μM TSA for 48 h. (B) FACS analysis of control or HP1βΔN stably expressing WI38 cells treated with or without 0.5 μM TSA for the indicated times. (C) TUNEL analysis of control or HP1βΔN stably expressing WI38 cells treated with 0.5 μM TSA for 30 h. (D) Time-lapse images of H2B-GFP-infected control or HP1βΔN stably expressing WI38 cells treated with or without 1 μM TSA. Yellow arrows point to the lost chromosomes in TSA-treated HP1βΔN stably expressing cells. Scale bars, 10 μm.

A dominant-negative HP1 mutant exacerbates the effect of chromatin-disrupting agents on kinetochore structure. (A and B) Detergent preextracted mock or HA-tagged HP1βΔN stably expressing WI38 cells were immunostained or Western blotted with the indicated antibodies. None of the anti-HP1 antibodies recognized truncated HP1βΔN (see Fig. S3A in the supplemental material). (C) Mock or HA-tagged HP1βΔN stably expressing WI38 cells treated with 1 μM TSA for 15 h and stained with antibodies to HA, ACA, and HP1β. (D) Mock or HP1βΔN stably expressing WI38 cells treated with or without 1 μM TSA for 15 h and stained with antibodies to ACA and hMis12. Scale bars, 10 μm.

Relocalization of macroH2A caused by chromatin-disrupting agents is mediated by the histone chaperone HIRA but is independent of histone H3 variant, H3.3. (A) WI38 cells were transfected with siRNAs to HIRA, H3.3, or a control luciferase siRNA by using nucleofection and then treated with 1 μM TSA for 8 h (siH3.3) or 5 μM AzaC for 72 h (siHIRA and siLuc) and subjected to immunostaining with the indicated antibodies. (B and C) A total of 100 cells from panel A were scored for colocalizing macroH2A1.2 and ACA foci. Comparable results were obtained, regardless of whether the cells were treated with TSA or AzaC. Scale bars, 10 μm.

Chromatin-disrupting agents cause relocalization of HP1 and macroH2A proteins to pericentromeres. (A) WI38 cells treated with or without TSA (1 μM) for 15 h and stained with the indicated antibodies. Yellow arrow marks weak colocalization between HP1α and ACA in control cells. (B) GFP-HP1γ-transfected WI38 cells were treated with or without TSA (1 μM) for 15 h, and GFP was directly visualized. (C) WI38 cells were treated with or without 1 μM TSA for 15 h and immunostained with anti-HP1β antibodies, followed by FISH to detect pericentromeric satellite III DNA sequences. (D) ChIP with control immunoglobulin G (IgG) or HP1γ antibody to detect the associated pericentromeric satellite III sequence in WI38 cells. (E) WI38 cells mock treated (control) or treated with TSA (5 μM) for 15 h or AzaC (5 μM) for 72 h and stained with the indicated antibodies. The large domain of macroH2A staining is the inactive X chromosome. Scale bars, 10 μm.

Recruitment of HP1 proteins to pericentromeres is mediated by the histone variant, histone H3.3. (A) WI38 cells were infected with retroviruses encoding HA-histone H3.1 and HA-histone H3.3, treated with or without TSA (1 μM), and then subjected to ChIP analysis with anti-HA or control antibodies to detect association with satellite III pericentromeric DNA. (B) WI38 cells were transfected, by nucleofection, with a pool of two siRNAs targeted to the histone H3.3A and H3.3B mRNAs or a control siRNA to luciferase. Expression of the indicated genes was analyzed by reverse transcription-PCR. (C) WI38 cells from panel B were treated with 1 μM TSA for 8 h and stained with antibodies to HP1β and ACA. (D) A total of 100 cells from panel C were scored for colocalizing HP1β and ACA foci. The mean of three independent experiments is given. Scale bars, 10 μm.

The recruitment of HP1 proteins to pericentromeres is mediated by the histone chaperone HIRA. (A) WI38 cells were infected with a retrovirus encoding HIRA(520-1017), treated for 8 h with TSA (1 μM), and stained with antibodies to ACA or HP1β as indicated. (B) A total of 100 cells from panel A, after treatment with TSA or sodium butyrate (NaBu), were scored for colocalizing ACA and HP1β foci. (C) WI38 cells were transfected with siRNAs to HIRA or luciferase by nucleofection, treated with TSA (1 μM) for 8 h, and then stained with antibodies to ACA or HP1β. (D) A total of 100 cells from panel C were scored for colocalizing HP1β and ACA foci. The mean of three independent experiments is given. Cell extracts were Western blotted with antibodies to HIRA and actin. siHIRA#2 and siHIRA#4 are two distinct siRNAs to HIRA. (E) WI38 cells were treated with TSA as indicated and subjected to ChIP analysis with antibodies to HIRA (WC15 and D34) or appropriate controls to detect association with satellite III pericentromeric DNA. Scale bars, 10 μm.

Chromatin-disrupting agents delay progression through M-phase, accompanied by defects in kinetochore structure and activation of the mitotic checkpoint. (A) WI38 cells were treated without and with TSA (1 μM) and Western blotted to detect total acetylated histone H3. (B) Same as in panel A, but sonicated chromatin was subjected to ChIP with anti-acetyl H3, anti-H3K9Me2, or anti-H3K9Me3 antibodies as indicated to detect associated pericentromeric satellite III DNA sequence. (C) FACS analysis of WI38 cells treated with 1 μM TSA and 8 μM apicidin for 24 h and 5 μM AzaC for 72 h in either 21% (ambient) or 2% (approximating physiological) oxygen. WI38 cells grow faster in 2% oxygen and so have a larger G₂/M population in the absence of CDAs. (D) Time-lapse images of H2B-GFP-expressing WI38 cells treated with or without TSA (1 μM). The yellow arrowhead points to an unaligned chromosome in metaphase, and the yellow arrow points to a lagging chromosome in anaphase. Time is expressed as “hours.minutes”. (E) hMis12 and Bub1 staining of mitotic WI38 cells treated with or without TSA (1 μM) for 15 h. The yellow arrowheads point to hMis12 foci at kinetochores. The yellow arrows point to the enriched Bub1 foci at the kinetochores of unaligned chromosomes. Scale bars, 10 μm.