Critical role of the ubiquitin ligase activity of UHRF1, a nuclear RING finger protein, in tumor cell growth

Abstract

Early cellular events associated with tumorigenesis often include loss of cell cycle checkpoints or alteration in growth signaling pathways. Identification of novel genes involved in cellular proliferation may lead to new classes of cancer therapeutics. By screening a tetracycline-inducible cDNA library in A549 cells for genes that interfere with proliferation, we have identified a fragment of UHRF1 (ubiquitin-like protein containing PHD and RING domains 1), a nuclear RING finger protein, that acts as a dominant negative effector of cell growth. Reduction of UHRF1 levels using an UHRF1-specific shRNA decreased growth rates in several tumor cell lines. In addition, treatment of A549 cells with agents that activated different cell cycle checkpoints resulted in down-regulation of UHRF1. The primary sequence of UHRF1 contains a PHD and a RING motif, both of which are structural hallmarks of ubiquitin E3 ligases. We have confirmed using an in vitro autoubiquitination assay that UHRF1 displays RING-dependent E3 ligase activity. Overexpression of a GFP-fused UHRF1 RING mutant that lacks ligase activity sensitizes cells to treatment with various chemotherapeutics. Taken together, our results suggest a general requirement for UHRF1 in tumor cell proliferation and implicate the RING domain of UHRF1 as a functional determinant of growth regulation.

Figure 1.

Identification of an antiproliferative fragment of UHRF1. The fragment (amino acid positions 506–642) was identified as a hit that showed both reduction of cell growth in the absence of doxycycline (Dox, a tetracycline analog) and normal cell growth in the presence of Dox. The position of the fragment in the full-length UHRF1 protein is indicated as an arrow and the amino acid sequence of the fragment is shown. The primary sequence of UHRF1 contains a UBQ domain (ubiquitin like, AA 14–89), a PHD domain (AA 330–379), a YDG/SRA domain (AA 427–599), and a RING finger domain (AA 724–762). A549.tTA cells were stained with the cell tracker dye, DiI, infected with tet-regulatable retroviruses expressing GFP or the GFP fused UHRF1 fragment, and incubated at 37°C for 5 d. The effect of the fragment on proliferation was assessed with comparison of the DiI intensity distribution between the GFP-positive population (gating on subpopulations consisting of the GFP-high expressers; blue lines and red lines in the figure) and the GFP-negative population (black lines) in the same sample. The localization of the GFP fused UHRF1 fragment and GFP alone in A549.tTA cells is shown in the right panels.

Figure 2.

Down-regulation of UHRF1 in H1299 cells results in G1 and G2/M cell cycle arrest. (A) Reduction of UHRF1 protein in H1299 cells treated with oligofectamine or transfected with siRNAs against either luciferase or UHRF1. Measurement was performed 24 h after transfection. Lactate dehydrogenase was used as the loading control. (B) Flow cytometric analysis of siRNA transfected H1299 cells pulsed with BrdU 72 h after transfection and then stained with FITC-conjugated α-BrdU and propidium iodide. DNA content is indicated on the x-axis and extent of BrdU incorporation is indicated on the y-axis. Arrows indicate increased G1 and G2/M phase cell populations for the UHRF1-transfected cells compared with the controls. Color coding in the plots goes in the order from blue to green to red, with red representing the highest cell density. (C) Graphical representation of flow cytometric data from B.

Figure 3.

Retroviral mediated delivery of UHRF1-specific shRNA is antiproliferative in A549, HeLa, and H1299 cells. (A–C) GFP positivity profiles for A549 (A), HeLa (B), and H1299 (C) cells infected with retrovirus expression either UHRF1-specific shRNA or control luciferase (Lux) shRNA. The number of GFP-positive cells at each time point is plotted relative to the %GFP-positive cells at day 3. (D) Reduction of UHRF1 protein levels in cells infected with either retrovirus expressing UHRF1-specific shRNA or retrovirus-encoding control luciferase shRNA. Three days after infection, cells were sorted using the GFP marker present in the retroviral vector and lysed in gel running buffer, and then Western blot analysis was performed for the presence of UHRF1 protein and the loading control, GAPDH.

Figure 4.

Quantitative analysis of UHRF1 mRNA in several tumor cell lines (A) and primary tumors (B and C). The real-time PCR method was used for quantification of UHRF1 mRNA, as described in Materials and Methods. Data are shown as a mean (indicated as bars or circles) ± SD of triplicate reactions. Each circle represents a RNA sample from an individual patient. Expression levels of RNA from tumor tissues and tumor-associated normal tissues are shown as closed circles and open circles, respectively.

Figure 5.

UHRF1 displays RING-dependent autoubiquitination activity in vitro. (A) 293T cells were transfected using a myc-GFP-UHRF1 expression construct. Forty-eight hours after transfection, cells were harvested and treated as described in Materials and Methods. The UHRF1-transfected lysates were split into four aliquots that were subjected to different combinations of enzymes required for the ubiquitination reaction. Recombinant APC2/APC11 was used as a positive control. Gel was probed using α-Flag antibody to look at extent of ubiquitination and also with α-Myc to look at protein expression levels. (B) 293T cells were transfected using myc-GFP-UHRF1 wild-type and RING finger mutant protein expression constructs and then treated as described in Materials and Methods. Gel was probed with α-Flag antibody to look at polyubiquitination (B, top panel) and also with α-Myc to look at protein expression levels (B, bottom panel). (C) Purified recombinant His₆-UHRF1 wild-type, H741A, and ΔRING proteins expressed in SF9 cells. (D) Recombinant His₆-UHRF1 proteins or APC2/APC11 were incubated with recombinant E1, UbcH5, and flag-ubiquitin in a 96-well Ni(II) coated plate. After washing to remove unbound proteins, incorporated flag-ubiquitin was detected using an anti-flag primary antibody and an HRP-conjugated secondary antibody.

Figure 6.

Overexpression of GFP-UHRF1 ΔRING mutant in A549 cells sensitizes cells to the effects of different chemotherapeutics. (A) GFP positivity profile for A549.tTA cells infected with retroviruses expressing GFP, GFP-UHRF1 wild-type, or GFP-UHRF1 ΔRING mutant proteins. The number of GFP-positive cells at each time point is plotted relative to the %GFP-positive cells at day 4. (B–E) GFP positivity profiles for A549.tTA cells infected with the same retroviruses and then treated with different drugs as described in Materials and Methods. The number of GFP-positive cells at each time point is plotted relative to the %GFP-positive cells in an untreated sample on the same day.

Figure 7.

Cellular survival upon etoposide treatment requires the RING domain of UHRF1. (A) Schematic diagram of deletion mutants used in domain analysis. (B) GFP positivity profile for A549.tTA cells infected with retroviruses expressing different GFP-fused deletion mutants of UHRF1. The number of GFP-positive cells at each time point is plotted relative to the %GFP-positive cells at day 3. (C) GFP positivity profiles for A549.tTA cells infected with the same retroviruses and then treated with etoposide as described in Materials and Methods. The number of GFP-positive cells at each time point is plotted relative to the %GFP-positive cells in an untreated sample on the same day.

Figure 8.

Both GFP-UHRF1 and GFP-UHRF1 ΔRING localize to the nucleus. A549.tTA cells were infected with either GFP-UHRF1 wild-type or GFP-UHRF1 ΔRING retrovirus. Three days postinfection, cells were fixed using 3.7% paraformaldehyde in PBS, stained with Hoechst 33342, washed with PBS, and imaged in a six-well dish using an inverted fluorescence microscope equipped with a CCD camera. Localization of GFP-UHRF1 is shown in A and E, localization of GFP-UHRF1 ΔRING is shown in C and G, and DNA staining is shown in B, D, F, and H.

Figure 9.

UHRF1 mRNA (A) and protein (B) is down-regulated in response to genotoxic and cytotoxic agents in A549 cells. A549 cells were treated with different agents and then 24 h after initiation of treatment, cells were harvested for either quantitative PCR analysis of UHRF1 mRNA or Western blot analysis of UHRF1 protein and the loading control, actin. Data for PCR analysis are shown as a mean ± SD of triplicate reactions. Cells were treated with 100 μM etoposide or 40 μM cis-platinum for 3 h at 37°C and washed with PBS, and then fresh medium was added. Other drugs were left in the medium until cells were harvested (20 nM taxol, 1 mM hydroxyurea, 330 nM nocodazole, and 3 mU/ml bleomycin).