The retinoblastoma protein regulates pericentric heterochromatin

Abstract

The retinoblastoma protein (pRb) has been proposed to regulate cell cycle progression in part through its ability to interact with enzymes that modify histone tails and create a repressed chromatin structure. We created a mutation in the murine Rb1 gene that disrupted pRb's ability to interact with these enzymes to determine if it affected cell cycle control. Here, we show that loss of this interaction slows progression through mitosis and causes aneuploidy. Our experiments reveal that while the LXCXE binding site mutation does not disrupt pRb's interaction with the Suv4-20h histone methyltransferases, it dramatically reduces H4-K20 trimethylation in pericentric heterochromatin. Disruption of heterochromatin structure in this chromosomal region leads to centromere fusions, chromosome missegregation, and genomic instability. These results demonstrate the surprising finding that pRb uses the LXCXE binding cleft to control chromatin structure for the regulation of events beyond the G(1)-to-S-phase transition.

FIG. 1.

Generation of the Rb1^ΔLXCXE mutant mouse strain. (A) The genomic structure of the B-pocket coding region of Rb1 is shown at the top. The targeting vector contained a LoxP-flanked PGK-neo^r cassette inserted into intron 20 and the ΔLXCXE mutations of I740A, N744A, and M748A in exon 22 (marked by a silent PstI site). Homologous recombination resulted in the Rb1^ΔLXCXE-NEO allele that is diagrammed in the middle. The locations of probes used for Southern blotting are shown (H, HindIII; B, BamHI; P, PetI; P^*, the PstI site that marks our mutation). The correctly recombined Rb1^ΔLXCXE allele was generated by breeding chimeric male mice with a Cre-expressing transgenic strain, and the structure of a correctly excised allele is shown at the bottom. (B) Southern blots of representative targeted ES clones are shown, along with DNA from the untargeted J1 cells. Restriction enzymes used to digest genomic DNA and the probes that were used to hybridize to each blot are shown below the autoradiographs. (C) PCR detection of the ΔLXCXE mutations in exon 22, presence of the EIIa-cre transgene, and correct deletion of the PGK-neo^r cassette are shown. Ethidium bromide-stained gels demonstrate amplification of sequences from control ES cell DNA and tails from F₁ founders. The sequence of primer 121 anneals specifically to the altered sequence in exon 22 of Rb1^ΔL. The asterisk next to 27C5 indicates that this DNA sample was supplemented with pMC1-cre to serve as a positive control. Clone 38E8 is an ES line that was generated by transfection with pMC1-cre to delete the neo^r marker and also serves as a positive control. (D) The genotypes of offspring resulting from Rb1^ΔL/+ crosses are shown, with genotype frequencies in brackets.

FIG. 2.

The Rb1^ΔL-encoded protein fails to interact with chromatin-regulating factors and is defective for transcriptional repression. (A) pRb, p107, and p130 protein expression levels were measured in wild-type, Rb1^ΔL/ΔL, and Rb1^−/− MEFs by Western blotting (top), and the ability of GST-E7 to interact with pocket proteins was tested in GST pull-down assays and detected by Western blotting (bottom). The asterisk denotes a cross-reactive band in p130 blots. (B) The ability of pRb^ΔL to interact with E2F transcription factors was detected by electrophoretic mobility shift assay. Extracts from wild-type, Rb1^ΔL/ΔL, and Rb1^−/− cells were incubated with a radiolabeled double-stranded DNA oligonucleotide containing a consensus E2F binding site. Gel shift complexes were competed with unlabeled wild-type and mutant oligonucleotides (lanes 3, 4, 8, 9, 13, and 14). Complexes containing pRb and E2Fs were identified by supershifting with an α-pRb monoclonal antibody (lanes 6, 11, and 16). (C) The identities of proteins coisolated with GST-Rb from nuclear extracts in an LXCXE binding cleft-dependent manner were determined by Western blotting. Nuclear extract is abbreviated as NE. (D) GST-Suv4-20h1 and -2 proteins were used to coprecipitate pRb from wild-type, Rb1^ΔL/ΔL, and Rb1^−/− extracts. Western blots of input and precipitated protein levels are shown. An asterisk denotes background caused by the high-molecular-weight GST-Suv4-20h1 protein. (E) Northern blots were performed on RNA extracted from serum-starved or proliferating MEFs to assess expression of E2F target genes. The blots were probed to quantify message levels for p107, Cyclin E1, Thymidylate synthase, Cyclin E2, Cyclin A2, and ARPP₀ (loading control). Asynch, asynchronous.

FIG. 3.

G₁ progression in Rb1^ΔL/ΔL MEFs. (A) Actively proliferating MEF cultures were pulse-labeled with BrdU, processed for flow cytometry, and analyzed for propidium iodide and BrdU staining. The proportion of cells in each phase of the cell cycle is indicated for each genotype. (B) The relative lengths of G₁ in Rb1^ΔL/ΔL and wild-type cells were compared by replating confluent cultures at low density and pulse-labeling them with BrdU at the indicated time points. The percentage of cells incorporating BrdU is shown for three independent experiments. (C) Forward-scatter analysis of the G₁ cells in panel A was used to compare the relative sizes of cells from the different Rb1 genotypes. The error bars indicate 1 standard deviation from the mean.

FIG. 4.

Mitotic defects in Rb1^ΔL/ΔL MEFs. (A) DAPI-stained mitotic figures from wild-type and Rb1^ΔL/ΔL cells are shown. The percentage of mitoses with lagging chromosomes was tabulated for at least 100 anaphases in each of three independent experiments. (B) Propidium iodide-stained cells were analyzed by flow cytometry for cells with greater than 4 N DNA content to identify aneuploid cells. The percentages of cells in this category are indicated above the bars. The error bars indicate 1 standard deviation from the mean.

FIG. 5.

The spindle checkpoint is unaffected by the Rb1^ΔL mutation. (A) Mad2 expression levels in wild-type, Rb1^−/−, and Rb1^ΔL/ΔL fibroblasts were measured by Western blotting. (B) Wild-type and Rb1^ΔL/ΔL MEFs were cultured in the presence of nocodazole (open symbols) or dimethyl sulfoxide (filled symbols) for the indicated periods of time before being processed for flow cytometry and analyzed for phospho-H3-S10 staining. The circles and triangles indicate wild-type and Rb1^ΔL/ΔL MEFs, respectively. The error bars indicate 1 standard deviation from the mean.

FIG. 6.

Centromere fusions between Rb1^ΔL/ΔL chromosomes. (A) Representative DAPI-stained chromosome spreads for wild-type, Rb1^ΔL/ΔL, and Rb1^−/− are shown. Chromosomes that are joined at the centromere are indicated by an arrow. (B) The number of centromere fusions per metaphase spread is plotted as a frequency histogram; 35 metaphase spreads were examined for each genotype. (C) Centromere and telomere FISH analyses of chromosome fusions. Metaphase chromosome spreads were hybridized with major satellite and telomere probes to visualize their locations in joined chromosomes. The arrow in the leftmost panel indicates a centromere fusion, while the arrow on the right locates the p-arm telomeres nearest to the fusion point.

FIG. 7.

Altered pericentric heterochromatin in Rb1^ΔL/ΔL fibroblasts. (A) Representative fluorescent micrographs of interphase nuclei stained with DAPI and antibodies against trimethylated H4-K20 are shown in the left column. Similarly stained unfixed chromosome spreads for wild-type, Rb1^ΔL/ΔL, and Rb1^−/− are shown on the right. The arrow indicates joined centromeres. (B) Chromatin immunoprecipitations were performed using the antibodies indicated below the gel. Major satellite repeat sequences were PCR amplified and analyzed by ethidium bromide staining of an agarose gel (a negative image of the gel is shown). PCR amplification of these repeats generated both the 74-bp and 308-bp bands. Labels above each gel lane indicate the genotype of chromatin used for that lane. IgG, immunoglobulin G. (C) Major satellite sequences in precipitated chromatin were also analyzed by real-time PCR to quantify differences between wild-type, Rb1^−/−, and Rb1^ΔL/ΔL. The error bars indicate 1 standard deviation from the mean. The asterisk indicates a statistically significant difference (t test; P < 0.001).