p21/Zbtb18 repress the expression of cKit to regulate the self-renewal of hematopoietic stem cells

Abstract

The maintenance of hematopoietic stem cells (HSCs) is a complex process involving numerous cell-extrinsic and -intrinsic regulators. The first member of the cyclin-dependent kinase family of inhibitors to be identified, p21, has been reported to perform a wide range of critical biological functions, including cell cycle regulation, transcription, differentiation, and so on. Given the previous inconsistent results regarding the functions of p21 in HSCs in a p21-knockout mouse model, we employed p21-tdTomato (tdT) mice to further elucidate its role in HSCs during homeostasis. The results showed that p21-tdT⁺ HSCs exhibited increased self-renewal capacity compared to p21-tdT⁻ HSCs. Zbtb18, a transcriptional repressor, was upregulated in p21-tdT⁺ HSCs, and its knockdown significantly impaired the reconstitution capability of HSCs. Furthermore, p21 interacted with ZBTB18 to co-repress the expression of cKit in HSCs and thus regulated the self-renewal of HSCs. Our data provide novel insights into the physiological role and mechanisms of p21 in HSCs during homeostasis independent of its conventional role as a cell cycle inhibitor.

Keywords

hematopoietic stem cells / self-renewal / p21 / Zbtb18 / cKit

References

[1] Aoki K, Meng G, Suzuki K et al. RP58 associates with condensed chromatin and mediates a sequence-specific transcriptional repression. J Biol Chem 1998;273:26698–26704.

[2] Baubet V, Xiang C, Molczan A et al. Rp58 is essential for the growth and patterning of the cerebellum and for glutamatergic and GABAergic neuron development. Development 2012;139:1903–1909.

[3] Bernitz JM, Kim HS, MacArthur B et al. Hematopoietic stem cells count and remember self-renewal divisions. Cell 2016;167:1296–1309.e10.

[4] Bosbach B, Deshpande S, Rossi F et al. Imatinib resistance and microcytic erythrocytosis in a KitV558Delta;T669I/+gatekeeper-mutant mouse model of gastrointestinal stromal tumor. Proc Natl Acad Sci U S A 2012;109:E2276–E2283.

[5] Cheng T. Cell cycle inhibitors in normal and tumor stem cells. Oncogene 2004;23:7256–7266.

[6] Cheng T, Rodrigues N, Shen H et al. Hematopoietic stem cell quiescence maintained by p21cip1/waf1. Science 2000;287:1804–1808.

[7] Cheng H, Zheng Z, Cheng T. New paradigms on hematopoietic stem cell differentiation. Protein Cell 2020;11:34–44.

[8] Czechowicz A, Kraft D, Weissman IL et al. Efficient transplantation via antibody-based clearance of hematopoietic stem cell niches. Science 2007;318:1296–1299.

[9] Delavaine L, La Thangue NB. Control of E2F activity by p21Waf1/Cip1. Oncogene 1999;18:5381–5392.

[10] Deshpande S, Bosbach B, Yozgat Y et al. KIT receptor gain-of-function in hematopoiesis enhances stem cell self-renewal and promotes progenitor cell expansion. Stem Cells 2013;31:1683–1695.

[11] Devgan V, Mammucari C, Millar SE et al. p21WAF1/Cip1 is a negative transcriptional regulator of Wnt4 expression downstream of Notch1 activation. Genes Dev 2005;19:1485–1495.

[12] Ding L, Saunders TL, Enikolopov G et al. Endothelial and perivascular cells maintain haematopoietic stem cells. Nature 2012;481:457–462.

[13] Dutto I, Tillhon M, Cazzalini O et al. Biology of the cell cycle inhibitor p21(CDKN1A): molecular mechanisms and relevance in chemical toxicology. Arch Toxicol 2015;89:155–178.

[14] Foudi A, Hochedlinger K, Van Buren D et al. Analysis of histone 2B-GFP retention reveals slowly cycling hematopoietic stem cells. Nat Biotechnol 2009;27:84–90.

[15] Georgakilas AG, Martin OA, Bonner WM. p21: a two-faced genome guardian. Trends Mol Med 2017;23:310–319.

[16] Grinenko T, Arndt K, Portz M et al. Clonal expansion capacity defines two consecutive developmental stages of long-term hematopoietic stem cells. J Exp Med 2014;211:209–215.

[17] Harper JW, Adami GR, Wei N et al. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 1993;75:805–816.

[18] Hirai S, Miwa A, Ohtaka-Maruyama C et al. RP58 controls neuron and astrocyte differentiation by downregulating the expression of Id1-4 genes in the developing cortex. EMBO J 2012;31:1190–1202.

[19] Itkin T, Gur-Cohen S, Spencer JA et al. Distinct bone marrow blood vessels differentially regulate haematopoiesis. Nature 2016;532:323–328.

[20] Ito K, Suda T. Metabolic requirements for the maintenance of self-renewing stem cells. Nat Rev Mol Cell Biol 2014;15:243–256.

[21] Janzen V, Forkert R, Fleming HE et al. Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a. Nature 2006;443:421–426.

[22] Karimian A, Ahmadi Y, Yousefi B. Multiple functions of p21 in cell cycle, apoptosis and transcriptional regulation after DNA damage. DNA Repair (Amst) 2016;42:63–71.

[23] Kiel MJ, Yilmaz OH, Iwashita T et al. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 2005;121:1109–1121.

[24] Kim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory requirements. Nat Methods 2015;12:357–360.

[25] Kreis NN, Louwen F, Yuan J. The multifaceted p21 (Cip1/Waf1/CDKN1A) in cell differentiation, migration and cancer therapy. Cancers (Basel) 2019;11:1220.

[26] Krivtsov AV, Twomey D, Feng Z et al. Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9. Nature 2006;442:818–822.

[27] LaBaer J, Garrett MD, Stevenson LF et al. New functional activities for the p21 family of CDK inhibitors. Genes Dev 1997;11:847–862.

[28] Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods 2012;9:357–359.

[29] Lara-Astiaso D, Weiner A, Lorenzo-Vivas E et al. Immunogenetics. Chromatin state dynamics during blood formation. Science 2014;345:943–949.

[30] Li Y, Zhao H, Huang X et al. Embryonic senescent cells re-enter cell cycle and contribute to tissues after birth. Cell Res 2018;28:775–778.

[31] Mansilla SF, de la Vega MB, Calzetta NL et al. CDK-independent and PCNA-dependent functions of p21 in DNA replication. Genes (Basel) 2020;11:593.

[32] Matsumoto A, Takeishi S, Kanie T et al. p57 is required for quiescence and maintenance of adult hematopoietic stem cells. Cell Stem Cell 2011;9:262–271.

[33] Matsuoka Y, Sasaki Y, Nakatsuka R et al. Low level of c-kit expression marks deeply quiescent murine hematopoietic stem cells. Stem Cells 2011;29:1783–1791.

[34] McCulloch EA, Siminovitch L, Till JE. Spleen-colony formation in anemic mice of genotype WW. Science 1964;144:844–846.

[35] Morita Y, Ema H, Nakauchi H. Heterogeneity and hierarchy within the most primitive hematopoietic stem cell compartment. J Exp Med 2010;207:1173–1182.

[36] Morrison SJ, Scadden DT. The bone marrow niche for haematopoietic stem cells. Nature 2014;505:327–334.

[37] Nakamura-Ishizu A, Takizawa H, Suda T. The analysis, roles and regulation of quiescence in hematopoietic stem cells. Development 2014;141:4656–4666.

[38] Okado H, Ohtaka-Maruyama C, Sugitani Y et al. The transcriptional repressor RP58 is crucial for cell-division patterning and neuronal survival in the developing cortex. Dev Biol 2009;331:140–151.

[39] Pietras EM, Warr MR, Passegue E. Cell cycle regulation in hematopoietic stem cells. J Cell Biol 2011;195:709–720.

[40] Pinho S, Frenette PS. Haematopoietic stem cell activity and interactions with the niche. Nat Rev Mol Cell Biol 2019;20:303–320.

[41] Qiu J, Takagi Y, Harada J et al. Regenerative response in ischemic brain restricted by p21cip1/waf1. J Exp Med 2004;199:937–945.

[42] Qiu J, Papatsenko D, Niu X et al. Divisional history and hematopoietic stem cell function during homeostasis. Stem Cell Rep 2014;2:473–490.

[43] Sanjuan-Pla A, Macaulay IC, Jensen CT et al. Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy. Nature 2013;502:232–236.

[44] Shah M, Kumar H, Qiu S et al. Low c-Kit expression identifies primitive, therapy-resistant CML stem cells. JCI Insight 2023;8:e157421.

[45] Shin JY, Hu W, Naramura M et al. High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias. J Exp Med 2014;211:217–231.

[46] Stier S, Cheng T, Forkert R et al. Ex vivo targeting of p21Cip1/Waf1 permits relative expansion of human hematopoietic stem cells. Blood 2003;102:1260–1266.

[47] Trapnell C, Williams BA, Pertea G et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 2010;28:511–515.

[48] van Os R, Kamminga LM, Ausema A et al. A limited role for p21Cip1/Waf1 in maintaining normal hematopoietic stem cell functioning. Stem Cells 2007;25:836–843.

[49] Wang Y, Fisher JC, Mathew R et al. Intrinsic disorder mediates the diverse regulatory functions of the Cdk inhibitor p21. Nat Chem Biol 2011;7:214–221.

[50] Wang Y, Lu T, Sun G et al. Targeting of apoptosis gene loci by reprogramming factors leads to selective eradication of leukemia cells. Nat Commun 2019;10:5594.

[51] Wang R, Bhatt AB, Minden-Birkenmaier BA et al. ZBTB18 restricts chromatin accessibility and prevents transcriptional adaptations that drive metastasis. Sci Adv 2023;9:eabq3951.

[52] Waskow C, Madan V, Bartels S et al. Hematopoietic stem cell transplantation without irradiation. Nat Methods 2009;6:267–269.

[53] Wilson A, Laurenti E, Oser G et al. Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair. Cell 2008;135:1118–1129.

[54] Wright PE, Dyson HJ. Intrinsically disordered proteins in cellular signalling and regulation. Nat Rev Mol Cell Biol 2015;16:18–29.

[55] Xiang C, Baubet V, Pal S et al. RP58/ZNF238 directly modulates proneurogenic gene levels and is required for neuronal differentiation and brain expansion. Cell Death Differ 2012;19:692–702.

[56] Yamazaki S, Iwama A, Takayanagi S et al. Cytokine signals modulated via lipid rafts mimic niche signals and induce hibernation in hematopoietic stem cells. EMBO J 2006;25:3515–3523.

[57] Yamazaki S, Ema H, Karlsson G et al. Nonmyelinating Schwann cells maintain hematopoietic stem cell hibernation in the bone marrow niche. Cell 2011;147:1146–1158.

[58] Yang S, Liu L, Cao C et al. USP52 acts as a deubiquitinase and promotes histone chaperone ASF1A stabilization. Nat Commun 2018;9:1285.

[59] Ye M, Zhang H, Amabile G et al. C/EBPa controls acquisition and maintenance of adult haematopoietic stem cell quiescence. Nat Cell Biol 2013;15:385–394.

[60] Yoshihara H, Arai F, Hosokawa K et al. Thrombopoietin/MPL signaling regulates hematopoietic stem cell quiescence and interaction with the osteoblastic niche. Cell Stem Cell 2007;1:685–697.

[61] Zhang Y, Liu T, Meyer CA et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol 2008;9:R137.

[62] Zhao M, Tao F, Venkatraman A et al. N-Cadherin-expressing bone and marrow stromal progenitor cells maintain reserve hematopoietic stem cells. Cell Rep 2019;26:652–669.e6.

[63] Zou P, Yoshihara H, Hosokawa K et al. p57(Kip2) and p27(Kip1) cooperate to maintain hematopoietic stem cell quiescence through interactions with Hsc70. Cell Stem Cell 2011;9:247–261.

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