The UBC9 E2 SUMO conjugating enzyme binds the PR-Set7 histone methyltransferase to facilitate target gene repression.

PR-Set7/Set8/KMT5a is a chromatin-modifying enzyme that specifically monomethylates lysine 20 of histone H4 (H4K20me1). In this study we attempted to identify PR-Set7-interacting proteins reasoning that these proteins would provide important insights into the role of PR-Set7 in transcriptional regulation. Using an unbiased yeast two-hybrid approach, we discovered that PR-Set7 interacts with the UBC9 E2 SUMO conjugating enzyme. This interaction was confirmed in human cells and we demonstrated that PR-Set7 was preferentially modified with SUMO1 in vivo. Further in vitro studies revealed that UBC9 directly binds PR-Set7 proximal to the catalytic SET domain. Two putative SUMO consensus sites were identified in this region and both were capable of being SUMOylated in vitro. The absence of either or both SUMO sites did not perturb nuclear localization of PR-Set7. By employing whole genome expression arrays, we identified a panel of genes whose expression was significantly altered in the absence of PR-Set7. The vast majority of these genes displayed increased expression strongly suggesting that PR-Set7 predominantly functions as a transcriptional repressor. Importantly, the reduction of UBC9 resulted in the consistent derepression of several of these newly identified genes regulated by PR-Set7. Our findings indicate that direct interaction with UBC9 facilitates the repressive effects of PR-Set7 at specific target genes, most likely by SUMOylating PR-Set7

The K110 and K131 SUMOylation sites are dispensable for nuclear localization of PR-Set7.

<p>HEK 293 cells transfected with GFP-PR-Set7 wild type, K110R, K131R or K110R/K131R mutants (green) were fixed, stained with DAPI (blue) and visualized using a Zeiss Axio Imager upright fluorescence microscope.</p

PR-Set7 is selectively modified by SUMO1 in cells and SUMOylated at K110 or K131 <i>in vitro</i>.

<p>(A) HEK 293 cells were co-transfected with the indicated expression plasmids. Cell extracts were prepared in the presence of NEM 2 days post-transfection, followed by immunoprecipitations using an anti-HA antibody. HA-bound samples were fractionated by SDS-PAGE prior to Western analysis using anti-FLAG or anti-HA antibodies. (B) Two putative PR-Set7 SUMOylation sites, one perfect and one partial, were identified using SUMOsp 2.0 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022785#pone.0022785-Ren1" target="_blank">[40]</a>. ClustalX was used to align the sequence surrounding the putative SUMO sites of PR-Set7 in various organisms. Conserved residues are shaded and red asterisks mark K110 and K131. (C) Recombinant wild type PR-Set7, N-terminal (aa1–191) or C-terminal (aa192–352) fragments were used as substrates in <i>in vitro</i> SUMOylation assays. Reactions were fractionated by SDS-PAGE gel followed by Western analysis using anti-His and anti-SUMO1 antibodies. Asterisk (*) denotes an unknown contaminating protein. (D) Recombinant wild type PR-Set7, K110R, K131R or K110/131R double mutant were used as substrates in <i>in vitro</i> SUMOylation assays. Reactions were fractionated by SDS-PAGE followed by Western analysis using anti-PR-Set7 antibodies.</p

UBC9 directly binds the N-terminal region of PR-Set7.

<p>(A) The indicated recombinant fusion proteins were expressed in <i>E. coli</i>, purified by affinity chromatography and fractionated by SDS-PAGE. (B) Purified recombinant S-tag-PR-Set7 was incubated with GST-UBC9 or GST alone prior to an S-tag immunoprecipitation. Western analysis of the bound material was performed using UBC9 and His antibodies. (C) ³⁵S-labeled PR-Set7 was incubated with GST-UBC9 or GST alone prior to GST pull downs. PR-Set7 binding was determined by autoradiography. Western analysis of the input (In) and bound (B) fractions were performed using GST antibodies to confirm equal loading. (D) Purified recombinant His-tagged N- and C-terminal PR-Set7 proteins were incubated with GST-UBC9 or GST alone prior to GST pull downs. Western analysis of the bound fractions was performed using His and GST antibodies.</p

Genes whose expression change >2-fold in cells lacking PR-Set7.

<p>Genes whose expression change >2-fold in cells lacking PR-Set7.</p

UBC9 transiently interacts with PR-Set7 in cells.

<p>(A) HEK 293 cell lysates expressing FLAG-PR-Set7 or FLAG-null were immunoprecipitated with an UBC9 antibody. Western analysis of the input (In) and bound (B) fractions were analyzed using FLAG or UBC9 antibodies. (B) HEK 293 cells co-transfected with the indicated plasmids were crosslinked using 10 µM BMH prior to FLAG-immunoprecipitations. Western analysis was performed using Myc or FLAG antibodies on the input (In), unbound (U) and bound (B) fractions.</p

Yeast two-hybrid screen identifies UBC9 as a PR-Set7-interacting protein.

<p>(A) Schematic representation of the different Gal4-DBD-PR-Set7 bait proteins used for the yeast two-hybrid screen and the recovered AD-UBC9. (B) The indicated plasmids were co-transformed into AH109 yeast strain and grown on medium lacking: tryptophan and leucine (DDO), and histidine (TDO) and adenine with addition of X-alpha-galactosidase (QDO). Growth on TDO and QDO indicates interaction between PR-Set7 and UBC9. (C) Western analysis of HA-immunoprecipitations from yeast co-transformants expressing the indicated plasmids using PR-Set7 and UBC9 antibodies on the input (In), unbound (U) and bound (B) fractions.</p

Concerted Activities of Distinct H4K20 Methyltransferases at DNA Double-Strand Breaks Regulate 53BP1 Nucleation and NHEJ-Directed Repair

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 nonhomologous 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