Identification of UBN1 and UBN2 as candidate Hpc2 orthologs. (A) Sequence alignment of S. cerevisiae Hpc2p and candidate orthologs from other species. Residues that are identical to Hpc2p are red, and those that are similar are yellow. At any position, at least 70% of residues must be similar for yellow to be applied. (B) Schematic full-length alignment of S. cerevisiae Hpc2p and human UBN1 and UBN2. The HRD from panel A is in pink. Other domains conserved between UBN1 and UBN2 are in yellow and blue. (C) UBN1 and UBN2 mRNAs are expressed in a panel of primary and transformed cell lines, based on RT-PCR analysis. Lane 1, primary WI38 fibroblasts; lane 2, primary IMR90 fibroblasts; lane 3, primary breast epithelial cells; lane 4, primary ovarian epithelial cells; lane 5, A498 renal carcinoma cells; lane 6, M14 melanoma cells; lane 7, MCF7 breast carcinoma cells; lane 8, OVCAR4, ovarian carcinoma cells; lane 9, HL60, leukemia cells. (D) Antibodies to UBN1 detect the UBN1 protein. Lanes 1 and 2, immunoprecipitation of cell extracts with control antibody or rabbit polyclonal pAbUBN1C; lanes 3 and 4, extracts from cells treated with the indicated siRNAs. All lanes were Western blotted with a mouse monoclonal antibody to UBN1. (E) Antibodies to ASF1a and UBN1 both (co)immunoprecipitate ASF1a, HIRA, and UBN1 from IMR90 fibroblasts. (F) Antibodies to UBN1 and HIRA both (co)immunoprecipitate UBN1, HIRA, and ASF1a from WI38 fibroblasts, hMPCs, and U2OS osteosarcoma cells. Input is 1/10 the amount of lysate used for immunoprecipitation. The bracket marks ASF1a, and the arrowhead marks the antibody light chain from the immunoprecipitation.

UBN1 interacts preferentially with HIRA. (A) IMR90 cells were nucleofected with siRNAs to luciferase, HIRA, or UBN1 as indicated. Cell extracts were Western blotted to detect UBN1, HIRA, and ASF1 (a mixture of anti-ASF1 polyclonal antibodies [pAb87 and -88] was used to detect both isoforms of ASF1, ASF1a and ASF1b). RNA was analyzed by RT-PCR for expression of HIRA, UBN1, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase). (B) In vitro-translated ³⁵S-labeled HA-tagged full-length HIRA or ASF1a was incubated with in vitro-translated ³⁵S-labeled UBN1 and immunoprecipitated with anti-HA antibodies.

Mapping of HIRA and UBN1 interaction domains. (A) In vitro-translated ³⁵S-labeled wild-type HA-tagged HIRA (lanes 2 and 7) or the indicated deletion mutants (lanes 3 to 5 and 8 to 10) were incubated with in vitro-translated ³⁵S-labeled full-length wild-type UBN1 or ASF1a and immunoprecipitated with anti-HA antibodies. (B) In vitro-translated ³⁵S-labeled wild-type HA-tagged UBN1 (lane 5) or the indicated UBN1 or UBN2 mutants (lanes 2 to 4) were incubated with in vitro-translated ³⁵S-labeled HIRA(WD) (residues 1 to 400) and immunoprecipitated with anti-HA antibodies. The asterisks mark the HA-UBN1 or HA-UBN2 fusion proteins.

The HRD is an evolutionarily conserved HIRA-binding domain. (A) Sf9 cells were singly or coinfected with baculoviruses to express recombinant His-HIRA(1-405) and/or His-UBN1(1-175). His-tagged proteins were recovered from clarified extracts by IMAC and resolved on a Superdex 200 10/30 size exclusion column. The indicated globular molecular masses (670, 158, 44, and 17 kDa) provide an indication of the apparent hydrodynamic size of the complex but not a measurement of actual molecular mass. The void elution volume is indicated. (B) Purified recombinant GST or GST-Hir1p(WD) (residues 1 to 400) was incubated with ³⁵S-labeled wild-type (Hpc2p) or mutant (Hpc2pΔHRD) Hpc2p as indicated. GST proteins and bound Hpc2p were isolated on glutathione beads.

Point mutations within the UBN1 HRD and the HIRA WD domain disrupt the HIRA/UBN1 and HIRA/UBN2 complexes. (A) The substitution mutants M1 to M8 that were tested for binding in panels B and C. Residues 124 to 170 of UBN1 are shown. (B) In vitro-translated ³⁵S-labeled wild-type HA-tagged UBN1(1-166) or the mutants M1 to M6 were incubated with in vitro-translated ³⁵S-labeled HIRA(WD) and immunoprecipitated with anti-HA antibodies. (C) As for panel B but with mutants M7 and M8 in HA-UBN1(1-166). (D) In vitro-translated ³⁵S-labeled full-length wild-type HA-tagged human HIRA or HA-HIRA(R227K) was incubated with in vitro-translated ³⁵S-labeled full-length UBN1 and ASF1a and then immunoprecipitated with anti-HA antibodies. (E) In vitro-translated ³⁵S-labeled wild-type HA-tagged UBN1 or UBN2 was incubated with in vitro-translated ³⁵S-labeled wild-type human HIRA(WD) or HIRA(WD, R227K) and immunoprecipitated with anti-HA antibodies.

UBN1 is localized to PML bodies in senescent cells, together with HIRA. (A) Growing and senescent (after extended growth in culture) IMR90 fibroblasts were stained with antibodies to HIRA and UBN1. (B) Senescent (after extended growth in culture) IMR90 fibroblasts were stained with antibodies to PML and UBN1. (C) Senescent (after extended growth in culture) IMR90 fibroblasts were stained with antibodies to UBN1 and SP100. (D) IMR90 cells were infected with a retrovirus encoding HA-tagged UBN1 and stained with antibodies to HA and PML. Yellow arrows mark HA-UBN1 in PML bodies. (E) IMR90 cells were nucleofected with an siRNA to UBN1 or a control siRNA (siLuc) and then stained to detect UBN1 and PML.

UBN1 binds to the cyclin A2 gene and HMTase activity. (A) ChIP analysis of the cyclin A2 gene in proliferating IMR90 cells, with control (purified rabbit anti-mouse), anti-acetylated histone H3, or affinity-purified UBN1 antibodies as indicated. (B) Proliferating IMR90 cells were infected with a control retrovirus or a virus encoding HA-tagged UBN1 prior to ChIP analysis of the cyclin A2 gene using anti-HA antibodies. (C) Proliferating IMR90 cells were infected with a control lentivirus or a lentivirus encoding an shRNA to UBN1 and then subjected to ChIP analysis of the cyclin A2 gene with antibodies to UBN1 or control antibodies (melon gel purified from rabbit preimmune serum [PI]). (D) Proliferating IMR90 cells were nucleofected with control siRNA or siRNA to UBN1 and then subjected to ChIP analysis of the cyclin A2 gene with antibodies to UBN1 or control antibodies (PI). (E) ChIP analysis of the cyclin A2 gene, with control and anti-ASF1a antibodies as indicated. (F) Extracts were prepared from IMR90 cells and immunoprecipitated with control or anti-UBN1 antibodies. Immunoprecipitates were incubated with ³H-labeled S-adenosylmethionine and wild-type GST-histone H3 or the indicated GST-histone H3 mutants. The left two lanes were incubated with or without purified recombinant SETDB1 methyltransferase, as indicated. Top panel, autoradiograph; bottom panel, Coomassie blue stain.

Ectopic expression of UBN1 induces senescence. (A) IMR90 cells were infected with a control retrovirus or a virus encoding HA-tagged wild-type UBN1. Infected cells were selected in puromycin. (B) Cells from panel A were stained for expression of senescence-associated (SA) β-galactosidase at 10 days after infection. (C) Quantitation of results from panel B. (D) Cells from panel A were stained with antibodies to HA and HIRA. (E) Quantitation of cells with HIRA foci from panel D. Error bars indicate standard deviations. (F) Cells from panel A were stained with DAPI. (G) Quantitation of cells with SAHF (DAPI foci) from panel F.

UBN1 is required for formation of SAHF. (A) IMR90 cells were infected with control retroviruses or viruses encoding activated Ras or shUBN1, as indicated, and then selected in puromycin. (B) Cells from panel A were scored for formation of SAHF. (C) Representative images of cells from panel A.