SSB1/NABP2 and SSB2/NABP1 Have Essential and Overlapping Roles in Maintaining Hematopoietic Stem and Progenitor Cells
Authors: Shi Wei , Vu Therese , Boyle Glen , Al-Ejeh Fares , Pandita Tej , Ginalski Krzysztof , Rowicka Maga , Lane Steven W. , Khanna Kum Kum 
Published: October 2019

Single-stranded DNA binding (SSB) proteins are essential for a variety of DNA metabolic processes and the maintenance of genomic stability. SSB1 and its homolog SSB2, share greater sequence and domain homology to the archaeal and bacterial SSBs than eukaryotic RPA. They form complexes with two other proteins, C9Orf80 and INTS3, and play roles in mediating transcription and DNA repair. SSB1 (also known as OBFC2B or NABP2) is recurrently mutated in various cancers, however the precise function in normal development is incompletely understood. We have previously shown that Ssb1 is required for skeletogenesis, telomeric homeostasis and genomic stability in vivo while Ssb2 knockout mice are viable and grow normally without any detectable phenotype. Interestingly, we observed pronounced upregulation of Ssb2 in response to Ssb1 deletion and modest up-regulation of Ssb1 in response to Ssb2 deletion, suggesting that Ssb1 and Ssb2 may have some overlapping functions.

To investigate the specific roles of both Ssb1 and Ssb2 in adult tissue homeostasis, we generated conditional double-knockout (DKO) mouse models of both genes. DKO in adult mice was achieved by using a tamoxifen-inducible Cre (Ssb1fl/fl Ssb2fl/fl R26-CreERT2), in which Ssb1 and Ssb2 are conditionally deleted by the administration of tamoxifen. Induced DKO mice become moribund within seven days featured with pancytopenia and dramatic loss of hematopoietic stem and progenitor cells (HSPCs), suggesting that Ssb1 and Ssb2 are required for the maintenance of haematopoietic stem and progenitors cells (HSPCs).

DKO bone marrow was markedly hypocellular with reduction in all lineages of haematopoietic development. Functionally, HSPCs in DKO mice show decreased quiescence at the early stage followed by decreased proliferation and increased cell loss due to apoptotic cell death at the later stage, suggesting the imbalanced bone marrow homeostasis upon DKO may eventually result in exhaustion of the stem cell pool in DKO mice. Furthermore, bona fide HSPC intrinsic functional deficiency caused by DKO was confirmed by competitive bone marrow transplant, where DKO bone marrows showed abolished differentiation capacity and failed to repopulate the bone marrows of recipient mice after induction of DKO in the established engraftments from the Ssb1fl/fl Ssb2fl/fl R26-CreERT2 donors. Gene expression of DKO HSPCs demonstrated an exacerbated p53/p21 DNA damage response and pronounced interferon response. Validating these findings, stabilization of p53 and increased apoptotic cell death were observed in DKO bone marrows and HSPCs and induction of cell cycle and expression of interferon target genes was confirmed by QPCR. DKO HSPCs have increased expression of IFN induced surface markers such as Sca1. The IFN response was intrinsic to HSPCs.

Mechanistically, DKO HSPCs manifest a profile of stalled replication forks on DNA combing analysis, unrepaired double strand breaks (increased gammaH2Ax foci and alkaline comet tail moment) and telomeric loss resulting in widespread chromosomal instability. DKO HSPC showed aberrant cytoplasmic accumulation of single stranded DNAs, with R-loop formation (DNA:RNA hybrid), driving this genetic instability and cell-intrinsic interferon response.

Altogether, these data provide strong evidence that Ssb1 and Ssb2 have essential functions in regulating haematopoiesis through repairing replication associated DNA damage as well as resolution of R-loop generated during transcription, to maintain genomic stability during normal HSPC homeostasis.

Disclosures

No relevant conflicts of interest to declare.