Nucleostemin deletion reveals an essential mechanism that maintains the genomic stability of stem and progenitor cells

Abstract

Stem and progenitor cells maintain a robust DNA replication program during the tissue expansion phase of embryogenesis. The unique mechanism that protects them from the increased risk of replication-induced DNA damage, and hence permits self-renewal, remains unclear. To determine whether the genome integrity of stem/progenitor cells is safeguarded by mechanisms involving molecules beyond the core DNA repair machinery, we created a nucleostemin (a stem and cancer cell-enriched protein) conditional-null allele and showed that neural-specific knockout of nucleostemin predisposes embryos to spontaneous DNA damage that leads to severe brain defects in vivo. In cultured neural stem cells, depletion of nucleostemin triggers replication-dependent DNA damage and perturbs self-renewal, whereas overexpression of nucleostemin shows a protective effect against hydroxyureainduced DNA damage. Mechanistic studies performed in mouse embryonic fibroblast cells showed that loss of nucleostemin triggers DNA damage and growth arrest independently of the p53 status or rRNA synthesis. Instead, nucleostemin is directly recruited to DNA damage sites and regulates the recruitment of the core repair protein, RAD51, to hydroxyurea-induced foci. This work establishes the primary function of nucleostemin in maintaining the genomic stability of actively dividing stem/progenitor cells by promoting the recruitment of RAD51 to stalled replication-induced DNA damage foci.DNA damage repair | homologous recombination | conditional knockout | replication fork stalling | neural development S tem and progenitor cells play critical roles in embryonic organogenesis, adult tissue regeneration, and tumor development. To maintain self-renewing proliferation, they must be protected from replication-induced DNA damage that limits the proliferative lifespan of most dividing cells. Replication-induced DNA damage may occur spontaneously as a result of stalled and collapsed replication forks, caused by the slowing of the DNA replication machinery over replication "trouble" zones or previously unrepaired damage sites (1-3). Alternatively, replication stalling can be triggered by drugs that deplete the endogenous nucleotide pool (e.g., hydroxyurea) or inhibit the activity of DNA replication (e.g., camptothecin). Prolonged replication stalling will lead to the collapse of replication machinery and doublestrand DNA breaks (DSBs), causing cell cycle arrest and genomic instability (4, 5). To date, it remains unclear how stem and progenitor cells survive the increased risk of replication-induced DNA damage during this hyperactive mitotic window of embryogenesis.Nucleostemin (NS) is a stem cell-enriched nucleolar protein (6). Its biological significance has been illustrated by the early embryonic lethal phenotype of NS germ-line knockout mice (7), which ineluctably hinders further analyses of its in vivo function beyond the blastula stage. To date, the mechanism of NS action in vivo remains unclear. Although some suggested a connection to the p53 pathway (6,(8)(9)(10), ...

Turning a new page on nucleostemin and self-renewal

“…Regardless, until such evidence is presented, it is scientifically inaccurate to refer to mammalian nucleostemin as a nucleolar GTPase or assume it functions as a GTPase enzyme. The biological importance of nucleostemin has never been in doubt, as it is well established that nucleostemin makes key contributions to blastocyst formation (Beekman et al, 2006;Zhu et al, 2006), embryogenesis (Meng et al, 2013), postnatal tissue regeneration Shugo et al, 2012), cancer development (Lin et al, 2010;Sijin et al, 2004;Tamase et al, 2009) and reprograming to pluripotency (Qu and Bishop, 2012).…”

“…One study has shown that nucleostemin is able to reduce telomere damage through modification of telomeric repeat Science (2014Science ( ) 127, 3885-3891 doi:10.1242 binding factor 1 (TRF1, also known as TERF1) and recruitment of promyelocytic leukemia protein isoform IV (PML-IV) in telomerase-negative human cancer cells (Hsu et al, 2012). Two reports have identified that it protects the genome integrity of stem or progenitor cells by promoting the recruitment of RAD51, the core component of the homologous recombination machinery, to stalled replication-induced DNA damage foci Meng et al, 2013) (Fig. 1).…”

Nucleolin and nucleophosmin: nucleolar proteins with multiple functions in DNA repair

“…uS3) has been reported to act as an apurinic/apyrimidinic endonuclease in both nuclear and mitochondrial base excision repair (Kim et al 2013), while nucleostemin/GNL3 has been implicated in the recruitment of RAD51 to stalled replication forks Meng et al 2013), and Nol12 has D r a f t 5 been identified as an important protective factor against oxidative DNA damage (Scott et al 2016). Most strikingly, two proteins -nucleophosmin (NPM1) and nucleolin (NCL) -are required for optimal activity of multiple independent DNA repair pathways, and their dysregulation has been reported in numerous cancers, in particular leukemias and other myeloproliferative disorders (Berger et al 2015;Colombo et al 2011;Federici et al 2013).…”

Distinct genome protective vs. ribosome synthetic functions of the paralogous nucleolar proteins nucleostemin and GNL3L

“…Thus, we believe that the conclusion reached by Romanova et al might be confounded by unsuspected issues surrounding the immediate versus longterm effects of nucleostemin depletion, as well as the interconnecting relationship between nucleolar function and cell cycle. Furthermore, while the present investigation was under way, a parallel study suggested that there is a direct molecular recruitment of nucleostemin to DNA damage sites, where it interacts with the DNA repair protein RAD51 (Meng et al, 2013). This also alerted us to the possibility that nucleostemin is involved in genome protection and not rRNA synthesis.…”

“…In this respect, it is noteworthy that effects on ribosome synthesis have been observed following two rounds of RNAi-mediated nucleostemin knockdown over a period of 5 days (Romanova et al, 2009a;Romanova et al, 2009b). Motivated by the recent discovery of a role for nucleostemin in protecting telomeres and the genome integrity of stem and progenitor cells (Hsu et al, 2012;Lin et al, 2013;Meng et al, 2013;Yamashita et al, 2013), we began to consider whether the accumulation of DNA damage, rather than impaired ribosome production, is actually the initial event following nucleostemin depletion. Using human breast cancer MDA-MB-231 cells, we found that DNA damage arises as early as 12 hours after the initiation of nucleostemin depletion, without incurring a significant effect on rRNA synthesis or nucleolar structure for up to 48 hours.…”