Werner syndrome protein positively regulates XRCC4-like factor transcription

XRCC4-like factor (XLF) is involved in non-homologous end joining-mediated repair of DNA double-strand breaks (DSBs). Mutations in the WRN gene results in the development of Werner syndrome (WS), a rare autosomal recessive disorder characterized by premature ageing and genome instability. In the present study, it was identified that XLF protein levels were lower in WRN-deficient fibroblasts, compared with normal fibroblasts. Depletion of WRN in HeLa cells led to a decrease of XLF mRNA and its promoter activity. Chromatin immunoprecipitation assays demonstrated that WRN was associated with the XLF promoter. Depletion of XLF in normal human fibroblasts increased the percentage of β-galactosidase (β-gal) staining-positive cells, indicating acceleration in cellular senescence. Taken together, the results suggest that XLF is a transcriptional target of WRN and may be involved in the regulation of cellular senescence.


Introduction
Our genomic DNA is constantly attacked and damaged by normal intracellular metabolic processes and a number of environmental factors, including ultraviolet (UV) light, ionizing radiation (IR), radiomimetic drugs and reactive oxygen species (ROS) (1,2). DNA damage also occurs spontaneously during DNA replication (2). To cope with these lesions, eukaryotic cells have evolved the complex DNA damage response (DDR) machinery (2). DDR senses damaged DNA and activates the cell cycle checkpoint to halt cell cycle progression, allowing time for DNA repair. The DDR process also induces apoptosis or senescence when damaged DNA fails to be repaired (2). Deficiency in DDR leads to mutations, genomic instability and cellular senescence, an enabling characteristic associated with cancer (3).
Of the various forms of DNA damage, DNA double-strand breaks (DSBs) are the most lethal (1). There are two main pathways that are independently responsible for DSB repair in mammalian cells, namely non-homologous end-joining (NHEJ) and homologous recombination (HR) (2). NHEJ is the predominant pathway, which induces the direct ligation of two broken DNA ends together (1,4). NHEJ repair occurs during G0 and G1 phase of the cell cycle, when the extensive sequence homology for repair is absent (1).
The NHEJ pathway is initiated by the rapid association of the Ku70/Ku80 heterodimer to the broken DNA ends (2). Ku70/Ku80 then recruits the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) to form a DNA-PK complex (5). This kinase complex phosphorylates the nuclease Artemis to facilitate the initial processing of ends, and provides protection of the ends required for the following DNA ligation by another complex containing DNA ligase IV, XRCC4 and XRCC4-like factor (XLF; also called Cernunnos or NHEJ1) (2,6,7). XLF is structurally similar to XRCC4 and physically interacts with XRCC4. It is recruited to the DSB site in a Ku-dependent manner at an early stage of NHEJ (8,9) and facilitates XRCC4-mediated joining of blunt ends and several types of mismatched ends, that are non-complementary or partially complementary.
Deficiency in DDR pathways, including the NHEJ pathway, lead to premature ageing (10). Ku70, Ku80 or double knockout mice exhibit a series of age-related phenotypes, shortened life span and accumulation of chromosomal instability (11). Increased spontaneous translocations have also been identified in XLF-deficient murine embryonic stem cells (12). However, the link between XLF and ageing remains to be established.
Werner syndrome (WS) is a rare autosomal recessive progeroid syndrome characterized by the premature onset of multiple age-related disorders, including atherosclerosis, cancer predisposition and premature ageing. The WRN gene, when mutated, causes WS. The majority of the known WRN mutations are nonsense, producing truncated proteins lacking the nuclear localization signal (13). The WRN protein possesses helicase (14) and exonuclease (15) activities, and is important in multiple DNA metabolism pathways including DNA repair, recombination, replication and telomere maintenance (16). During the process of NHEJ, WRN is physically and functionally associated with, and regulated by, the major NHEJ factors, including the Ku70/Ku80 heterodimer, DNA-PKcs and the DNA ligase IV/XRCC4 complex, to optimize DNA endprocessing (17)(18)(19)(20)(21).
WRN also functions in DNA transcription. It promotes RNA polymerase I-dependent transcription of ribosomal RNA (22) and is important in RNA polymerase II (RNA pol II)-dependent transcription (23). Transcription alterations have been identified in human fibroblasts from WS patients (24) and in the cells with RNAi-based short-term knockdown of WRN (25). The efficiency of RNA pol II transcription is reduced by ~50% in WRN-deficient cells compared with that in normal cells. The gene expression profiles in those cells resemble that of fibroblasts derived from old donor patients (24,25). The transcription alterations in WS are specific to sets of certain genes involved in different biological pathways, including DNA repair, DNA replication and cell cycle control (25), and may thus contribute to the development of the WS phenotype.
In the present study, it was identified that XLF was positively modulated by WRN and involved in the regulation of cellular senescence.

Materials and methods
Cell lines, siRNA oligos and antibodies. The human osteosarcoma cell line U2OS, human normal fibroblast cell line WI38, human normal fibroblast cell line GM00637 and human WRN-deficient fibroblast cell line AG11395 were purchased from the American Type Culture Collection (Manassas, VA, USA). The cell lines were cultured in DMEM medium with 10% fetal bovine serum (FBS; Hyclone Laboratories, Inc., Logan, UT, USA) and grown at 37˚C in the presence of 5% CO 2 .
Cell growth assay. WI38 cells were transfected with si-XLF or si-CTR with RNAiMAX. Cells were trypsinized 24 h following transfection and transferred into 6-well plates (1x10 4 cells/well). The cell number was counted every day for 5 days, with triplicated wells being used at each time point.
Western blot analysis. Cell lysates were prepared in 0.5% NP-40 lysis buffer [50 mmol/l Tris (pH 8.0), 250 mmol/l NaCl, 5 mmol/l EDTA, 0.5% NP40] containing protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). The protein concentration was determined using an DC assay kit (Bio-Rad, Hercules, CA, USA). Equal amounts of proteins were resolved on 4-18% gradient SDS-PAGE gels and transferred onto nitrocellulose membranes (Bio-Rad). The blots on nitrocellulose were blocked with 5% non-fat milk in PBST (PBS with 0.05% Tween-20) and were sequentially incubated with primary antibodies as indicated and horseradish peroxidase-conjugated secondary antibodies in 5% non-fat milk in PBST. Blots were washed with PBST following each incubation. The immunoreactive bands were visualized by Amersham Biosciences ECL reagents ((Piscataway, NJ, USA) following the manufacturer's instructions.
Transfections and dual luciferase reporter assays. siRNA oligonucleotide duplex at a final concentration of 40 µM, was transfected into U2OS cells twice with a 24 h interval using oligofectamine (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. Transfectants were used for further experiments 24 h following the secondary transfection. For dual luciferase reporter assays, on the day prior to siRNA transfection, 5x10 4 cells were seeded into each well of 24-well plates. Following the secondary siRNA transfection (4 h), a firefly luciferase reporter construct under the control of the XLF promoter (1 µg) and a Renilla luciferase reporter construct under the control of the TK promoter for normalization of transfection efficiency (10 ng) were co-transfected into cells in triplicate using FuGENE6 (Roche Diagnostics) at a ratio of 1 µg of plasmid to 3 µl of FuGENE6. Luciferase activity was determined with the dual luciferase assay system (Promega Corporation, Madison, WI) 48 h following the first siRNA transfection. Relative light units were determined using a luminometer (microtiter plate luminometer). Experiments were performed at least three times independently and each combination was tested in triplicate wells.

Reverse-transcription PCR and real-time reverse-transcription PCR.
Total RNA was extracted from U2OS cells using the TRIzol reagent (Invitrogen Life Technologies). Reverse transcription (RT)-PCR was performed using the Access Quick RT-PCR system (Promega Corporation) essentially according to the manufacturer's instructions. Real-time PCR was performed using the QuantiTect SYBR-Green PCR kit (Qiagen, Hilden, Germany) and the iQ5 thermal cycler (Bio-Rad). Primers used for amplification of GAPDH were GAPDH: sense, TGG TAT CGT GGA AGG ACT CA and antisense, CCA GAT GAG GCA GGG ATG AT. Primers used for amplification of XLF were XLF: sense, GAG TCC ACG GGT ACT TCA GG and antisense, GGG CCT GTC AAC ATC AAC TT.
Chromatin immunoprecipitation (ChIP). ChIP assays were performed as previously described (27). The ChIP-enriched DNA was amplified by PCR with primer pairs that are specific for the XLF promoter sequence. XLF promoter first fragment corresponds to the position from -596 to -337, primers used were forward: GGT ACC GAA GGG ATA ATG AAT TCT GAT TGG GGA CAG and reverse: GGA TCC TCC GAC CTC ATC CTT TAC CTC TCC TGC TTC. XLF promoter second fragment corresponds to the position from -360 to -90, primers used were forward: GGT ACC GGA GAG GTA AAG GAT GAG GTC GGA CTA TG and reverse: GGA TCC GGC TAG TAG AAG GGT AGT GGC GCG TCT TG. XLF promoter third fragment corresponds to the position from -81 to +169, primers used were forward: GGT ACC GGC CTC TCC TCC ACT TAC CCT GGC CAC TG and reverse: GGA TCC GAC TCG AAC GCG ATT CCA CCT ACC GTC AG.

Results and Discussion
XLF is a transcriptional target of WRN. A candidate gene approach was employed to identify DDR factors in the human fibroblast cell line AG11395A, which was originally isolated from a WRN patient bearing a nonsense mutation in the WRN gene. It was identified that the endogenous XLF protein level was lower in AG11395A cell line than in the control fibroblast cell line GM00637G (Fig. 1A). In U2OS cells, inhibition of WRN expression by two independent siRNA oligos resulted in a decrease of XLF protein levels (Fig. 1B), which were not restored by treatment with the proteasome inhibitor MG132 (Fig. 1C). Real-time RT-PCR assays demonstrated that the mRNA levels of XLF were significantly decreased upon depletion of WRN by siRNA (Fig. 1D). Taken together, these WRN positively regulates the XLF promoter activity. To determine if WRN regulates the promoter activity of XLF, the putative XLF promoter region from -596 to +169 (relative to the putative transcription start site described in the Ensembl protein-coding gene ENSG00000187736) was cloned into the pGL3 basic luciferase reporter vector. The resulting pGL3-XLFpr was identified to have pronounced luciferase reporter activity in U2OS cells (data not shown). It was identified that the XLF promoter activity was significantly downregulated when WRN expression was inhibited by siRNA in U2OS cells ( Fig. 2A). The antibodies against WRN were used for ChIP on cross-linked chromatin fragments prepared from U2OS cells. The ChIP-enriched DNA was subjected to PCR analysis using three pairs of primers for amplification of three fragments within the XLF promoter region. The data revealed that the second fragment (from -360 to -90) of the XLF promoter region was detected in the anti-WRN immunoprecipitates (Fig. 2B). These results suggest that WRN resides on the XLF promoter region, positively regulating its activity.
Depletion of XLF promotes cellular senescence in normal human fibroblasts. The prominent biological function of XLF is to repair DSBs by NHEJ, while the central effect of WRN deficiency is premature cellular senescence. It is well known that defects in NHEJ-mediated DSB repair contribute to cellular senescence. Therefore, we hypothesized that defects in XLF would lead to premature senescence. Indeed, inhibition of XLF expression by transfection with two independent siRNA oligos resulted in a decrease of cell growth in the normal human fibroblasts WI38 ( Fig. 3A and B), while an increase in the percentage of β-gal-positive cells as compared with the mock transfectants (Fig. 3C). Therefore, it was concluded that XLF is critical in promoting cell proliferation and suppressing cellular senescence. WRN is known to physically and functionally interact with the DNA-PK kinase complex and the XRCC4/Ligase IV complex, two key complexes involved in NHEJ. The DNA-PK kinase complex has been demonstrated to phosphorylate WRN in vivo and in vitro, and this phosphorylation inhibits the WRN helicase and exonuclease activities. By contrast, the interaction between XRCC4 and WRN, stimulates WRN's exonuclease activity, enabling it to serve as a DNA end-processing factor during NHEJ. In the present study, the results reveal a novel mechanism of the functional interplay between WRN and the NHEJ process, in which WRN positively regulates XLF at the transcription level. Furthermore, these data provide evidence for the first time, to the best of our knowledge, of the functional link of XLF to cellular senescence.