LRIF1 interacts with HP1α to coordinate accurate chromosome segregation during mitosis

Abstract

Heterochromatin protein 1α (HP1α) regulates chromatin specification and plasticity during cell fate decision. Different structural determinants account for HP1α localization and function during cell division cycle. Our earlier study showed that centromeric localization of HP1α depends on the epigenetic mark H3K9me3 in interphase, while its centromeric location in mitosis relies on uncharacterized PXVXL-containing factors. Here, we identified a PXVXL-containing protein, ligand-dependent nuclear receptor-interacting factor 1 (LRIF1), which recruits HP1α to the centromere of mitotic chromosomes and its interaction with HP1α is essential for accurate chromosome segregation during mitosis. LRIF1 interacts directly with HP1α chromoshadow domain via an evolutionarily conserved PXVXL motif within its C-terminus. Importantly, the LRIF1-HP1α interaction is critical for Aurora B activity in the inner centromere. Mutation of PXVXL motif of LRIF1 leads to defects in HP1α centromere targeting and aberrant chromosome segregation. These findings reveal a previously unrecognized direct link between LRIF1 and HP1α in centromere plasticity control and illustrate the critical role of LRIF1-HP1α interaction in orchestrating accurate cell division.

Figure 1

LRIF1 is a novel interacting partner of HP1α protein. (A) Thymidine-synchronized interphase HeLa cells (I) or nocodazole-treated mitotic HeLa cells (M) were extracted with Triton X-100-containing buffer. Clarified cell lysates were subjected to immunoprecipitation with LRIF1 antibody and control rabbit IgG, and separated by SDS-PAGE followed by western blotting with corresponding antibodies. Note that LRIF1 immunoprecipitation (IP) brought down HP1α (lane 5 and 6). (B) Schematic drawing of LRIF1 truncation mutants. (C) GST-HP1α recombinant protein on glutathione-agarose was used as an affinity matrix to absorb MBP-tagged LRIF1 and its deletion mutants. Samples were fractionated by SDS-PAGE, followed by Coomassie Brilliant Blue (CBB) staining (top panel). Western blotting using an anti-MBP antibody confirmed that both full-length (FL) and C-terminal (CT) LRIF1 proteins directly bound to HP1α (bottom panel).

Figure 2

LRIF1 is required for accurate chromosome segregation in mitosis. (A) HeLa cells were transfected with LRIF1 siRNA, TIP60 siRNA (as a positive control), or negative control siRNA and mCherry-H2B for live cell imaging. Representative phenotypes were shown. Scale bar, 10 μm. (B–E) Quantification of mitotic phenotypes in A. Cells exhibiting unaligned chromosomes and failing to align at the metaphase plate within 60 min after nuclear envelope breakdown (NEBD) were considered to be misaligned. Scatter plot of the time from NEBD to anaphase onset was shown. At least 26 cells per group (n = 26, control siRNA; n = 35, LRIF1 siRNA; n = 35, TIP60 siRNA) were examined from three independent experiments. Data represent mean ± SEM and statistical significance was tested by two-sided t-test and represented by asterisks corresponding to *P < 0.05, **P < 0.01.

Figure 3

LRIF1 colocalizes with HP1α at centromere in mitosis. (A) HeLa cells were transfected with LRIF1 siRNA and synchronized with nocodazole followed by chromosome spread, fixation, and immunofluorescence staining. Scale bar, 10 μm. (B) Quantification of LRIF1 fluorescence intensity (normalized to ACA) at kinetochores in LRIF1-depleted cells. Data represent mean ± SEM and were examined with two-sided t-test. A total of 50 kinetochores were examined from three independent experiments. ***P < 0.001. (C) HeLa cells were synchronized with nocodazole followed by chromosome spread, fixation, and immunofluorescence staining with antibodies of LRIF1 (red), Hec1 (green), and ACA (blue). Scale bar, 10 μm. (D) Plot profile of LRIF1, ACA, and Hec1 fluorescence intensity across the kinetochore pair. (E) HeLa cells were synchronized with nocodazole followed by chromosome spread, fixation, and immunofluorescence staining with antibodies of LRIF1 (red), HP1α (green), and ACA (blue). Scale bar, 10 μm. (F) Plot profile of LRIF1 and HP1α fluorescence intensity across the kinetochore pair.

Figure 4

LRIF1–HP1α interaction is essential for accurate mitosis. (A) Real-time imaging of chromosome movement in cells expressing LRIF1 siRNA and RNAi-resistant GFP-LRIF1 FL (full-length) or its mutant GFP-LRIF1 FLM (full-length mutant). To visualize chromosome movement with a DeltaVision system, cells were co-transfected with mCherry-H2B. Scale bar, 10 μm. (B) Statistical analysis of chromosome bridges in A. At least 26 cells per group (n = 30, LRIF1 FL; n = 26, LRIF1 FLM) were examined from three independent experiments. (C) Statistical analysis of mitotic delay judged by the intervals between NEBD and anaphase onset in A (n = 30, LRIF1 FL; n = 26, LRIF1 FLM). Data represent mean ± SEM and statistical significance was tested by two-sided t-test and represented by asterisks corresponding to *P < 0.05, **P < 0.01.

Figure 5

Perturbation of LRIF1–HP1α interaction compromises the accuracy of chromosome segregation. (A) CBB staining of SDS-PAGE gel showed the quality and quantities of the purified recombinant TAT-GFP and TAT-GFP-LRIF1^Δ (amino acids 565–600). (B) HeLa cells expressing FLAG-HP1α and GFP-LRIF1 were subjected to immunoprecipitation (IP) with FLAG antibody in the presence of TAT-GFP or TAT-GFP-LRIF1^Δ for 4 h and immunoblotted with FLAG and GFP antibodies, respectively. Note that the endogenous HP1α–LRIF1 interaction was perturbed by the addition of TAT-GFP-LRIF1^Δ. (C) HeLa cells expressing mCherry-H2B were synchronized with thymidine and released for 8 h to reach G2/M phase. Cells were cultured in DMEM with 2.5 μM TAT-GFP (upper panels) or TAT-GFP-LRIF1^Δ (lower panels) at 37°C for 30 min before image collection. Live cell images were taken every 3 min. Note that TAT-GFP-LRIF1^Δ-treated cells failed to fulfill accurate chromosome alignment and segregation. Scale bar, 10 μm. (D and E) Quantitative analyses of the mitotic progression as a function of LRIF1–HP1α association. The accurate chromosome segregation was compromised in cells treated with TAT-GFP-LRIF1^Δ (2.5 μM), which is comparable to what was seen in LRIF1-suppressed cells. At least 35 cells per group (n = 40, TAT-GFP; n = 35, TAT-GFP-LRIF1^Δ) were examined from three independent experiments. Data represent mean ± SEM and were examined with two-sided t-test. **P < 0.01.

Figure 6

LRIF1 is essential for HP1α localization to the centromere. (A) Representative immunofluorescence images of HeLa cells transfected with control or LRIF1 siRNA for 48 h. Scale bar, 10 μm. (B) Quantitative analyses of the fluorescence intensity of HP1α in the centromere compared with that of ACA. A total of 100 kinetochores were examined from three independent experiments. Data represent mean ± SEM and were examined with two-sided t-test. **P < 0.01. (C) Model for LRIF1-mediated HP1α recruitment at the centromeres during mitosis. See Discussion section for details.