Introduction
Maintenance of a stable genome is critical for cells to faithfully pass on the correct genetic information to the next generation. Unfortunately, long chain nucleotide macromolecules that form our genome are constantly threatened by mutagenic origins that arise from both exogenous and endogenous sources (
De Bont and van Larebeke, 2004;
Gonzalo, 2014). These mutagens include UV light, ionized irradiations, chemical agents, endogenous estrogens, metabolism generated reactive oxygen species (ROS), etc.
(De Bont and van Larebeke, 2004;
Friedberg et al., 2004;
Helleday et al., 2014). Moreover, additional DNA damages can also arise from disruption of cellular activities such as DNA replication, transcription and telomere protection (
Mirkin E V and Mirkin S M, 2007;
Longhese, 2008 ; Haffner et al., 2011; Olcina et al., 2013). It is estimated that over 10
5 DNA lesions are created per human cell per day (
Gonzalo, 2014), including mispaired DNA bases, pyrimidine dimers, inter-strand cross-links (ICLs), single-strand DNA breaks (SSBs) and double-strand DNA breaks (DSBs) (
Ciccia and Elledge, 2010). If improperly or inefficiently handled, these lesions will result in DNA mutations, chromatin deletions or rearrangements and may eventually cause a variety of pathological phenotypes such as tumorigenesis and premature aging (
Hoeijmakers, 2009;
Gonzalo, 2014). To preserve genome integrity and avoid mutations, cells develop DNA damage response (DDR), a signal transduction pathway mediated by PIKKs family member (primarily ATM, ATR and DNAPKcs), to detect DNA damages and to orchestrate downstream cellular activities (
Ciccia and Elledge, 2010). The execution of DNA damage repair mechanisms is stimulated and tightly regulated by DDR (
Sirbu and Cortez, 2013). In preparation for DNA damage repair, DDR inhibits cell cycle progression by activating the checkpoint kinases Chk1 or Chk2 (
Zhou and Elledge, 2000), which usually become gradually turned off with the repair of DNA damages (
Polo and Jackson, 2011). However, under abnormal circumstances upon excessive DNA damages, compromised DDR or misregulated DDR, DNA damage will accumulate in cells to an extent that exceeds their handling capacity and eventually lead to genome instability. To eliminate or restrict unwanted genomes, cells undergo either programmed cell death (apoptosis) or permanent cell cycle arrest (cellular senescence), both of which undermine organismal homeostasis and contribute to physiological aging over time (
Norbury and Zhivotovsky, 2004;
Roos and Kaina, 2006;
Chen et al., 2007;
Schmitt et al., 2007;
d’Adda di Fagagna, 2008; Garinis et al., 2008;
Childs et al., 2014). In this regard, genome instability induced by misregulated or defective DDR is widely considered to contribute to the accelerated aging phenotypes in multiple diseases including Werner syndrome (WS), Ataxia telangiectasia (A-T), Fanconi’s anemia (FA) and Hutchinson-Gilford progeria syndrome (HGPS) (
Oshima et al., 1993;
D’Andrea and Grompe, 2003;
Garinis et al., 2008;
Sahin and Depinho, 2010;
Gonzalo, 2014;
Brosh et al., 2016).
HGPS is a devastating and ultra-rare premature aging disease, with an estimated incidence rate of 1 in every 21 million of a population (
Gonzalo et al., 2016) and data from Progeria Research Foundation). HGPS patients, despite very young, are characterized by a variety of aging associated clinical phenotypes, including loss of subcutaneous fat, loss of hair, bone joint abnormalities and severe cardiovascular disorders (
Gordon et al., 2007;
Merideth et al., 2008; Capell and Collins, 2016). HGPS patients start to display these features at the age of 12-24 months old and die at an average age of 13-14 years old due to myocardial infarction or stroke (
Gordon et al., 2007). Interestingly, unlike many other accelerated aging diseases such as WS, A-T or FA, which are caused by mutations that directly interfere with DDR, most HGPS cases arise from a
de novo single point mutation in the
LMNA gene, a gene that mainly encodes two type V intermediate filament proteins: lamin A and lamin C (
Oshima et al., 1993;
D’Andrea and Grompe, 2003;
Eriksson et al., 2003;
Capell and Collins, 2006;
Mathew, 2006;
Lavin, 2008;
Musich and Zou, 2009;
Zhang et al., 2015). This mutation (C1824T) lies in the exon 11 of the
LMNA gene and activates a cryptic splice donor site, yielding a 50 amino acid truncated mutant protein termed progerin (Fig. 1) (
Eriksson et al., 2003). Progerin lacks an internal Zmpste24 cleavage site and undergoes an abnormal post-translational modification, being permanently farnesylated at the C terminus. This farnesyl tail anchors it to the inner nuclear membrane (INM) and disrupts the structure and function of nuclear lamina meshwork, an important polymeric structure that regulates nuclear morphology, chromatin organization and various cellular processes (
Eriksson et al., 2003;
Capell and Collins, 2006;
Gonzalo, 2014). Progerin accumulation induces multiple pathological phenotypes such as lobulated nuclear morphology, disrupted heterochromatin structure, altered gene expression profile, premature cellular senescence and genome instability (
Goldman et al, 2004; Liu et al., 2005;
Capell and Collins, 2006;
Shumaker et al., 2006;
McCord et al., 2013). Among these phenotypes, genome instability is closely related to premature aging, leaving us an important question regarding how progerin perturbs genome integrity (
Lombard et al., 2005). Over the past decade, researchers addressed this question from various angles and have achieved significant progress. In this review, we will first briefly describe phenotypes related to genome instability in HGPS and then discuss some of the recent findings to synthesize a set of molecular mechanisms to explain progerin’s role in genome instability. Finally, we will summarize current and potential HGPS therapeutic approaches targeting HGPS pathological pathways that include but not limited to genome instability.
Deficiencies in DNA damage responses (DDR) & DNA double-strand break (DSB) repair in HGPS
In this section, we will give a brief introduction about how cells sense, signal and repair DSBs, the most toxic type of DNA damage, followed by listing phenotypes related genome instability in HGPS.
DSBs can be detected by protein sensors such as PARP1/2, the Ku70-Ku80 complex and the Mre11-Rad50-Nbs1 (MRN) complex in a sequence-independent manner (
Polo and Jackson, 2011). Once recruited, the MRN complex serves as a scaffold to dock and activate signaling proteins including ATM, the master kinase that phosphorylates and activates a variety of factors involved in DNA damage repair and checkpoint activation (
Lee and Paull, 2004,
2005; Gonzalo, 2014). One important substrate of ATM is the histone H2A variant H2AX (
Burma et al., 2001). Upon phosphorylation, H2AX turns into gH2AX, a widely recognized DSB marker, and attracts MDC1, which in turn further recruits MRN complex and ATM (
Stewart et al., 2003; Chapman and Jackson, 2008;
d’Adda di Fagagna, 2008). Additional ATM mediates the phosphorylation of adjacent H2AX molecules, thereby evoking a positive feedback loop and amplifying DDR signals (
d’Adda di Fagagna, 2008;
Polo and Jackson, 2011). The above signaling process enriches MDC1 at DSBs, which facilitates the recruitment of downstream DSB repair factors (
Kolas et al., 2007;
Mailand et al., 2007;
Kinner et al., 2008;
Fradet-Turcotte et al., 2013). Among these factors, BRCA1 and 53BP1 are of significance, given their roles in regulating the balance between the homologous recombination (HR) and the non-homologous end joining (NHEJ) DSB repair pathways (
Gonzalo, 2014). HR and NHEJ are the two major DSB repair pathways that antagonize with each other at DSB break ends (
Polo and Jackson, 2011;
Chapman et al., 2012;
Gonzalo, 2014;
Gonzalo and Kreienkamp, 2015). HR utilizes sister chromatid as a template to fix DSBs in an error-free fashion, while NHEJ simply rejoins DSB break ends together and therefore is likely to introduce DNA mutations, chromatin deletions or rearrangements (
Polo and Jackson, 2011). BRCA1 favors HR by promoting DSB end resection and preparing for downstream homology search, whereas 53BP1 promotes NHEJ and inhibits HR by restricting end resection (
Chapman et al., 2012;
Zimmermann et al., 2013). As stated above, DDR and DSB repair are highly complex and spatial-temporally regulated cellular responses, and each step is strictly dependent on the previous one. The presence of progerin may disrupt multiple steps during the process via different mechanisms and yield a variety of defective phenotypes.
The defective DSB repair in HGPS cells was initially described by
Liu et al. in 2005. In that study, skin fibroblasts from HGPS patients displayed a significantly delayed recruitment of 53BP1 and Rad51, two important players in NHEJ and HR respectively, suggesting a delay of both NHEJ and HR (
Liu et al., 2005). Similar delays were also observed in mouse embryonic fibroblasts (MEFs) from Zmpste24
−/− mice (a progeroid mouse model which accumulates the unprocessed prelamin A and mimics HGPS phenotypes) (
Liu et al., 2005). Consequently, both HGPS fibroblasts and Zmpste24
−/− MEFs showed much higher sensitivity to various DNA damage inducing agents such as UV light, camptothecin (CPT), etoposide and mitomycin C (MMC) (
Liu et al., 2005). Along with these observations, additional studies reported a basal accumulation of gH2AX foci in HGPS fibroblasts as well as MEFs from
LmnaG609G mouse (a progeroid mouse model which carries the classic HGPS mutation), potentially due to their delayed DSB repair (
Liu et al., 2008;
Osorio et al., 2011).
Delays in repair lead to accumulation of DSBs and prolonged activation of upstream DDR signals (
Malaquin et al., 2015). This was confirmed by a persistent activation of the ATM/ATR kinases and checkpoint pathways in HGPS cells (
Liu et al., 2006;
Musich and Zou, 2009). As a result, HGPS fibroblasts displayed a strong activation of Chk1 and Chk2, limited proliferation potential and premature senescence (
Liu et al., 2006;
Musich and Zou, 2009). Ectopic expression of progerin in HeLa cells has been shown to induce ATR nuclear foci and impair 53BP1 recruitment upon UV light or ciplastin treatment, implying a direct negative impact of progerin on DDR and DSB repair (
Manju et al., 2006;
Musich and Zou, 2009;
Musich and Zou, 2011). Together, the above studies provided strong evidence showing the defects within both DDR and DSB repair in HGPS cells. The rest of this article will separately discuss the molecular mechanisms underlying DSB repair as well as DDR defects in HGPS.
Defective DSB repair in HGPS
Progerin impairs DSB repair by interfering with normal lamin A/C functionality
Over the past several years, researchers started to identify lamin A/C as important players in DSB repair. MEFs from
Lmna−/− mice displayed elevated accumulation of gH2AX, aneuploidy and increased chromatin breaks, suggesting genome instability (
Gonzalez-Suarez et al., 2009;
Redwood et al., 2011). Moreover,
Lmna−/− MEFs also bear increased sensitivity to DNA interstrand crosslinks (ICLs) and replication stress (
Singh et al., 2013). When treated with ICL agents,
Lmna−/− MEFs showed decreased recruitment of the FANCD2 repair factor and delayed gH2AX clearance, accompanied by increased aberrant chromosomes (
Singh et al., 2013). Similar defects were also observed upon hydroxyurea-induced replication stress (
Singh et al., 2013). In addition, one recent study on murine cells reported that lamin A engaged into chromatin through interacting with histone variant H2AX and stabilized DNA damage repair foci, suggesting a structural role of lamin A in DSB repair (
Mahen et al., 2013).
Lamin A/C favors both NHEJ and HR by maintaining their repair protein levels. Depletion or shRNA-induced suppression of lamin A results in cysteine protease Cathepsin L (CTSL) mediated degradation of the NHEJ promoting factor 53BP1 and transcriptional reduction of BRCA1 and RAD51, two key HR regulators (
Gonzalez-Suarez et al., 2009;
Redwood et al., 2011). Consequently, deletion of lamin A/C inhibits both NHEJ and HR (
Redwood et al., 2011 ; Singh et al., 2013). Mechanistically, 53BP1 binds to lamin A/C via its Tudor domain in human dermal fibroblasts, suggesting that lamin A/C maintains a nucleoplasmic pool of 53BP1 (
Gibbs-Seymour et al., 2015). The transcriptional downregulation of BRCA1 and Rad51 in
Lmna null cells is mediated by the formation of a p130/E2F4 repressor complex, which is increased upon loss of lamin A (
Redwood et al., 2011).
Recent studies performed by Zhou’s group highlighted lamin A’s capacity to tether a variety of DNA damage response factors to the nuclear matrix. For example, lamin A interacts with and facilitates the nuclear matrix localization of histone acetyltransferase Mof (
Krishnan et al., 2011). Mof is an important protein involved in genome integrity maintenance and loss of Mof results in G2/M cell cycle arrest, massive chromatin aberrations and defective repair of DSBs (
Li et al., 2010). Lamin A was also shown to interact with and facilitate the localizations and activities of the SIRT1 and SIRT6: two important enzymes involved in genome stability maintenance (
Mostoslavsky et al., 2006;
Oberdoerffer et al., 2008;
Liu et al., 2012;
Dobbin et al., 2013 ; Ghosh et al., 2015). Collectively, the above studies indicated an indispensable role of lamin A/C in DSB repair. Interestingly, mice that harbored a lamin A mutant that lacks exon 9 (
LmnaD9/D9) showed progeroid phenotypes without apparent defects in DSB repair and genome stability, suggesting that other lamin A mutant might induce premature aging without perturbing genome integrity and also that there were specific domains within lamin A that played a functional role in DSB repair (
Das et al., 2013).
Progerin is a dominant negative mutant of lamin A and may disrupt normal lamin A functionality in DSB repair (
Schreiber and Kennedy, 2013). For example, unprocessed prelamin A (retains the C terminus farnesyl tail and possesses toxicities similar to progerin) displayed a significantly lower Mof affinity compared to wild-type lamin A (
Krishnan et al., 2011). Hence accumulation of prelamin A in Zmpste24
−/− MEFs interfered Mof-lamin A interaction, disrupted the retention of Mof at the nuclear matrix and impaired 53BP1 foci formation at DNA lesion (
Krishnan et al., 2011). Moreover, prelamin A and progerin were both shown to disrupt the interaction between lamin A and SIRT1. As a result, the subcellular localization and deacetylase activity of SIRT1 were significantly disrupted in Zmpste24
−/− MEFs as well as in HGPS fibroblasts (
Liu et al., 2012). The presence of progerin also disrupted SIRT6 activation and compromised SIRT6 dependent DSB repair (DNA-PKcs recruitment, CtIP deacetylation, and PARP1 mono-ADP ribosylation) (
Ghosh et al., 2015). Taken together, these reports suggest that progerin may jeopardize DSB repair by disrupting the normal lamin A’s functionality.
Altered epigenetic modifications impair DSB repair in HGPS
Abnormal heterochromatin structure and histone modifications are both hallmarks of HGPS associated epigenetic alterations. In 2004, Goldman et al. initially revealed a significant loss of peripheral heterochromatin in late passage HGPS fibroblasts (
Goldman et al., 2004). Subsequent studies uncovered a variety of altered histone modifications in HGPS fibroblasts and HEK293 cells overexpressing progerin (
Shumaker et al., 2006). These alterations include loss of facultative heterochromatin marker histone H3 lysine 27 trimethylation (H3K27me3), loss of pericentric constitutive heterochromatin marker histone H3 lysine 9 trimethylation (H3K9me3) and enrichment of constitutive heterochromatin marker histone H4 lysine 20 trimethylation (H4K20me3) (
Shumaker et al., 2006). Using chromatin immunoprecipitation followed by sequencing (ChIP-seq), our group identified genome-wide complex alterations of H3K27me3, which was associated with gene density (
McCord et al., 2013). Genome-wide chromosome conformation capture (Hi-C) uncovered a global loss of spatial compartmentalization of chromatin in late passage HGPS fibroblasts, suggesting additional chromatin structure abnormalities (
McCord et al., 2013).
In recent years, researchers started to connect the abnormal histone modifications and defective DSB repair in HGPS cells. The general concept was that aberrant histone modifications rendered a more condensed chromatin structure and created a physical barrier preventing efficient DNA repair in HGPS cells. For instance, Krishnan et al. showed that Zmpste24
−/− MEFs bear H4K16 hypo-acetylation due to the dissociation of the histone acetyltransferase Mof from the nuclear matrix (
Krishnan et al., 2011). As a result, chromatin became more condensed, leading to delayed 53BP1 recruitment upon irradiation induced DSBs. Overexpressing Mof or treating cells with HDAC inhibitor restored normal 53BP1 recruitment and ameliorated the premature senescence phenotype in Zmpste24
−/− MEFs (
Krishnan et al., 2011).
A more recent study from the same group revealed that unprocessed prelamin A or progerin exhibited an enhanced binding capacity to the H3K9me3 methyltransferase SUV39h1. This protected SUV39h1 from proteasome degradation in Zmpste24
−/− MEFs and HGPS fibroblasts, leading to increased accumulation of H3K9me3 (Liu et al., 2013). Interestingly, these findings are contradictory to the previously established knowledge of H3K9me3 loss in HGPS (
Shumaker et al., 2006). This discrepancy might reflect the differences regarding the cell donors or cellular passages. Nevertheless, the authors observed a passage dependent reduction of H3K9me3, which was consistent with previous findings (Liu et al., 2013). They speculated that accumulation of H3K9me3 might build up a barrier and block effective DNA damage repair. Notably, depletion of SUV39h1 in Zmpste24
−/− mice (Zmpste24
−/− SUV39H1
−/− ) was able to significantly reduce H3K9me3, improve DSB repair and alleviate cellular senescence (Liu et al., 2013). At the organismal level, Zmpste24 and SUV39h1 double knockout mice displayed higher bodyweight, increased bone mass and extended lifespan in comparison to Zmpste24
−/− mice (Liu et al., 2013).
In addition to Mof and SUV39h1, progerin also disturbs other chromatin modifiers. For example, HGPS fibroblasts and wild type skin fibroblasts overexpressing progerin both displayed significantly reduced protein levels of the retinoblastoma binding protein 4 and 7 (RBBP4 & RBBP7), both of which are components of the nucleosome remodeling deacetylase (NuRD) complex (
Pegoraro et al., 2009). Interestingly, loss of RBBP4 and RBBP7 was able to recapitulate part of HGPS associated phenotypes in non-progeria cells. HeLa cells transfected with RBBP4/7 targeting siRNAs displayed a significant loss of H3K9me3 (
Pegoraro et al., 2009). Moreover, normal cells depleted of RBBP4/7 showed increased counts of gH2AX foci at basal level (
Pegoraro et al., 2009). These findings suggested that progerin might affect chromatin structure and DSB repair at least partially through downregulating RBBP4 and RBBP7.
Progerin misregulates HR/NHEJ pathway choice
The choice between HR and NHEJ DSB repair pathways is regulated by the interplay between BRCA1 and 53BP1 and is dependent on cell cycle progression (
Polo and Jackson, 2011;
Chapman et al., 2012;
Escribano-Díaz et al., 2013). In G0/G1 phase, 53BP1 promotes NHEJ and inhibits HR by blocking DSB end resection (
Chapman et al., 2012;
Escribano-Díaz et al., 2013). This is carried out through its interaction with local H4K20me2 around DSBs, which forms a chromatin barrier and blocks DNA resection machinery (
Bothmer et al., 2010;
Gupta et al., 2014). When cells enter S phase, however, cyclin-dependent kinases (CDKs) phosphorylate the CtBP interacting protein (CtIP), which then binds to BRCA1. The CtIP/BRCA1 complex is able to antagonize with 53BP1, promoting DNA end resection and HR (
Escribano-Díaz et al., 2013;
Panier and Boulton, 2014). Unlike HR, which carries out homology-based error free DSB repair, NHEJ simply re-ligates DNA break ends and mediates rapid, but error-prone DSB repair (
Polo and Jackson, 2011;
Chapman et al., 2012). The balance between HR and NHEJ is critical for cells to maintain genome integrity and cellular homeostasis. Mis-regulation of this process could result in chromatin relocation, genome instability and cell death (
Bunting et al., 2010;
Patel et al., 2011).
Our recent study pointed out that smooth muscle cells (SMCs) differentiated from HGPS iPSCs displayed a misregulated HR/NHEJ pathway choice, resulting in inappropriate DSB fix and mitotic defects (
Zhang et al., 2014). We discovered that there was a strong suppression of HR in HGPS SMCs during S/G2 phase as indicated by an almost complete removal of Rad51 recruitment (
Zhang et al., 2014). Moreover, overexpression of progerin elicited a strong downregulation of PARP1, a protein that inhibits NHEJ (
Patel et al., 2011;
Zhang et al., 2014). PARP1 downregulation and HR deficiency together triggered an over activation of the error-prone NHEJ during S/G2 phase. This was demonstrated by a strong recruitment of 53BP1 in HGPS SMCs upon low dosage (mainly creates DSBs in S/G2 phase) CPT treatment (
Ray Chaudhuri et al., 2012; Zhang et al., 2014). As a result, over-activated NHEJ led to chromatin rearrangements, aneuploidy/polyploidy karyotypes, mitotic arrest and eventual mitotic cell death (
Zhang et al., 2014). These results provided a molecular mechanism that potentially contributed to the vascular SMC loss phenotype in HGPS (
Varga et al., 2006 ; Zhang et al., 2014). Similar to HGPS SMCs, late passage HGPS fibroblasts also displayed PARP1 downregulation and abnormal HR during S/G2 phase ((
Zhang et al., 2014) and unpublished data). As a result, HGPS fibroblasts displayed lagging mitotic chromosomes and micronuclei, confirming a defective mitosis (
Cao et al., 2007). Additional results demonstrated the presence of lagging chromosomes in mitotic HeLa cells overexpressing progerin, suggesting a direct involvement of progerin in HGPS mitotic defects (
Cao et al., 2007). Consistent with these results, aneuploidy was also observed in bone marrow cells from Zmpste24
−/− mice (
Liu et al., 2005). Interestingly, although an over-activation of NHEJ in S/G2 phase HGPS SMCs was shown, we didn’t see a strong NHEJ activation in S/G2 phase HGPS fibroblasts ((
Zhang et al., 2014) and unpublished data). In fact, this NHEJ efficiency was comparable to that in S/G2 phase normal controls, suggesting that the severity of pathway choice disruption is cell type dependent (unpublished data). Moreover, this difference may also explain why we didn’t observe apparent cell death in HGPS fibroblasts.
A variety of previous studies reported a delayed recruitment of 53BP1 in HGPS fibroblasts, suggesting a disrupted NHEJ (
Liu et al., 2005,
2013a,
2013b;
Krishnan et al., 2011). However, we found that NHEJ was over-activated in S/G2 phase HGPS SMCs, raising the possibility that delays of NHEJ were predominantly present in G0/G1 phase. Indeed, we observed a significantly delayed recruitment of RIF1 (a NHEJ promoting protein downstream of 53BP1) in serum synchronized G0/G1 phase HGPS fibroblasts (unpublished data). Delayed NHEJ in G0/G1 phase may result in accumulation of DNA damage, prolonged DDR and premature senescence in HGPS.
Together, the above results implied a complete disruption of HR/NHEJ pathway choice in HGPS (Fig. 2). In G0/G1 phase, the presumably prevailing NHEJ is significantly delayed, causing insufficient DSB repair. In S/G2 phase, HR was drastically impaired whereas NHEJ was over-active (at least in HGPS SMCs), leading to inappropriate error-prone repair of DSBs, chromatin abnormalities, and genome instability.
Disrupted DDR in HGPS
Progerin or unprocessed prelamin A inhibits ATM activation upon DSBs
The ataxia telangiectasia mutated (ATM) is the master regulator in DDR. In response to DSBs, it is recruited by the MRN complex to damaged DNA lesions, where it undergoes self-activation through auto-phosphorylation (
Bakkenist and Kastan, 2003;
Lee and Paull, 2005). ATM activation requires Nbs1 (a component of the MRN complex) and the histone acetyltransferase Tip60 (
Lee and Paull, 2007). Deletion of Tip60 diminished ATM auto-phosphorylation, suggesting that its activity was critical for ATM activation (
Sun et al., 2005;
Shiloh and Ziv, 2013). Activated ATM then phosphorylates numerous down-stream substrates and orchestrates chromatin remodeling, DNA damage repair and cell fate determination (
Shiloh and Ziv, 2013). Deficiencies in ATM usually result in aberrant DNA damage repair, chromatin translocations and genome instability (
Tubbs and Sleckman, 2014).
A study from Liu et al. showed that ATM was persistently activated in HGPS fibroblasts at a basal level without additional mutagenic treatment. This further activated Chk2 and led to cell cycle arrest (
Liu et al., 2006). Interestingly, in contrast to Liu et al.’s results, a more recent study on Zmpste24
−/− MEFs revealed an attenuated ATM activation in response to ionized irradiation (
Liu et al., 2013a). In support of their findings, we also observed a reduced activation of ATM in HGPS fibroblasts treated with doxorubicin (Dox, a topoisomerase II inhibitor), suggesting impaired ATM activation upon DSBs (unpublished data). These conflicting findings were probably due to differential experimental conditions. Liu et al.’s mainly performed their study on HGPS cells without any additional treatment and discovered a basal activation of ATM, which was caused by unrepairable DSBs, whereas we focused on the immediate ATM activation in response to exogenous DSBs ((
Liu et al., 2006) and unpublished data). Mechanistically, we found that the defective ATM activation was correlated with progerin accumulation and the loss of H3K9me3 in HGPS fibroblasts (unpublished data). H3K9me3 is an important epigenetic mark, which has been previously shown to recruit Tip60 and activate ATM (
Sun et al., 2009). Loss of H3K9me3 by SUV39h1/2 depletion resulted in ATM inactivation, suggesting a critical role of H3K9me3 in ATM activity (
Sun et al., 2009). Therefore, we speculate that loss of H3K9me3 may disrupt ATM activation and contribute to genome instability in HGPS.
Progerin disrupts DNA damage induced histone modifications
Upon DSBs, cells carry out a variety of histone modifications (methylation, acetylation, phosphorylation, ubiquitination, etc.) to drive chromatin structure remodeling, DNA break exposure, DDR signal amplification and DSB repair factor recruitment (
Bird et al., 2002;
Mailand et al., 2007;
Kuo and Yang, 2008;
Price and D’Andrea, 2013 ; Ayrapetov et al., 2014). One of the most profound and well-characterized histone modifications upon DSB is the rapid phosphorylation of histone variant H2AX, mediated by PIKKs family members: ATM, ATR or DNAPKcs (
Burma et al., 2001;
Ward and Chen, 2001;
Stiff et al., 2004). gH2AX, MDC1 and ATM create a positive feedback signal amplification loop and serve to recruit and activate downstream DSB repair factors (
Paull et al., 2000; Kinner et al., 2008). Another important type of DSB dependent histone modification is the ubiquitination of histone H2A and its variants (
Mailand et al., 2007;
Mattiroli et al., 2012). RNF8 mediated H2A ubiquitination orchestrates the recruitment of 53BP1 and BRCA1. siRNA-mediated suppression of RNF8 was sufficient to abolish 53BP1 and BRCA1 foci formation upon DSBs (
Kolas et al., 2007;
Mailand et al., 2007), indicating a critical role of histone ubiquitination in DSB repair. In a more recent study,
Ayrapetov et al. (2014) described a transient enrichment of H3K9me3 around DSB breaks. This modification was mediated by a newly defined complex that consists of Kap-1, HP1, and SUV39h1. In their model, SUV39h1 methylates H3K9 around DSBs creating H3K9me3. The nascent H3K9me3 further recruits Kap-1/HP1/SUV39h1 complex through its interaction with HP1 and mediates the methylation of adjacent H3K9, creating a positive feedback loop (
Ayrapetov et al., 2014). H3K9me3 recruits histone acetyltransferase Tip60, which down the road acetylates both ATM and histone H4, and promotes DSB repair (
Sun et al., 2009;
Ayrapetov et al., 2014).
The potential impact of progerin on DSB dependent histone modifications is under appreciated. Recently, our group demonstrated that there was a passage dependent reduction of gH2AX response in HGPS fibroblasts upon treatment with dox and CPT, suggesting that progerin accumulation might impair gH2AX signals (unpublished data). The reduced gH2AX signal strength was associated with less recruitment of 53BP1 and RIF1, in agreement with its critical roles in DSB repair (
Kinner et al., 2008). Consistently, we observed weaker but more persistent gH2AX foci in HGPS fibroblasts. Mechanistically, we found that the weak gH2AX responses were caused by a reduced activation of ATM and loss of H3K9me3 in HGPS fibroblasts (unpublished data), providing a link between the previously established H3K9me3 loss and the DSB repair defects in HGPS. Interestingly, the reduction of ATM activation and gH2AX signals were mainly observed in G0/G1 phase but not in S/G2 phase HGPS cells, providing an upstream mechanism to explain the misregulated NHEJ in HGPS cells (see discussion in the section “Progerin misregulates HR/NHEJ pathway choice”).
It has been previously reported that HGPS fibroblasts bear reduced level of the heterochromatin protein 1α (HP1α), a heterochromatin binding protein critical for the maintenance of H3K9me3 (
Scaffidi and Misteli, 2005;
Shumaker et al., 2006;
Zhang et al., 2015). Moreover, HP1α was implied to participate in the transient H3K9me3 modification at DSB break ends. Overexpression of a chromatin domain deleted mutant of HP1α was shown to disrupt the recruitment of Kap-1/HP1/SUV39h1 upon DSBs (
Ayrapetov et al., 2014). Thus, although the direct evidence is lacking, reduction of HP1α in HGPS cells may also impair the transient enrichment of H3K9me3 at local DNA lesions and hinder DSB repair.
Progerin disrupts DNA damage-induced chromatin remodeling
A study on Zmpste24
−/− MEFs showed that DNA damage triggered chromatin remodeling was impaired by unprocessed prelamin A (
Liu et al., 2013a). Due to reduced ATM activation, ATM-dependent phosphorylation of Kap-1 was compromised in Zmpste24
−/− MEFs. Un-phosphorylated Kap-1 constantly associates with MNase-resistant heterochromatin and blocks DNA damage-induced chromatin de-condensation and remodeling, hindering the recruitment of DSB repair factors (
Liu et al., 2013a). siRNA-mediated suppression of Kap-1 was able to rescue the defective chromatin remodeling, improve 53BP1 recruitment and alleviate cellular senescence in Zmpste24
−/− MEFs (
Liu et al., 2013a).
Progerin undermines genome stability by increasing ROS levels
Genome instability can be triggered either by functional deficiencies in DNA damage repair or by an increased amount of DNA damaging agents. The above discussions mainly focused on progerin’s effects delaying DNA damage repair. In this section, we address the potential of progerin to elevate the levels of endogenous DNA damage agents. ROS (O
2−∙, H
2O
2, OH∙, etc.) are metabolic byproducts generated during biochemical reactions and are the major endogenous agents accounting for oxidative DNA damage (
De Bont and van Larebeke, 2004). By reacting with the double bonds of DNA bases, ROS are able to create more than 20 types of DNA damages including oxidized bases, SSBs and DSBs (
Cooke et al., 2003;
De Bont and van Larebeke, 2004; Lin and Beal, 2006;
Murphy, 2009). Under physiologic conditions, these toxic effects of ROS are antagonized by defense mechanisms such as DNA damage repair. However, oxidative stress will emerge under pathological conditions, when ROS production outcompetes the capacity of cellular defense mechanisms and impairs the intactness of genome (
De Bont and van Larebeke, 2004). In 2009, Viteri et al. reported that HGPS cells bear significantly increased accumulation of ROS (1.4 to 4 folds) and oxidatively modified proteins compared to age-matched normal controls. Notably, it seemed that HGPS cells displayed normal levels of ROS scavenging proteins, indicating that progerin affected ROS levels by expanding their origins (
Viteri et al., 2010). Elevated ROS in HGPS was later on confirmed by Richards et al. in 2010. Significantly, they found that high levels of ROS were associated with the accumulation of DSBs and the impaired cellular proliferation potential in HGPS fibroblasts (
Richards et al., 2011). Moreover, HGPS cells displayed higher sensitivity to oxidative stress as evidenced by inefficient repair of DSBs induced by ROS insults (
Richards et al., 2011). Treatment with ROS scavenger N-acetyl cysteine (NAC) alleviated their background DNA damage level and extended their population doubling times, confirming a negative role of ROS in HGPS genome stability (
Richards et al., 2011). These findings suggested that progerin might perturb genome stability by introducing extra ROS and sensitizing cells to oxidative stress.
ROS production is tightly correlated with mitochondria functionality (
Murphy, 2009). Disruption of mitochondrial function can result in oxidative damage under a variety of pathological situations (
Lin and Beal, 2006;
Murphy, 2009;
Pagano et al., 2014). Studies by our group and others have revealed progerin-induced mitochondrial deficiencies in HGPS fibroblasts (
Xiong et al., 2015). To begin with, an SILAC (stable isotope labeling with amino acid) analysis revealed that mitochondrial oxidative phosphorylation proteins were reduced in HGPS patients in comparison to normal subjects (
Rivera-Torres et al., 2013). Consistently, mitochondrial ATP synthesis was also found to be reduced HGPS fibroblasts (
Viteri et al., 2010;
Xiong etal., 2016). Mitochondrial defects were also observed in fibroblasts obtained from adult progeroid mouse models: Zmpste24
−/− and
LmnaG609/G609 (
Osorio et al., 2011 ; Peinado et al., 2011). Recently, our group reported that HGPS fibroblasts bear swollen and fragmented mitochondria (
Xiong et al., 2016). Overexpression of progerin in normal fibroblasts was able to recapitulate these abnormal mitochondria, suggesting that progerin negatively impacts mitochondrial integrity and function (
Xiong et al., 2016). Our study provided the structural base for HGPS associated mitochondrial defects. Function wise, HGPS fibroblasts and progerin-expressing normal controls displayed significantly delayed mitochondrial movements (
Xiong et al., 2016). Moreover, mitochondrial membrane potential (MMP) was shown to be disrupted in HGPS fibroblasts and HGPS-iPSC differentiated SMCs (
Xiong et al., 2016). Consistent with previous reports, we observed an obvious elevation of mitochondrial specific ROS in HGPS fibroblasts (
Xiong et al., 2015). Together, these findings revealed mitochondria as an important factor that progerin targets to increase cellular ROS.
Progerin affects genome integrity by perturbing DNA replication
The second source of endogenous DNA damage comes from collapsed DNA replication forks (
Shrivastav et al., 2008). DNA replication is a fundamental biologic process that allows cells to accurately duplicate their genetic materials (
Mazouzi et al., 2014). Replicating genomes are especially susceptible to mutations as endogenous and exogenous agents may threaten their integrity by interfering with the replication fork stabilization, progression, and reestablishment (
Branzei and Foiani, 2005;
Mazouzi et al., 2014). For example, DSBs may occur due to replication fork collapse when the replication fork encounters DNA lesions (SSBs, inter-strand crosslinks or pyrimidine dimers) (
Shrivastav et al., 2008). In this regard, elevated ROS induced by progerin may create DNA lesions on replicating nucleotide chains and introduce DSBs in S phase HGPS cells.
DNA replication is spatial-temporally orchestrated by the replisome, a multiple-component protein complex that consists of replicative helicases, DNA polymerases and their accessory factors such as the clamp loader, replication factor C (RFC) and the clamp itself, proliferation cell nuclear antigen (PCNA) and topoisomerases (
Branzei and Foiani, 2010). Given the complexities of this machinery and its functions, it is reasonable to speculate that genetic or functional compromise of its components will negatively affect replication fork and genome stability. Despite lacking direct evidence, several studies implied that progerin or prelamin A may perturb the stability of DNA replication fork machinery (
Liu et al., 2008;
Musich and Zou, 2009;
Tang et al., 2012). For example, Liu et al. reported that the sliding clamp ring protein PCNA formed discrete foci in early passage S phase HGPS cells, but not in late passage HGPS cells, suggesting a redistribution of PCNA occurred during the accumulation of progerin (
Liu et al., 2008). PCNA tethers the DNA polymerase d and ε to ensure rapid DNA synthesis (
Kelman, 1997). Its disassociation from the replications forks not only renders unprotected single strand (ss) or double strand (ds) DNA junctions but also inhibits DNA replication, resulting in stalled DNA replication fork and replication fork collapse (
Moir et al., 2000;
Liu et al., 2008;
Musich and Zou, 2009). In this sense, progerin-induced redistribution of PCNA may result in replication fork stalling and subsequent DSBs. This notion was supported by Liu et al.’s finding that xeroderma pigmentosum group A (XPA), a protein with high affinity to ss/ds DNA junctions, was co-localized with DSB marker gH2AX in late passage HGPS cells (
Liu et al., 2008). Subsequent studies by Tang et al. discovered that the large subunit of replication factor C (RFC) was proteolytically degraded in HGPS fibroblasts, leading to a truncated RFC of around 75kD and defective loading of PCNA and polymerase d to the replication fork. These results provided a mechanistic base underlying how progerin perturbed PCNA distribution and introduced DSBs during DNA replication (
Tang et al., 2012).
As discussed above, progerin may cause replicative stress by introducing excessive ROS and interfering with replication fork progression. Under physiologic conditions with limited exogenous genotoxic stimuli, endogenous DSBs caused by collapsed replication fork are considered as an important contributor to genome instability. Unfortunately, HR, the predominant DSB repair pathway in S/G2 phase, was found to be disrupted in HGPS cells, implying that HGPS cells were vulnerable to replicative stress induced DSBs (
Liu et al., 2005;
Zhang et al., 2014). The exact molecular mechanisms underlying the HGPS-related HR deficiency remain unclear. The intermediate single strand DNA structure (ssDNA) generated by replicative fork collapse activates ATR (ATM- and Rad3- related) kinase, which is located to the damaged DNA lesion, triggers DNA damage checkpoint response and facilitates homologous recombination and replication fork restart (
Flynn and Zou, 2011). Liu et al. has previously reported that HGPS cells exhibited persistent CHK1 phosphorylation at serine 345 and nuclear ATR foci formation at a basal level, suggesting ATR activation and cell cycle arrest (
Liu et al., 2006; Musich and Zou, 2009). However, the above study was performed in unsynchronized cells. It remains unclear whether ATR is able to properly respond to replicative stress and fully carry out its functions in HGPS cells during S/G2 phase. Moreover, we failed to observe ATR activation in HGPS cells in response to Doxorubicin treatment (unpublished data). Therefore, additional studies are required to elucidate ATR activation in HGPS cells upon DNA damage.
Therapeutic approaches to HGPS
Approaches targeting progerin level and toxicity
Progerin retains a permanent farnesyl tail, which anchors it to the INM and triggers a variety of cellular phenotypes. One classic strategy to neutralize progerin toxicity is to block its farnesylation with farnesyltransferase inhibitors (FITs). Treating HGPS fibroblasts with FTI was able to improve HPGS nuclear morphology (
Capell et al., 2005). Moreover, in vivo studies on two HGPS mouse models (BACG608G and
LmnaHG/+ ) demonstrated that FTI treatment was sufficient to improve a variety of HGPS associated phenotypes such as short lifespan, growth retardation and bone abnormalities (tested on
LmnaHG/+ ) and loss of vascular SMCs (tested on BACG608G) (
Varga et al., 2006;
Capell et al., 2008; Yang et al., 2006,
2008; Zhang et al., 2013). Significantly, clinical trial on HGPS patients with FTI showed that FTI treatment was able to improve HGPS patients' cardiovascular status and extend the lifespan of HGPS patients by 1.6 years (
Gordon et al., 2012,
2014). FTI is by far the most extensively studied and widely applied treatment toward HGPS. It shows considerable beneficial effects on HGPS both
in vitro and
in vivo. Suspects on FTI mainly focus on its specificity as it may influence multiple cellular pathways that involve farnesyltransferase. Indeed, FTI treatment was shown to induce centrosome separation defects and causes “donut-shaped” nuclei, calling that attentions should be paid when adopting FTI treatment as a therapeutic strategy (
Verstraeten et al., 2011).
The second type aims at reducing the absolute level of progerin and thereby reduce toxicities. One representative of these strategies is the sequence specific morpholino antisense oligonucleotides (AONs), which blocks the cryptic splicing donor site on prelamin A premRNA. Scaffidi et al. showed that AONs were able to improve the nuclear morphology and restore gene expression profile in HGPS fibroblasts (
Scaffidi and Misteli, 2005).
In vivo study performed by Osorio et al. also confirmed the beneficial effects of AONs on
LmnaG609G transgenic mice (
Osorio et al., 2011). However, attentions should be paid to off-target effects since the dosages of AONs were relatively high (>10 mM) (
Scaffidi and Misteli, 2005;
Osorio et al., 2011).
In 2011, Cao et al. reported rapamycin, a macrolide-antibiotic, as a promising chemical to reduce the progerin level in HGPS fibroblasts. Mechanistically, rapamycin was able to stimulate the autophagy pathway and remove insoluble progerin aggregates in HGPS cells. In vitro characterization confirmed that rapamycin treatment improved HGPS nuclear morphology, delayed HGPS cellular senescence and promoted HGPS proliferation (
Cao et al., 2011). Despite that rapamycin benefits HGPS fibroblasts,
in vivo studies are necessary to validate its effect on HGPS animal models.
Approaches targeting HGPS genome instability
Genome instability is considered as a driving force of the premature aging phenotypes in HGPS. Thus approaches aiming to maintain genome integrity were also proposed to treat HGPS. One of these methods is to use drugs that target histone modifiers to remove the abnormal epigenetic alterations in HGPS and rescue DSB repair defects. For example, Krishnan et al. showed that HDAC inhibitor sodium butyrate was able to increase H4K16ac, restore 53BP1 recruitment and alleviate premature senescence in Zmpste24
−/− MEFs (
Krishnan et al., 2011). Another study from the same group proposed that resveratrol was able to enhance SIRT1 activity by reinforcing the interaction between SIRT1 and lamin A (
Liu et al., 2012).
Progerin may also induce genome instability by disrupting mitochondrial functionality and increasing oxidative stress (
Rivera-Torres et al., 2013). Using ROS scavengers to alleviate intracellular oxidative stress is an alternative way to reduce DNA damage in HGPS. Intriguingly, in our recent study, we showed that methylene blue, a mitochondrial targeting anti-oxidant was able to enhance mitochondrial functionality and reduce ROS levels in HGPS fibroblasts (
Xiong et al., 2015). Consequently, methylene blue delayed HGPS premature senescence and significantly extended its proliferation capacity (
Xiong et al., 2015).
Conclusion marks
Progerin can be recognized not only as a dominant negative lamin A mutant but also as a gain of function protein, which may impact genome integrity through unknown complex mechanisms. This feature makes mechanistic study especially challenging. Based on our above discussions, mechanisms underlying HGPS genome instability can be divided into three groups: disrupted DDR, defective DSB repair and increased source of DNA damage. These groups can be further sub-categorized according to their specific defects. These defects are associated with lamin A functionality, epigenetic modifications, HR/NHEJ pathway choice, ATM activation; DSB induced histone modifications and chromatin remodeling, ROS balance and DNA replication (Fig. 3). These mechanisms are multi-dimensional but interconnected and together they are reflected on the complex HGPS pathological phenotypes. Despite significant mechanistic advances, important questions remain to be addressed. For example, it is unknown how progerin negatively regulates HR during S/G2 phase. Moreover, how progerin triggers the cell cycle-dependent inactivation of ATM and gH2AX responses is still an open question. Thanks to the mechanistic advances, novel drugs are identified and extremely consummate current FTI based therapy. These approaches mainly target HGPS genome instability and include the adoption of HDAC inhibitors and ROS scavengers. Despite promising, these methods are still under development and additional studies are indispensable to evaluate their effects and risks.
Higher Education Press and Springer-Verlag Berlin Heidelberg
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