ABSTRACT 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

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SIMILAR CONTENT BEING VIEWED BY OTHERS AGE-ASSOCIATED IMBALANCE IN IMMUNE CELL REGENERATION VARIES ACROSS INDIVIDUALS AND ARISES FROM A DISTINCT SUBSET OF STEM CELLS Article Open access 24

October 2024 TAZ PROTECTS HEMATOPOIETIC STEM CELLS FROM AN AGING-DEPENDENT DECREASE IN PU.1 ACTIVITY Article Open access 03 September 2022 LOSS OF SIRT1 INHIBITS HEMATOPOIETIC STEM CELL

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Protoc._ 1, 2979–2987 (2007). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS 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. AUTHOR INFORMATION Author notes * Jianwei Wang Present address: Present address: Institute for Stem Cell Biology and Regenerative Medicine, Tsinghua University, Beijing 100084,

China., AUTHORS AND AFFILIATIONS * Research Group on Stem Cell Aging, Leibniz Institute on Aging – Fritz Lipmann Institute (FLI), Beutenbergstr. 11, 07745 Jena, Germany Jianwei Wang, Yohei

Morita, Bing Han & K. Lenhard Rudolph * Institute for Medical Microbiology, University Hospital Münster, Domagkstraße 10, 48149 Münster, Germany Silke Niemann * Institute of Medical

Microbiology, Jena University Hospital (UKJ), 07747 Jena, Germany Bettina Löffler * Research Group on Molecular Aging, Faculty of Medicine, Friedrich-Schiller-University, 07745 Jena, Germany

K. Lenhard Rudolph Authors * Jianwei Wang View author publications You can also search for this author inPubMed Google Scholar * Yohei Morita View author publications You can also search

for this author inPubMed Google Scholar * Bing Han View author publications You can also search for this author inPubMed Google Scholar * Silke Niemann View author publications You can also

search for this author inPubMed Google Scholar * Bettina Löffler View author publications You can also search for this author inPubMed Google Scholar * K. Lenhard Rudolph View author

publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS J.W. performed most of the experiments, Y.M. performed FACS analysis and wrote the manuscript; B.H.

performed ELISA measurements, B.L. and S.N. performed the food pad assay and K.L.R. designed and supervised the project, and wrote the manuscript. CORRESPONDING AUTHOR Correspondence to K.

Lenhard Rudolph. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. INTEGRATED SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURE 1 VERIFICATION OF

SHRNAS OF CANDIDATE GENES. (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. SUPPLEMENTARY FIGURE 4 _PER2_ DELETION RESCUES MYELOID/LYMPHOID SKEWING IN THE CONTEXT

OF DNA DAMAGE. (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. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Information (PDF

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ARTICLE CITE THIS ARTICLE 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). https://doi.org/10.1038/ncb3342 Download citation * Received: 11 March 2015 * Accepted: 14 March 2016 * Published: 18 April 2016 * Issue Date: May 2016 *

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