Divergent FUS phosphorylation in primate and mouse cells following double-strand DNA damage

Abstract

Fused in sarcoma (FUS) is a RNA/DNA protein involved in multiple nuclear and cytoplasmic functions including transcription, splicing, mRNA trafficking, and stress granule formation. To accomplish these many functions, FUS must shuttle between cellular compartments in a highly regulated manner. When shuttling is disrupted, FUS abnormally accumulates into cytoplasmic inclusions that can be toxic. Disrupted shuttling of FUS into the nucleus is a hallmark of ~10% of frontotemporal lobar degeneration (FTLD) cases, the neuropathology that underlies frontotemporal dementia (FTD). Multiple pathways are known to disrupt nuclear/cytoplasmic shuttling of FUS. In earlier work, we discovered that double-strand DNA breaks (DSBs) trigger DNA-dependent protein kinase (DNA-PK) to phosphorylate FUS (p-FUS) at N-terminal residues leading to the cytoplasmic accumulation of FUS. Therefore, DNA damage may contribute to the development of FTLD pathology with FUS inclusions. In the present study, we examined how DSBs effect FUS phosphorylation in various primate and mouse cellular models. All cell lines derived from human and non-human primates exhibit N-terminal FUS phosphorylation following calicheamicin γ1 (CLM) induced DSBs. In contrast, we were unable to detect FUS phosphorylation in mouse-derived primary neurons or immortalized cell lines regardless of CLM treatment, duration, or concentration. Despite DNA damage induced by CLM treatment, we find that mouse cells do not phosphorylate FUS, likely due to reduced levels and activity of DNA-PK compared to human cells. Taken together, our work reveals that mouse-derived cellular models regulate FUS in an anomalous manner compared to primate cells. This raises the possibility that mouse models may not fully recapitulate the pathogenic cascades that lead to FTLD with FUS pathology.

Proximity-based labeling reveals DNA damage-induced N-terminal phosphorylation of fused in sarcoma (FUS) leads to distinct changes in the FUS protein interactome

“…We genetically fused APEX2 to the N-terminus of three FUS protein variants via a (GGGS) 3 linker to generate three Twin-Strep-tagged® constructs: 1) wild-type human FUS (FUS WT), 2) phosphomimetic FUS (FUS PM), and 3) the ALS-linked P525L mutant FUS (FUS P525L) (Figure 1A). FUS PM was generated by substituting the 12 consensus S/T_Q residues, which are phosphorylated by DNA-PK following DSB, with the negatively charged amino acid aspartate (Deng et al ., 2014b; Johnson et al ., 2020). The FUS P525L mutation was first identified in 2012 and causes a severe form of juvenile ALS (Conte et al , 2012; Zhou et al , 2020).…”

“…We wanted to ensure that FUS PM bound proteins in a similar manner to biological relevant N-terminally phosphorylated FUS. Thus, we also confirmed that endogenous UPF1 binds preferentially to FUS treated with calicheamicin γ1 (CLM), a known inducer of N-terminal FUS phosphorylation (Deng et al ., 2014b; Johnson et al ., 2020; Rhoads et al ., 2018) (Supplementary Figure 4). Lastly, we confirmed the interaction of three novel binding partners, VPS35, MOV10 and CLTA, to our three FUS variants (Figure 4A, red bar).…”

“…Although hypomethylated FUS accumulates in in FTLD-FUS inclusions, genetic mutations, or cellular stressors, have not been discovered that explain this phenomenon (Dormann et al ., 2012; Ravenscroft et al , 2013). Unlike methylation, our lab has shown that a biologically relevant stressor, double strand DNA breaks (DSBs), triggers the DNA dependent protein kinase (DNA-PK) to phosphorylate FUS at 12 key S/T_Q residues in the N-terminal SYGQ-low complexity domain (Deng et al ., 2014b; Johnson et al ., 2020; Monahan et al ., 2017; Murray et al ., 2017; Rhoads et al ., 2018). Phosphorylated FUS (p-FUS) then accumulates in the cytoplasm of the cell (Deng et al , 2014a; Johnson et al ., 2020).…”

“…Thus, these previous models do not address how non-genetic mediators of FUS pathology, such as post-translational modifications, may shift FUS function. Previous non-genetic models demonstrate that cytoplasmic accumulation of FUS can be triggered by other, non-genetic mechanisms including loss of transportin-1/FUS interaction, cellular stressors, and/or altered post-translational modifications (Bowden & Dormann, 2016; Darovic et al ., 2015; Dormann et al ., 2012; Higelin et al ., 2016; Johnson et al ., 2020; Sama et al ., 2013; Scaramuzzino et al ., 2013; Singatulina et al ., 2019). Although hypomethylated FUS accumulates in in FTLD-FUS inclusions, genetic mutations, or cellular stressors, have not been discovered that explain this phenomenon (Dormann et al ., 2012; Ravenscroft et al , 2013).…”

“…FUS can be phosphorylated at multiple N-and C-terminal residues, but the functional consequence of these modifications remain largely unexplored (Darovic et al ., 2015; Deng et al , 2014b; Droppelmann et al , 2014; Monahan et al , 2017; Murray et al , 2017). Our lab discovered that phosphorylation of 12 specific N-terminal residues in FUS by the DNA-dependent protein kinase (DNA-PK) causes the cytoplasmic accumulation of phosphorylated FUS (Deng et al ., 2014b; Johnson et al , 2020; Singatulina et al ., 2019). This cascade is triggered by double strand DNA breaks (DSB).…”