SMC5/6 is required for replication fork stability and faithful chromosome segregation during neurogenesis

Abstract

Mutations of SMC5/6 components cause developmental defects, including primary microcephaly. To model neurodevelopmental defects, we engineered a mouse wherein Smc5 is conditionally knocked out (cKO) in the developing neocortex. Smc5 cKO mice exhibited neurodevelopmental defects due to neural progenitor cell (NPC) apoptosis, which led to reduction in cortical layer neurons. Smc5 cKO NPCs formed DNA bridges during mitosis and underwent chromosome missegregation. SMC5/6 depletion triggers a CHEK2-p53 DNA damage response, as concomitant deletion of the Trp53 tumor suppressor or Chek2 DNA damage checkpoint kinase rescued Smc5 cKO neurodevelopmental defects. Further assessment using Smc5 cKO and auxin-inducible degron systems demonstrated that absence of SMC5/6 leads to DNA replication stress at late-replicating regions such as pericentromeric heterochromatin regions. In summary, SMC5/6 is important for completion of DNA replication prior to entering mitosis, which ensures accurate chromosome segregation. Thus, SMC5/6 functions are critical in highly proliferative stem cells during organism development.

Pathogenic variants in SLF2 and SMC5 cause segmented chromosomes and mosaic variegated hyperploidy

“…To investigate the two principal underlying causes of microcephaly, slowed cell cycle progression and/or increased apoptosis in the developing brain 2 , 26 – 28 , fixed whole-mount slf2 and smc5 depleted zebrafish embryos were stained with markers of cell cycle stage (G2/M: phospho-histone H3 serine-10) and apoptosis (TUNEL). F0 CRISPR embryos injected with either slf2 or smc5 sgRNA with recombinant Cas9 (Fig.…”

Time is of the essence: the molecular mechanisms of primary microcephaly

“…Therefore, activation of the MSP is only one of several possible pathways that can result in reduced cortical size. In fact, it was shown that microcephaly caused by loss of SMC5 in the brains of mice is independent of the MSP and thus cannot be rescued by knocking out the MSP signaling components 53BP1 and USP28 ( Atkins et al 2020 ; Phan et al 2021 ). However, this same microcephaly model can be rescued by knocking out CHK2 or P53, indicating that DNA damage signaling is predominantly responsible for the impaired cortical growth in these animals ( Atkins et al 2020 ).…”

“…In fact, it was shown that microcephaly caused by loss of SMC5 in the brains of mice is independent of the MSP and thus cannot be rescued by knocking out the MSP signaling components 53BP1 and USP28 ( Atkins et al 2020 ; Phan et al 2021 ). However, this same microcephaly model can be rescued by knocking out CHK2 or P53, indicating that DNA damage signaling is predominantly responsible for the impaired cortical growth in these animals ( Atkins et al 2020 ). Identifying which microcephaly mutations activate the MSP and which trigger other pathogenic pathways is an important area of future research.…”

A One-step strategy to target essential factors with auxin-inducible degron system in mouse embryonic stem cells

“…It evades the irreversibility of gene knockout strategies, and is not hindered by incomplete silencing and off-target effects ( Jackson et al, 2003 ; Kaelin, 2012 ). In addition, the AID system provides a powerful research tool for studying highly dynamic biological processes, such as the cell cycle, stem cell differentiation and neural activity ( Nora et al, 2017 ; Ng et al, 2019 ; Atkins et al, 2020 ). Unlike other gene silencing strategies, AID-mediated degradation rapid depletion of proteins of interest (POI) within minute or hourly timescales can directly attribute the observed phenotypes to POI function rather than secondary effects arising from the actions of indirect targets ( Weiss et al, 2007 ; Wood et al, 2016 ).…”