Although the PR-Set7/Set8/KMT5a histone H4 Lys 20 monomethyltransferase (H4K20me1) plays an essential role in mammalian cell cycle progression, especially during G2/M, it remained unknown how PR-Set7 itself was regulated. In this study, we discovered the mechanisms that govern the dynamic regulation of PR-Set7 during mitosis, and that perturbation of these pathways results in defective mitotic progression. First, we found that PR-Set7 is phosphorylated at Ser 29 (S29) specifically by the cyclin-dependent kinase 1 (cdk1)/cyclinB complex, primarily from prophase through early anaphase, subsequent to global accumulation of H4K20me1. While S29 phosphorylation did not affect PR-Set7 methyltransferase activity, this event resulted in the removal of PR-Set7 from mitotic chromosomes. S29 phosphorylation also functions to stabilize PR-Set7 by directly inhibiting its interaction with the anaphase-promoting complex (APC), an E3 ubiquitin ligase. The dephosphorylation of S29 during late mitosis by the Cdc14 phosphatases was required for APC cdh1 -mediated ubiquitination of PR-Set7 and subsequent proteolysis. This event is important for proper mitotic progression, as constitutive phosphorylation of PR-Set7 resulted in a substantial delay between metaphase and anaphase. Collectively, we elucidated the molecular mechanisms that control PR-Set7 protein levels during mitosis, and demonstrated that its orchestrated regulation is important for normal mitotic progression.[Keywords: PR-Set7; APC; Cdk1; Cdc14; cell cycle; chromatin] Supplemental material is available at http://www.genesdev.org.

Increasing evidence indicates that the post-translational modifications of the histone proteins play critical roles in all eukaryotic DNA-templated processes. To gain further biological insights into two of these modifications, the mono- and trimethylation of histone H4 lysine 20 (H4K20me1 and H4K20me3), ChIP-chip experiments were performed to identify the precise genomic regions on human chromosomes 21 and 22 occupied by these two modifications. Detailed analysis revealed that H4K20me1 was preferentially enriched within specific genes; most significantly between the first approximately 5% and 20% of gene bodies. In contrast, H4K20me3 was preferentially targeted to repetitive elements. Among genes enriched in H4K20me3, the modification was typically targeted to a small region approximately 1 kb upstream of transcription start. Our collective findings strongly suggest that H4K20me1 and H4K20me3 are both physically and functionally distinct. We next sought to determine the role of H4K20me1 in transcription since this has been controversial. Following the reduction of PR-Set7/Set8/KMT5a and H4K20me1 in cells by RNAi, all H4K20me1-associated genes analyzed displayed an approximately 2-fold increase in gene expression; H4K20me3-associated genes displayed no changes. Similar results were obtained using a catalytically dead dominant negative PR-Set7 indicating that H4K20me1, itself, is essential for the selective transcriptional repression of H4K20me1-associated genes. Furthermore, we determined that the H4K20me1-associated DNA sequences were sufficient to nucleate H4K20me1 and induce repression in vivo. Our findings reveal the molecular mechanisms of a mammalian transcriptional repressive pathway whereby the DNA sequences within specific gene bodies are sufficient to nucleate the monomethylation of H4K20 which, in turn, reduces gene expression by half.

SUMMARY Although selective binding of 53BP1 to dimethylated histone H4 lysine 20 (H4K20me2) at DNA double strand breaks (DSBs) is a necessary and pivotal determinant of non-homologous end joining (NHEJ)-directed repair, the enzymes that generate H4K20me2 at DSBs were unclear. Here we determined that the PR-Set7 monomethyltransferase (H4K20me1) regulates de novo H4K20 methylation at DSBs. Rapid recruitment of PR-Set7 to DSBs was dependent on the NHEJ Ku70 protein and necessary for NHEJ-directed repair. PR-Set7 monomethyltransferase activity was required, but insufficient, for H4K20me2 and 53BP1 nucleation at DSBs. We determined that PR-Set7-mediated H4K20me1 facilitates Suv4-20 methyltransferase recruitment and catalysis to generate H4K20me2 necessary for 53BP1 binding. The orchestrated and concerted activities of PR-Set7 and Suv4-20 were required for proficient 53BP1 nucleation and DSB repair. This report identifies PR-Set7 as an essential component of NHEJ and implicates PR-Set7 as a central determinant of NHEJ-directed repair early in mammalian DSB repair pathway choice.

PR-Set7/Set8/KMT5a is the sole histone H4 lysine 20 monomethyltransferase (H4K20me1) in metazoans and is essential for proper cell division and genomic stability. We unexpectedly discovered that normal cellular levels of monomethylated histone H3 lysine 9 (H3K9me1) were also dependent on PR-Set7, but independent of its catalytic activity. This observation suggested that PR-Set7 interacts with an H3K9 monomethyltransferase to establish the previously reported H4K20me1-H3K9me1 trans-tail ‘histone code’. Here we show that PR-Set7 specifically and directly binds the C-terminus of the Riz1/PRDM2/KMT8 tumor suppressor and demonstrate that the N-terminal PR/SET domain of Riz1 preferentially monomethylates H3K9. The PR-Set7 binding domain was required for Riz1 nuclear localization and maintenance of the H4K20me1-H3K9me1 trans-tail ‘histone code’. Although Riz1 can function as a repressor, Riz1/H3K9me1 was dispensable for the repression of genes regulated by PR-Set7/H4K20me1. Frameshift mutations resulting in a truncated Riz1 incapable of binding PR-Set7 occur frequently in various aggressive cancers. In these cancer cells, expression of wild-type Riz1 restored tumor suppression by decreasing proliferation and increasing apoptosis. These phenotypes were not observed in cells expressing either the Riz1 PR/SET domain or PR-Set7 binding domain indicating that Riz1 methyltransferase activity and PR-Set7 binding domain are both essential for Riz1 tumor suppressor function.

Multiple myeloma (MM) is an incurable malignancy of plasma cells. Although multiple myeloma patients often respond to initial therapy, the majority of patients will relapse with disease that is refractory to further drug treatment. Thus, new therapeutic strategies are needed. One common mechanism of acquired drug resistance involves a reduction in the expression or function of the drug target. We hypothesized that the cytotoxic activity of topoisomerase II (topo II) poisons could be enhanced, and drug resistance overcome, by increasing the expression and activity of the drug target, topo II in myeloma cells. To test this hypothesis, we evaluated the cytotoxicity of the anthracenecontaining topo II poison, ethonafide (AMP-53/6-ethoxyazonafide), in combination with the proteasome inhibitor bortezomib (PS-341/Velcade). Combination drug activity studies were done in 8226/S myeloma cells and its drug resistant subclone, 8226/Dox1V. We found that a 24-hour treatment of cells with bortezomib maximally increased topo IIα protein expression and activity, and consistently increased the cytotoxicity of ethonafide in the 8226/S and 8226/Dox1V cell lines. This increase in cytotoxicity corresponded to an increase in DNA double-strand breaks, as measured by the neutral comet assay. Therefore, increasing topo IIα expression through inhibition of proteasomal degradation increased DNA double-strand breaks and enhanced the cytotoxicity of the topo II poison ethonafide. These data suggest that bortezomib-mediated stabilization of topo IIα expression may potentiate the cytotoxic activity of topo II poisons and thereby, provide a strategy to circumvent drug resistance.