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FANCD2 Alleviates Physiologic Replication Stress in Fetal Liver HSC

Makiko Mochizuki-Kashio, Young me Yoon, Theresa Menna, Markus Grompe, Peter Kurre

Preprint posted on October 01, 2020 https://www.biorxiv.org/content/10.1101/2020.09.30.320796v1?ct=

In proliferating hematopoietic stem cells, FA proteins keep replication stress at bay.

Selected by Ram

Background

Fanconi Anemia (FA) pathway constituting 22 genes supports cellular DNA damage response during replication stress. Biallelic germline mutations in FA pathway genes cause FA (the most frequent bone marrow failure syndrome), predisposes individuals to different cancers and incurs anomalies in the hematopoietic stem cell (HSC) compartment, all of which attributed to DNA damage accumulation and sensitivity to DNA damaging agents1. In adults, HSCs are maintained in quiescence for a future activation to match the demand of blood cell lineages. While many studies reinforce the importance of the FA pathway proteins to thwart exogenous sources of replication stress, it is not clear what physiological role FA pathway proteins play to maintain the integrity of the HSC pool. Therefore, the authors of the current preprint set out to investigate this.

Key findings

  1. The authors first demonstrate that HSCs derived from Fancd2-/- (a FA gene) show functional deficits in a later stage of mouse development (after E12.5) compared to wild-type. Furthermore, to resolve the precise cell cycle deficiencies often observed in Fancd2-/-, the authors exploit a sequential double stain of EdU followed by BrdU to segregate HSC pool that are in different cell cycle stages: G2/M (EdU+), S (EdU+/BrdU+), and entering S (BrdU+) (fig. a). Based on this data, they suggest that fetal liver HSCs derived from Fancd2-/- mice manifest delayed S-phase entry or progression, albeit they are not quiescent as well.
  2. As Fancd2-/- increases susceptibility to DNA damaging agents, the authors tested if lack of Fancd2 would inherently induce replication stress leading to the delayed S-phase entry they observed earlier. To this end, they detected higher levels of replication stress markers like phosphorylated Replication protein A (pRPA32 S4/S8), phosphorylated Checkpoint kinase 1 (pChk1 S345), and phosphorylated Minichromosome Maintenance complex (pMcm2 S53/S108) in fetal liver HSCs derived from Fancd2-/-.
  3. They further demonstrated exaggerated Cdkn1a (also called p21) nuclear localization in fetal liver HSCs derived from Fancd2-/- (fig. b), suggestive of p53-independent activation of cell cycle delay. Interestingly, chemical inhibition (SD208) of TGF-β (a key signaling molecule in HSC) was sufficient to reverse p21 nuclear localization, reinforcing a p53-independent activation of cell cycle delay.

    (a) Kinetic cell cycles studies adapting a sequential EdU/BrdU injection protocol with sequential EdU/BrdU injection at E13.5, (b) Immunofluorescence of Cdkn1a(p21) (WT n=4 pups, 84 cells, Fancd2-/-; n=4 pups, 120 cells), (c) Molecular mechanism of replication stress-mediated HSC integrity in Fancd2-/-. Taken directly from Muchizuki-Kashio M et. al., 2020 under a CC-BY 4.0 international license.

Conclusion

The authors report a physiologic role for Fancd2 to counter endogenous replication stress in proliferating HSCs in vivo, a mechanism challenged in FA individuals (fig. c). They further report that Fancd2 supports rapidly proliferating HSCs in the fetal liver to sustain lifelong HSC supply.

Acknowledgments: Thanks to all the authors, especially Makiko Mochizuki-Kashio and Peter Kurre for openly sharing unpublished data and replying promptly.

References:

  1. https://doi.org/10.1016/j.cub.2017.07.043
  2. https://dx.doi.org/10.1016%2Fj.stemcr.2016.09.005
  3. https://doi.org/10.1038/s41467-019-12271-w
  4. https://doi.org/10.1371/journal.pgen.1005674
  5. https://doi.org/10.1016/j.molcel.2015.09.012

Tags: dna damage, fanconi anemia, hematopoietic stem cells, replication stress

Posted on: 14th October 2020

doi: https://doi.org/10.1242/prelights.25076

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Author's response

Makiko Mochizuki-Kashio (MK) and Peter Kurre (PK) shared

1) The authors suggest that suboptimal levels of endogenous Fancd2 are sufficient to induce replication stress in HSCs. They clearly showed this using several protein markers and alkaline comet assay2. However, I was wondering if the authors have tried DNA fiber assay to visualize the DNA replication fork defects?

MK and PK: The DNA fiber assay is indeed a gold standard for replication stress and depending on the protocol used and outcomes measured can provide additional detail. Given previous high impact papers demonstrating experimentally induced replication stress in FANCD2 defective HSPC, we chose to focus our experiments on validated downstream outcomes instead. In future work, timing and specifics of physiologic fork stall in fetal Fancd2-/- HSC will be important to resolve

2) The lack of γH2AX levels in your current and earlier work2 could be explained by delayed S-phase entry or progression. But your data reporting lesser R-loops (RNA-DNA hybrids formed during transcription) in Fancd2-/- cells seem to contradict earlier studies3-5. Moreover, you also demonstrate that Fancd2-/- cells have higher γH2AX levels when induced by camptothecin (a drug that is known to cause R-loop mediated DNA damage). Would you consider evaluating R-loop levels in your samples using other methods (like dot blot)? How do the authors reconcile their data in light of this evidences?

MK and PK: Excellent point. The R-loop observation, i.e. the lack of difference between the genotypes puzzled us as well and colleagues we consulted. We performed additional experiments involving camptothecin (CPT) in KO and WT fetal liver cells (below). Results confirm the predicted assay performance (increase in WT HSC under CPT challenge) and validate the lack of R-loop increase in the KO genotype under these circumstances. I think there are a number of strategies that might be helpful to address this, but the dot blot would be difficult given limited cell HSC numbers. We felt that this discrepancy did not contradict the key observations in the data.

R-loop (S9.6) immunofluorescence quantification (shared by the authors).

3) The authors’ cell cycle analysis clearly demonstrates fetal liver HSCs are proliferative than bone marrow HSCs, as expected. I might have miss understood, but do the authors also suggest that bone marrow HSCs are also less quiescent in Fancd2-/-?

MK and PK: Others showed that Fancd2-/- BM HSC are less quiescent than WT BM HSC and in G1. However, I do think that more detail is needed in resolving cell cycle progression and checkpoint activation in adult FA HSC during homeostasis and under stress, where conventional assays of dye incorporation and Ki67 activity may not paint a full picture. For all the reasons that Dr. Connie Eaves’ elegant work points to the differences between fetal- and adult phenotype HSC one has to be careful to extrapolate our observations to BM HSC.

4) Last but not least, it would be interesting to hear the authors’ ideas to mitigate the challenge of alleviating the pathological defects in FA patients?

MK and PK: A validation in patients is critical, but any interventional strategies will have to be carefully considered. While TGF-b inhibition rescued the Cdkn1a (p21) nuclear localization in fetal FA HSPC, the concurrent increase in pChk1 would limit the benefit in patients. Rather, our model provides opportunities for compound screening and to test rescue strategies under physiological conditions. Those could then be selectively validated in cord blood and young patients where the HSC phenotype switch is thought to occur 1-2 years after birth.

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