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.

Expression of the kinetochore protein Hec1 during the cell cycle in normal and cancer cells and its regulation by the pRb pathway

“…In starvation experiments, observed in RB or RB silenced cells. 33,34,36,37 In this scenario, Hec1 overexpression may contribute to the reported capacity of pRb depleted cells to undergo chromosome missegregation as a consequence of defects in centromere/kinetochore structure that are responsible for kinetochore-microtubule misattachments, 38 such as merotelic attachments. 6,39 Consistently, recent work has demonstrated that loss of pRB causes centromere dysfunction associated with premature loss of centromeric cohesion, impaired localization of the condensin II subunit CAP-D3 to centromeres and formation of lagging chromosomes at anaphase.…”

The retinoblastoma protein and PML collaborate to organize heterochromatin and silence E2F-responsive genes during senescence

“…Surprisingly, pRB-LXCXE interactions are critical for stress-responsive growth arrest, but not in reversible growth arrest or cell cycle arrest in development, even though each paradigm of proliferative control is pRB-dependent. 10,29,30 Notably, senescent Rb1 ΔL/ΔL MEFs can re-initiate DNA synthesis in response to ectopic E2F1 expression, whereas wildtype cells are resistant. 10 Furthermore, serially cultured Rb1 ΔL/ΔL MEFs escape senescence more readily than their wild-type counterparts, suggesting that defective silencing of proliferative genes can compromise the long-term stability of senescence arrest.…”

“…36,37 We don't favor this interpretation, because our previous studies have indicated that HDACcontaining complexes interact with pRB in a GST-RB pull-down assay irrespective of the growth state of the cells. 29 Based on this line of reasoning, we expect that PML-pRB interactions are likely quite direct.…”

“…29 The Rb1 ΔL allele encodes I746A, N750A, and M754A substitutions, and is detected by PCR genotyping as previously reported. 29 All cells were cultured in growth medium that contained Dulbecco modified Eagle medium (DMEM) supplemented with L-glutamine, streptomycin, penicillin, and 10% fetal bovine serum. Cells were maintained in a humidified chamber at 37 °C with 5% CO 2 .…”

Folding of a Cyclin Box

“…Cellular factors containing the LXCXE motif such as SWI/SNF complexes, DNMT1, Rbp2, and HDAC1/2 mediate transcriptional repression and chromatin regulation (21,24) by interacting with the LXCXE cleft located in the B subdomain (18) (see Fig. 1).…”