Progerin-Induced Replication Stress Facilitates Premature Senescence in Hutchinson-Gilford Progeria Syndrome

Abstract

Hutchinson-Gilford progeria syndrome (HGPS) is caused by a mutation in LMNA that produces an aberrant lamin A protein, progerin. The accumulation of progerin in HGPS cells leads to an aberrant nuclear morphology, genetic instability, and p53-dependent premature senescence. How p53 is activated in response to progerin production is unknown. Here we show that young cycling HGPS fibroblasts exhibit chronic DNA damage, primarily in S phase, as well as delayed replication fork progression. We demonstrate that progerin binds to PCNA, altering its distribution away from replicating DNA in HGPS cells, leading to ␥H2AX formation, ATR activation, and RPA Ser33 phosphorylation. Unlike normal human cells that can be immortalized by enforced expression of telomerase alone, immortalization of HGPS cells requires telomerase expression and p53 repression. In addition, we show that the DNA damage response in HGPS cells does not originate from eroded telomeres. Together, these results establish that progerin interferes with the coordination of essential DNA replication factors, causing replication stress, and is the primary signal for p53 activation leading to premature senescence in HGPS. Furthermore, this damage response is shown to be independent of progerin farnesylation, implying that unprocessed lamin A alone causes replication stress.KEYWORDS HGPS, progerin, senescence, aging, p53, telomere T he study of children with rare accelerated aging (progeria) syndromes has provided insight into the normal process of aging. Hutchinson-Gilford progeria syndrome (HGPS) is caused by a heterozygous, autosomal dominant mutation in LMNA (1), the gene that encodes lamin A, a key structural protein in the nuclear lamina. The lamina, a protein network that lines the inner nuclear membrane, provides structural support for the nucleus and is important for chromatin attachment, DNA replication, nuclear organization, and gene transcription in ways that are not completely understood. Farnesylation of prelamin A at the C terminus allows targeting to the inner nuclear membrane, where it is subsequently cleaved by the zinc metalloproteinase Zmpste24 to release mature lamin A into the lamina and nucleoplasm. In HGPS, the most common LMNA mutation (G608G) activates a cryptic splice donor site in exon 11, resulting in prelamin A mRNA with a 150-bp internal deletion, leading to a 50-amino-acid truncation in a region that contains the Zmpste24 cleavage site (reviewed in reference 2). The mutant lamin, termed progerin, retains the farnesyl lipid anchor, interferes with the integrity of the nuclear lamina, and causes the formation of misshapen nuclei. The retention of progerin on the inner nuclear membrane interferes with the function and normal distribution of lamin A, causing a loss of peripheral heterochromatin, increased DNA damage, impaired recruitment of DNA repair proteins to sites of DNA damage (3), and persistent activation of DNA damage response proteins (4). In addition to its

Heterochromatin loss as a determinant of progerin‐induced DNA damage in Hutchinson–Gilford Progeria

“…Importantly, we demonstrate that heterochromatin loss by itself is not sufficient for progerin to induce DNA damage: progerin‐expressing cells also have to undergo DNA replication to accumulate DNA damage, as suggested earlier (Wheaton et al, ). Moreover, we provide evidence that the onset of progerin‐induced DNA damage occurs exclusively during the late stages of DNA replication, prior to chromosome condensation and mitosis.…”

“…Progerin‐induced DNA damage necessitates cell proliferation (Hilton et al, ; Wheaton et al, ), arises in cells with lower levels of heterochromatin and is prevented by TERT. Since fragile sites like telomeres at the nuclear periphery and heterochromatin are all replicated late in S‐phase (Arnoult et al, ; Rhind & Gilbert, ), we asked whether the onset of DNA damage could coincide with the timing of normal heterochromatin replication in late S‐phase.…”

“…Previous studies reported that progerin expression leads to mitotic defects (Cao, Capell, Erdos, Djabali, & Collins, ; Dechat et al, ), whereas more recent findings suggested that both progerin and prelamin A may trigger DNA damage during DNA replication (Cobb, Murray, Warren, Liu, & Shanahan, ; Hilton et al, ; Wheaton et al, ). However, deciphering the causal and temporal links between the different progerin‐induced phenotypes remains challenging as the majority of studies have been conducted in patient‐derived cells, or cells constitutively expressing progerin, where immediate consequences of progerin expression and secondary effects arising from progerin‐induced senescence cannot be distinguished.…”

Targeting the phospholipase A2 receptor ameliorates premature aging phenotypes

“…PLA2R1 regulates senescence through induction of a mitochondrial program leading to ROS production, DNA damage, and the p53 pathway (Augert et al, 2009; Griveau et al, 2016). Progerin is able to induce DNA damage accumulation, eventually through ROS generation (Musich, & Zou, 2011; Richards, Muter, Ritchie, Lattanzi, & Hutchison, 2011; Wheaton et al, 2017). Our data support a role of PLA2R1 in progerin‐induced DNA damage as its knockdown reverts accumulation of DNA damage marks such as γH2AX and P‐ATM.…”

Diminished Canonical β-Catenin Signaling During Osteoblast Differentiation Contributes to Osteopenia in Progeria

“…Normal and two HGPS(I) and HGPS(II) fibroblast cell lines (HGADFN168, HGADFN164, HGADFN167) were obtained from the Progeria Research Foundation cell bank (Peabody, MA, USA). These cell lines have been fully characterized, including the karyotypes . Induced pluripotent stem cells (iPSCs) were generated by reprogramming with KLF4 , SOX2 , OCT4 , and C‐MYC Yamanaka factors, as we have described previously .…”