Ageing-associated impairments in haemato-lymphopoiesis are associated with DNA damage accumulation and reduced maintenance of lymphoid-biased (Ly-biased) compared with myeloid-biased (My-biased) haematopoietic stem cells (HSCs). Here, in vivo RNAi screening identifies period circadian clock 2 (Per2) as a critical factor limiting the maintenance of HSCs in response to DNA damage and ageing. Under these conditions, Per2 is activated predominantly in Ly-biased HSCs and stimulates DNA damage signalling and p53-dependent apoptosis in haematopoietic cells. Per2 deletion ameliorates replication stress and DNA damage responses in haematopoietic cells, thereby improving the maintenance of Ly-biased HSCs, lymphopoiesis, and immune function in ageing mice without increasing the accumulation of DNA damage. Per2-deficient mice retain Batf/p53-dependent induction of differentiation of HSCs in response to DNA damage and exhibit an elongated lifespan. Together, these results identify Per2 as a negative regulator of Ly-biased HSCs and immune functions in response to DNA damage and ageing.
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This work was supported by the European Union (advanced ERC grant to K.L.R., grant 323136—StemCellGerontoGenes), within the e:Med program (HaematoOpt) of the German Federal Ministry of Education and Research (BMBF), the German Research Foundation (DFG—RU745-10), the Baden-Württemberg-Stiftung (P1301029), the Leibniz association, and the State of Thuringia (FZ-12001-514). We thank Z.-Q. Wang from the Leibniz Institute on Aging for supplying ATM-deficient mice.
The authors declare no competing financial interests.
Integrated supplementary information
(A) The representative Western blot shows the efficiency of knockdown (by shRNAs) and over-expression (O/E) of Per2. Cell lysates obtained from lineage bone marrow cells that were infected by lentiviral vectors carrying two different shRNA (shRNA1 or shRNA2) targeting Per2 or Per2-cDNA or an empty control vector were loaded to detect PER2 protein expression. Data represent 1 out 3 independent experiments. (B) The histogram shows the mRNA level of Pcdh20 in lineage bone marrow cells of WT mice infected by lentiviruses carrying two different shRNAs against Pcdh20 or a scramble shRNA control vector. N = 3 repeat experiments, values are means ± s.e.m. (C) The histogram depicts the number of shRNAs detected by deep sequencing in the shRNA plasmid pool as well as in Lin cells derived from mTerc+/+ and G3 mTerc−/− donors after two round of transplantation as depicted in Fig. 1a. (D,E) Freshly isolated KSL cells from G3 mTerc−/− mice or mTerc+/+ mice were infected with 2 independent single shRNA constructs targeting (D) Per2 or (E) Pcdh20, or a scrambled shRNA control. Infected cells were transplanted into lethally irradiated mice along with non-infected cells from the same culture. The graphs show the changes in the percentage of infected cells (GFP+) in peripheral blood of primary recipients at indicated time points after transplantation. N = 5 recipients/group, values are means ± s.e.m., multiple t test was used to calculate P values.
Supplementary Figure 2 Per2 deletion improves self-renewal of HSCs in response to replicative stress.
(A–C) Cohorts of 3-month-old Per2−/− mice and Per2+/+ mice were sub-lethally irradiated (4 Gy) to induce premature aging and kept for 1 year. At this time point mice were sacrificed to analyse hematopoietic cells in bone marrow (8 Per2+/+ mice and 7 Per2−/− mice). (A,B) The histograms show the absolute number of (A) Ly-biased HSCs (marked by CD150lo CD34 KSL) and (B) My-biased HSCs (marked by CD150hi CD34 KSL), values are shown as mean ± s.e.m., multiple t test was used to calculate P values. (C) Representative FACS plots showing an increase in Ly-biased HSCs (CD150lo, CD34) in bone marrow of Per2−/− mice versus Per2+/+ mice. FACS was repeated on biological replicates (8 times for Per2+/+ mice and 7 times for Per2−/− mice).
Supplementary Figure 3 Per2 contributes to the activation of apoptosis signalling in hematopoietic cells in response to DNA damage but not to DNA damage sensing.
(A) Immunofluorescence staining of γH2AX in freshly isolated HSCs (CD34 LSK) from irradiated (4 Gy, 6 h after IR) and non-irradiated (nIR), 2–3-month-old Per2+/+ mice or Per2−/− mice. Representative data derived from 200 analysed cells for 1 out of 5 mice per genotype are shown. Chi-square test was used to generate P values. Note, there is no impact of Per2 gene status on the induction of γH2AX foci. (B) 8000 freshly isolated KSL cells from 2-month-old Per2+/+ and Per2−/− mice were cultured for 12 h followed by sub-lethal irradiation (4 Gy). The numbers of living cells were counted 24 h and 36 h after IR by automatic cell counter. This histogram depicts the number of living hematopoietic stem and progenitor cells (KSL) at indicated time points after sub-lethal irradiation (4 Gy). N = 5 repeat experiments per genotype, values are shown as mean ± s.e.m. and multiple t test was used to calculate P value.
(A–C) 3-month-old Per2−/− and Per2+/+ mice were exposed to 4 Gy γ-irradiation (IR) or non-irradiated (nIR) and were analysed one year later (same as in Supplementary Fig. 2). (8 Per2+/+ mice and 7 Per2−/− mice per group), values are shown as mean ± s.e.m. Multiple t test was used to calculate P value. (A) This histogram shows the percentage of B220+ and CD11b+ cells in peripheral blood. (B) This histogram shows the percentage of B220+ and CD11b+ cells in bone marrow. (C) Representative FACS plots showing the B220+ and CD11b+ cells in bone marrow of mice of the indicated genotype and treatment.
Supplementary Figure 5 Per2 deletion does not change B-lymphocyte production of stimulated B cell progenitors from young mice.
(A) 1000 freshly purified EBPs from 2-month-old Per2−/− and Per2+/+ mice were cultured and exposed to IL-7 at the indicated concentrations. FACS analysis determined the total numbers of B220+CD19+ cells after 4-day culture. N = 5 mice per group, dots represent individual mice, lines depict mean values. In contrast to the results on EBPs from old mice (Fig. 5b), Per2 gene status does not affect B-lymphocyte production rates of stimulated B-cell progenitors from young mice.
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Wang, J., Morita, Y., Han, B. et al. Per2 induction limits lymphoid-biased haematopoietic stem cells and lymphopoiesis in the context of DNA damage and ageing. Nat Cell Biol 18, 480–490 (2016) doi:10.1038/ncb3342
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