A de novo G608G mutation in LMNA is the predominant cause of Hutchinson-Gilford progeria syndrome (HGPS) (Eriksson et al., 2003). Lamin A is first synthesized as prelamin A and ZMPSTE24 metalloproteinase is required for its maturation (Pendas et al., 2002). Mice lacking Zmpste24 recapitulate many of the progeroid features in HGPS (Pendas et al., 2002). HGPS cells and Zmpste24−/− MEFs undergo accelerated senescence, attributable to delayed recruitment of repair proteins and defective DNA repair (Liu et al., 2005, 2006). However, the underlying molecular mechanism remains largely unknown.
Upon DNA damage, chromatin opens up for the loading and retention of repair proteins to DNA lesions (Iijima et al., 2008). To determine the chromatin accessibility, a micrococcal nuclease sensitivity assay was employed. In wild-type cells, chromatin accessibility peaked 30 min after γ-irradiation, reflected by the highest amount of low molecular weight oligonucleosomal fragments and the concurrent disappearance of high molecular weight DNA fragments (Fig. 1A,B). Thereafter, higher molecular weight DNA fragments gradually increased whereas the lower molecular weight fragments concurrently decreased, suggesting recondensation and restoration of chromatin. However, maximal chromatin accessibility was significantly delayed to 2 h and the chromatin started to recondense approximately 4 h after γ-irradiation in Zmpste24−/− cells (Fig. 1A,B and Fig. S1A, lane 6-10). Delayed chromatin remodeling in Zmpste24−/− cells was likely a consequence of accumulated prelamin A, as ectopic prelamin A in HEK293 cells caused similar defects (Fig. S2).
In response to DNA damage, ATM phosphorylates KAP-1 at Ser 824 (pS824-KAP-1), which weakens the binding of KAP-1 to MNase-resistant heterochromatin fraction and releases CHD3 from chromatin, leaving the heterochromatin de-condensed for loading essential repair proteins (Ziv et al., 2006; Goodarzi et al., 2008, 2011; Noon et al., 2010). To understand the mechanisms behind the defective chromatin remodeling in Zmpste24−/− MEFs, pS824-Kap-1 level was examined. The level of pS824-Kap-1 peaked around 30 min after γ-irradiation and was decreased gradually thereafter in wild-type cells (Fig. 1C,D), while it was significantly reduced in Zmpste24−/− MEFs. Consistently, in wild-type cells, the level of Kap-1 associated with MNase-resistant fraction was significantly reduced at 30 min after γ-irradiation in a dose-dependent manner, whereas it was hardly changed in Zmpste24−/− cells (Fig. 1E). As ATM is the only kinase responsible for pS824-Kap-1, and loss of ATM in MEFs also leads to defective chromatin remodeling upon DNA damage (Fig. S3), we asked whether ATM itself is affected in progeroid cells. Indeed, we found a significant reduction in the level of pS1981-ATM in Zmpste24−/− MEFs compared with wild-types (Fig. 1F and Fig. S4). Consistently, as a direct target of ATM (Shiloh, 2006), the level of pS343-Nbs1 was also significantly decreased in Zmpste24−/− MEFs in response to DNA damage (Fig. 1F and Fig. S4). Thus, these data indicate that defective ATM-Kap-1 signaling might underlie the defective chromatin remodeling in Zmpste24−/− MEFs.
We next tested whether knocking down Kap-1 could rescue the impaired chromatin remodeling, defective DNA repair, and early senescence in Zmpste24−/− MEFs. As shown, knocking down Kap-1 restored chromatin relaxation at 30 min and subsequent recondensation around 2 h after DNA damage in Zmpste24−/− MEFs (Fig. 2A,B and Fig. S5). Concurrently, the delayed recruitment of 53BP1 was restored and the sustained 53BP1 foci staining at 24 h after DNA damage were substantially reduced in Zmpste24−/− MEFs (Fig. 2C). Moreover, knocking down Kap-1 rescued the early senescence in Zmpste24−/− MEFs determined by senescence-associated β-galactosidase assay (Fig. 2D,E, 74 ± 5% positively stained cells with scramble vs. 35 ± 3% with Kap-1 siRNA in Zmpste24−/− cells, Mean ± SEM, P < 0.05). Thus, defective ATM-Kap-1 signaling underlies defective chromatin remodeling, defective DNA repair, and early senescence in Zmpste24−/− MEFs.
Collectively, we found that the defective DNA repair in laminopathy-based progeria was attributable to compromised ATM-Kap-1 signaling and delayed global chromatin remodeling. Knocking down Kap-1 rescues the defective DNA repair and early senescence in progeroid cells, suggesting an important role of chromatin remodeling in laminopathy-based premature aging. The delayed yet completely relaxed chromatin in Zmpste24−/− MEFs implies the existence of a potential backup mechanism that mediates late chromatin remodeling in progeroid cells. Indeed, it has been reported that BRIT1 (BRIT-repeat inhibitor of hTERT expression), Brca1 and cofactor COBRA1, E2F1, and p53 regulate global chromatin relaxation/remodeling (Peng et al., 2009; Ye et al., 2001). This could be a backup mechanism regulating global chromatin remodeling in Zmpste24−/− MEFs, in response to the defective ATM-Kap-1 signaling.
We recently showed that sodium butyrate (NaB) and trichostatin A (TSA) induced up-regulation of global histone acetylation and feeding progeria mice with NaB increased the acetylation of H4K16, rescued the delayed recruitment of 53BP1, and extended lifespan in progeroid mice (Krishnan et al., 2011). NaB and TSA belong to class I and II HDAC inhibitors (Bolden et al., 2006). TSA treatment activates ATM upon DNA damage in addition to increasing histone acetylation (Bakkenist & Kastan, 2003). Interestingly, preincubation of NaB also rescued defective chromatin remodeling upon DNA damage in Zmpste24−/− MEFs (see Fig. S1). Thus, in addition to local H4K16 acetylation surrounding DNA lesions, NaB treatment may directly enhance ATM activity thus rescuing defective global chromatin remodeling and DNA repair as well as early senescence in progeria mice.