The retinoblastoma 1 (RB1) protein is a well-studied tumour suppressor protein. It regulates the transcription of cell cycle genes through physical interaction with the E2F family of transcription factors and by recruiting chromatin-modifying enzymes to specific promoters. A new study by Blasco and colleagues in the April issue of Nature Cell Biology now shows that RB1 also maintains constitutive pericentric and telomeric heterochromatin — the inactive form of chromatin — with the potential of non-selectively repressing gene expression. This adds a new and more global tool to the repertoire of tumour-suppressor functions of RB1.

To address the role of the RB1 family — which comprises RB1, retinoblastoma-like 1 (RBL1) and RBL2 — in genome stability and chromosome structure, the authors used mouse embryonic fibroblasts (MEFs) from single-, double- (DKO) and triple-knockout (TKO) animals. After several passages, aneuploid cells were already evident in DKO and TKO MEFs, and after 20 passages, TKO cells established tetraploidy. These chromosome segregation defects were confirmed by fluorescence in situ hybridization (FISH) analysis, which showed chromosomes with four sister chromatids ('butterfly chromosomes').

Investigating changes in the chromatin structure of TKO cells in more detail, Blasco and colleagues found reduced DNA methylation, increased acetylation of histone 3 (H3), and decreased tri-methylation of histone 4 at lysine 20 (H4K20). As hypermethylation of DNA and histones, as well as the hypoacetylation of histones, are hallmarks of constitutive heterochromatin, the authors investigated the effect of RB1 proteins on pericentric and telomeric heterochromatin. Indeed, they found decreased DNA methylation and tri-methylation of H4K20 at pericentric heterochromatin in TKO cells. Using a dominant-negative mutant of E2F, the authors excluded a possible involvement of transcriptional changes caused by the absence of an RB1-E2F interaction.

As tri-methylation of H4K20 at pericentric chromatin is known to be mediated by the histone methyltransferases (HMTases) Suv4-20h1 and Suv4-20h2, the authors focused on potential interactions of these enzymes with RB1. First, they showed that a lack of RB1 didn't alter the expression levels of Suv4-20h. By overexpressing fluorescently tagged HMTases they confirmed that these enzymes were properly recruited to constitutive heterochromatin in TKO cells. These overexpressed HMTases could rescue the decreased H4K20 tri-methylation, so the authors concluded that this phenotype was the result of decreased Suv4-20h activity, indicating that RB1 proteins stabilize H4K20 tri-methylation by these HMTases. Finally, the authors showed direct binding of RB1, RBL1 and RBL2 to Suv4-20h1 and Suv4-20h2 in vitro, but not in vivo.

Altogether, this new study impressively shows that RB1 proteins are not only transcriptional repressors of specific target genes, but also have a role in the assembly of constitutive heterochromatin. Loss of RB1 therefore not only results in de-repression of specific promoters, but also in the global loss of a repressive chromatin state. Given this, RB1 seems to have made a quantum jump from a specific to a global tumour suppressor.