Expression and localization of Werner syndrome protein is modulated by SIRT1 and PML
Introduction
Two human RecQ helicases, WRN and BLM, localize in nucleoli and in nuclear structures called PML bodies (Marciniak et al., 1998, Ishov et al., 1999, Yankiwski et al., 2000, Yankiwski et al., 2001, Johnson et al., 2001). Mutant genes coding these proteins cause human cancer prone syndromes (Werner and Bloom syndromes, respectively; Harrigan and Bohr, 2003). The autosomal recessive Werner syndrome is a segmental progeria characterized by the accelerated appearance of many, but not all, aging symptoms (e.g., cataracts, osteoporosis, atherosclerosis, and diabetes). Cells from Werner syndrome patients show genomic instability and accelerated replicative senescence. WRN is a pleiotropic protein involved in the maintenance of genomic stability, especially at telomeric DNA, in the gene transcription, including the transcription of ribosomal RNA genes and in the regulation of apoptosis (Bachrati and Hickson, 2003, Bohr, 2005 and references therein).
The nuclear localization of WRN protein shows a very complex pattern of changes. It was examined using various antibodies as well as the recombinant WRN protein tagged with the green fluorescent protein (GFP). In most of the examined human cell lines, the endogenous WRN is localized in nucleoli (Gray et al., 1998, Marciniak et al., 1998). After inhibition of DNA replication or transcription (e.g., by UV-radiation, hydroxyurea, camptothecin), WRN exits from nucleoli and localizes in the nucleoplasm, frequently showing conspicuous nucleolar exclusion (Brosh et al., 2001, Shiratori et al., 2002, Karmakar and Bohr, 2005). This change in the localization is accompanied by posttranslational modifications of WRN including phosphorylation (Cheng et al., 2003), SUMOylation (Woods et al., 2004) and acetylation (Blander et al., 2002). The nucleoplasmic staining of WRN is often associated with an increased frequency of WRN foci and bodies (Constantinou et al., 2000, Sakamoto et al., 2001). Some of these WRN-containing structures were shown to contain PML protein, the defining constituent of PML bodies (Johnson et al., 2001, Blander et al., 2002).
Studies of colocalization between WRN and PML proteins were performed on the endogenous proteins (Johnson et al., 2001) or GFP-tagged, ectopically expressed WRN with the detection of the endogenous PML (Blander et al., 2002). These studies showed that endogenous WRN colocalized with endogenous PML protein in nucleoplasmic bodies in some cell lines (Johnson et al., 2001). The ectopically expressed GFP-WRN partially colocalized in large nucleoplasmic foci with the endogenous PML protein in UV-irradiated U-2 OS cells (Blander et al., 2002).
The PML protein positively regulates many anticarcinogenic processes, e.g., cell cycle arrest, apoptosis and cellular senescence (Salomoni and Pandolfi, 2002). It acts by controlling gene transcription. Several hypotheses have been proposed to explain the mechanism of this control (Zhong et al., 2000, Ching et al., 2005 and references therein). According to one of them, PML indirectly post-translationally modifies transcription factors, altering their affinity to the target sequences within gene regulatory elements. This is well-exemplified by the role of PML in activating the p53 tumor suppressor protein. The senescence-inducing signal promotes the formation of the p53, PML and CBP acetyltransferase complex accompanied by p53 acetylation at Lys 382. This acetylation does not take place in PML-null fibroblasts (Pearson et al., 2000, Bischof et al., 2002). The overexpression of the PML isoform IV, but not isoform I or III, also induces p53 acetylation at Lys382, which is accompanied by an increase in the activation of a p53-dependent gene promoter (Bischof et al., 2002).
PML has many isoforms derived from the alternative splicing. For many years, researchers usually used only one isoform to study PML (Jensen et al., 2001 and references therein). Only recently, the isoforms have been studied simultaneously using the same experimental system. They showed significant functional differences. For example, of five isoforms tested (I–V), only isoform IV induced cellular senescence when expressed in primary fibroblasts (Bischof et al., 2002).
One of the proteins recruited to PML nuclear bodies is SIRT1-a NAD-dependent, type III, histone/protein deacetylase (Langley et al., 2002). It is a human orthologue of yeast Sir2 protein. In yeasts, it is required for chromatin modification associated with transcriptional silencing of the mating-type loci and with the suppression of recombination at ribosomal DNA and telomere repeats (reviewed in Guarente, 2000, Anastasiou and Krek, 2006). Human SIRT1 binds p53 protein and promotes its deacetylation in vivo and in vitro. It represses p53-mediated transactivation of gene promoters, antagonizes PML IV-induced cellular senescence and DNA damage-induced apoptosis. Thus, SIRT1 can be viewed as an antisenescence and antiapoptotic protein (Luo et al., 2001, Vaziri et al., 2001, Langley et al., 2002). Apart from p53, SIRT1 has other non-histone substrates, e.g., Ku70 DNA repair protein (reviewed by Anastasiou and Krek, 2006). The SIRT1 homologue extends the lifespan of many organisms, including yeasts and worms (Tissenbaum and Guarente, 2002). It plays a major role in stress signaling, anti-apoptosis, calorie restriction response as well as glucose, fat and insulin biochemistry (reviewed by Anastasiou and Krek, 2006).
In our work, we further characterized the regulation of WRN localization. First, we labeled WRN with monomeric red fluorescent protein (mRFP1) to create a useful tool for protein colocalization studies and we tested in what experimental conditions its localization is consistent with the localization of the endogenous WRN. Second, we identified PML isoforms associating with nuclear bodies formed by mRFP-WRN. Third, we examined the influence of SIRT1 protein on the cellular localization of mRFP-labeled and native WRN. Our study shows, for the first time, the functional relationship between SIRT1 and WRN and may have implications for a better understanding of these two antisenescent proteins.
Section snippets
Plasmid construction
The mRFP-WRN expression plasmid was constructed based on the pcDNA3.1/HisC-WRN plasmid provided by Vilhelm Bohr (National Institute on Aging, Baltimore, MD, USA) and a plasmid coding for the monomeric red fluorescent protein (mRFP1) provided by Roger Tsien (Campbell et al., 2002). The mRFP coding sequence was amplified using primers with Acc65I (sense) and NotI (antisense) restriction sites, and was ligated into the Acc65I and NotI sites of pcDNA3.1/HisC-WRN plasmid. The resulting expression
Results
The mRFP-WRN localization was examined in U-2 OS cells, because others used them in localization studies of endogenous (Brosh et al., 2001) as well as the ectopically expressed, GFP-labeled WRN (von Kobbe et al., 2002, Blander et al., 2002, von Kobbe and Bohr, 2002). As expected, the mRFP-WRN protein localized in nucleoli or diffusely in the entire nuclei (see Supplementary Fig. 1A, a and b). However, this protein also formed large nuclear bodies, some of them were donut-shaped (Supplementary
Discussion
We report that SIRT1 overexpression is associated with the nucleolar exclusion of the mRFP (or EGFP)-labeled WRN, decreased frequency of cells with mRFP-WRN nuclear bodies and increased frequency of cells with cytoplasmic localization of the labeled WRN. This effect does not require the deacetylase activity of the SIRT1. Moreover, we did not find evidence of acetylation of mRFP-WRN protein fraction forming the nuclear bodies (Fig. 3). However, our results are not inconsistent with the report by
Acknowledgements
This work was supported by the Polish State Committee for Scientific Research (KBN) grants No. 3P04A/004/23 to M.R. and 2P05A/125/28 to D.B. R.V. was a fellow of the Fellowship Program at Department of Tumor Biology, totally supported by the National Cancer Institute–Office for International Affairs, NIH, Bethesda, MD, USA. The technical assistance of Mrs. Iwona Matuszczyk and as well as the editorial assistance of Mrs. Dorothea Dudek-Creaven are highly appreciated.
References (47)
- et al.
DNA damage-induced translocation of the Werner helicase is regulated by acetylation
J. Biol. Chem.
(2002) Deficient DNA repair in the human progeroid disorder, Werner syndrome
Mutat. Res.
(2005)- et al.
p53 Modulates the exonuclease activity of Werner syndrome protein
J. Biol. Chem.
(2001) - et al.
Werner helicase is localized to transcriptionally active nucleoli of cycling cells
Exp. Cell Res.
(1998) - et al.
Human diseases deficient in RecQ helicases
Biochimie
(2003) - et al.
Cellular dynamics and modulation of WRN protein is DNA damage specific
Mech. Ageing Dev.
(2005) - et al.
Negative control of p53 by Sir2alpha promotes cell survival under stress
Cell
(2001) - et al.
Mammalian SIRT1 represses forkhead transcription factors
Cell
(2004) - et al.
Cloning and characterization of RECQL, a potential human homologue of the Escherichia coli DNA helicase RecQ
J. Biol. Chem.
(1994) - et al.
The role of PML in tumor suppression
Cell
(2002)
Association of BRCA1 with Rad51 in mitotic and meiotic cells
Cell
Model organisms as a guide to mammalian aging
Dev. Cell
hSIR2(SIRT1) functions as an NAD-dependent p53 deacetylase
Cell
Colocalization, physical, and functional interaction between Werner and Bloom syndrome proteins
J. Biol. Chem.
Functional interaction of p53 and BLM DNA helicase in apoptosis
J. Biol. Chem.
Intracellular localization of proteasomes
Int. J. Biochem. Cell Biol.
p14 Arf promotes small ubiquitin-like modifier conjugation of Werners helicase
J. Biol. Chem.
SIRT1: linking adaptive cellular responses to aging-associated changes in organismal physiology
Physiology (Bethesda)
RecQ helicases: suppressors of tumorigenesis and premature aging
Biochem. J.
Deconstructing PML-induced premature senescence
EMBO J.
A monomeric red fluorescent protein
Proc. Natl. Acad. Sci. U.S.A.
Nucleolar components involved in ribosome biogenesis cycle between the nucleolus and nucleoplasm in interphase cells
J. Cell Biol.
Werner syndrome protein phosphorylation by abl tyrosine kinase regulates its activity and distribution
Mol. Cell Biol.
Cited by (35)
Acetylation of Werner protein at K1127 and K1117 is important for nuclear trafficking and DNA repair
2019, DNA RepairCitation Excerpt :It is observed that upon exogenous DNA damage, WRN is acetylated by p300/CBP at multiple lysine residues present between the N-terminus and C-terminus ends and then translocates from the nucleolus to the nucleoplasm [13]. Other studies indicated that sirtuins, primarily SIRT1(silent mating type information regulation 2 homolog 1), a member of the class III Histone deacetylase family, is involved in deacetylation of WRN [3,13–15]. It was predicted that ectopically expressed WRN may get acetylated at K366, K887 (N-terminal) and K1127, K1117, K1389, K1413 (C-terminal), with K1117, K1389, and K1413 appearing to be the primary acetylation sites [3].
Circulating markers of ageing and allostatic load: A slow train coming
2017, Practical Laboratory MedicineCitation Excerpt :The question remains, however, as to how one can extrapolate markers of cellular ageing to the ageing of the whole organism and whether these markers remain reflective of allostatic load, or reflect ongoing aspects of more local disease processes? This is particularly pertinent in the context of segmental ageing, where individual genes, such as those that give rise to unimodal progerias (e.g. WRN, Lamin A, BLM [25,26]) or genetic pathways that extend lifespan (e.g. mechanistic target of rapamycin [mTOR]) are likely to have non-uniform and non-cell-autonomous effects on the decline in tissue and organ function with increasing age. By extension, this implies that there is a direct interconnection between separate systems for tissue/organ and organismal ageing.
Stem cell aging in adult progeria
2015, Cell RegenerationCitation Excerpt :Such interaction is interrupted in progeroid cells, leading to prominent decline of adult stem cells in the progeria mouse model [45]. For WS, expression and localization of WRN is modulated by SIRT1 and PML [46]. SIRT1 is reported to deacetylate WRN [47].
Perspectives on translational and therapeutic aspects of SIRT1 in inflammaging and senescence
2012, Biochemical PharmacologyCitation Excerpt :Apart from FOXO3, several other protein substrates for SIRT1, which are involved in cell stress response signaling and cellular senescence, have been identified. This includes Ku70/Ku80, Wnt/β-catenin, Notch, and Werner syndrome protein [45–49] (Fig. 1). A recent study has identified XRCC5/Ku80 (Ku86) as a potential COPD-susceptibility gene through the multi-study fine-mapping strategy [50].
SIRT1 as a therapeutic target in inflammaging of the pulmonary disease
2012, Preventive MedicineCitation Excerpt :It remains to be seen whether SIRT1 regulates these genes and proteins in response to CS exposure. Several other SIRT1 protein substrates involved in cell stress response signaling and cellular senescence have been identified, including Ku70, Wnt/β-catenin, Notch, and Werner syndrome protein (Guarani et al., 2011; Holloway et al., 2010; Li et al., 2008; Uhl et al., 2010; Vaitiekunaite et al., 2007) (Table 1). Hence, the study on these molecules in condition of oxidative stress/cigarette smoke will further enhance the understanding of inflammaging in the pathogenesis of COPD.
Human cytomegalovirus UL97 kinase prevents the deposition of mutant protein aggregates in cellular models of Huntington's disease and Ataxia
2011, Neurobiology of DiseaseCitation Excerpt :To test the effect of UL97 on the formation of WRN aggregates, cells were transfected with WRN–RFP in the presence of either an empty plasmid, a plasmid encoding UL97 or a plasmid encoding the kinase-dead UL97/K355M. Consistent with previous studies (Vaitiekunaite et al., 2007), expression of the WRN–RFP protein resulted in the presence of numerous “donut-shaped” nuclear inclusions (Fig. 1A). In clear contrast to these observations, when WRN–RFP was expressed in the presence of UL97, nuclear WRN–RFP aggregates were rarely observed but instead WRN–RFP fluorescence appeared diffuse throughout the nucleoplasm (Fig. 1B).