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Ssb1 and Ssb2 cooperate to regulate mouse hematopoietic stem and progenitor cells by resolving replicative stress

Wei Shi, Therese Vu, Didier Boucher, Anna Biernacka, Jules Nde, Raj K. Pandita, Jasmin Straube, Glen M. Boyle, Fares Al-Ejeh, Purba Nag, Jessie Jeffery, Janelle L. Harris, Amanda L. Bain, Marta Grzelak, Magdalena Skrzypczak, Abhishek Mitra, Norbert Dojer, Nicola Crosetto, Nicole Cloonan, Olivier J. Becherel, John Finnie, Jeffrey R. Skaar, Carl R. Walkley, Tej K. Pandita, Maga Rowicka, Krzysztof Ginalski, Steven W. Lane and Kum Kum Khanna

Key points

  • Combined loss of Ssb1/Ssb2 induces rapid lethality due to replication stress associated loss of haematopoietic stem and progenitor cells.

  • Functionally, loss of Ssb1/Ssb2 activates p53 and IFN pathways causing enforced cell cycling in quiescent HSPCs and apoptotic cell loss.

Abstract

Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The two single-stranded DNA binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however their overlapping roles during normal physiology are incompletely understood. We generated mice where both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, while conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2. Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double strand breaks (DSBs) enriched at Ssb binding regions and CpG islands, together with the accumulation of R-loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon pathways which enforced cell cycling in quiescent HSPCs resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in-vitro cultured cDKO-HSCs, which was significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPCs function including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability.

  • Submitted June 30, 2016.
  • Accepted February 26, 2017.