Stem/Progenitor Cell Pool Essential for Maintaining the Hematopoietic

Histone Acetyltransferase Cofactor Trrap Is Essential for Maintaining the Hematopoietic Stem/Progenitor Cell Pool

This information is current as Joanna I. Loizou, Gabriela Oser, Vivek Shukla, Carla Sawan, of September 26, 2021. Rabih Murr, Zhao-Qi Wang, Andreas Trumpp and Zdenko Herceg J Immunol 2009; 183:6422-6431; Prepublished online 30 October 2009;

doi: 10.4049/jimmunol.0901969 Downloaded from http://www.jimmunol.org/content/183/10/6422

Supplementary http://www.jimmunol.org/content/suppl/2009/10/30/jimmunol.090196 Material 9.DC1 http://www.jimmunol.org/ References This article cites 39 articles, 11 of which you can access for free at: http://www.jimmunol.org/content/183/10/6422.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Histone Acetyltransferase Cofactor Trrap Is Essential for Maintaining the Hematopoietic Stem/Progenitor Cell Pool1

Joanna I. Loizou,*† Gabriela Oser,‡ Vivek Shukla,*§ Carla Sawan,* Rabih Murr,*¶ Zhao-Qi Wang,ʈ Andreas Trumpp,# and Zdenko Herceg2*

The pool of hematopoietic stem/progenitor cells, which provide life-long reconstitution of all hematopoietic lineages, is tightly controlled and regulated by self-renewal and apoptosis. Histone modifiers and chromatin states are believed to govern establish- ment, maintenance, and propagation of distinct patterns of gene expression in stem cells, however the underlying mechanism remains poorly understood. In this study, we identified a role for the histone acetytransferase cofactor Trrap in the maintenance of hematopietic stem/progenitor cells. Conditional deletion of the Trrap gene in mice resulted in ablation of bone marrow and increased lethality. This was due to the depletion of early hematopoietic progenitors, including hematopoietic stem cells, via a cell-autonomous mechanism. Analysis of purified bone marrow progenitors revealed that these defects are associated with in- Downloaded from duction of p53-independent apoptosis and deregulation of Myc transcription factors. Together, this study has identified a critical role for Trrap in the mechanism that maintains hematopoietic stem cells and hematopoietic system, and underscores the importance of Trrap and histone modifications in tissue homeostasis. The Journal of Immunology, 2009, 183: 6422–6431.

tem cells constitute a minority of cell populations in play an essential role in establishing and propagating distinct

adult tissues, yet they play a key role in development, patterns of gene expression required for maintenance of stem http://www.jimmunol.org/ S tissue homeostasis, and recovery from injury, and their cell pools (3–6). The importance of chromatin states in the deregulation may lead to cancer. Probably, the most well-char- maintenance of stem/progenitor cell pluripotency is further il- acterized adult stem cells are hematopoietic stem cells (HSCs),3 lustrated by the fact that virtually all cell types can be converted which give rise to all cells of hematopoietic lineages and have into more immature cell stages with wider differentiation po- the capacity to provide life-long reconstitution of these lineages tentials (7). Although the unique characteristics of HSCs are (1, 2). A pool of hematopoietic stem/progenitor cells that can likely to be a result of chromatin modifications, the mechanisms undergo several developmental fates including self-renewal and and molecular players involved in the maintenance and differ- differentiation, and the balance between different developmen- entiation fates of hematopoietic stem/progenitor cells remain by guest on September 26, 2021 tal fates is believed to be tightly controlled and regulated by largely unknown. proliferation and apoptosis (2). Chromatin marking occurs at the level of histones that are the A fundamental question for understanding stem cell biology targets for several posttranslational covalent modifications in- is how the pool of stem/progenitor cells are maintained and cluding acetylation and methylation (8). Acetylation of specific renewed. Recent studies indicated that histone modifications lysine residues within the amino-terminal tails of core histones is mediated by several transcription regulatory cofactors that have histone acetyltransferase (HAT) activity (8–11). The HAT † *International Agency for Research on Cancer (IARC), Lyon, France; Mammalian cofactor TRRAP (TRansformation/tRanscription domain-Asso- Genetics Laboratory, Cancer Research U.K., London Research Institute, Lincoln’s Inn Fields Laboratories, London, U.K.; ‡Department of Biomedicine, University Hos- ciated Protein) and its yeast homologue Tra1 are common com- pital Basel, Basel, Switzerland; §Mammalian Department of Biochemistry, Beadle ponents of many HAT complexes in yeast and mammalian cells. Center, Lincoln, NE 68588; ¶Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland; ʈLeibniz Institut for Age Research Fritz Lipmann Institut, Jena, TRRAP has been identified as a subunit common to p300/CBP- Germany; and #Deutsches Krebsforschungszentrum (DKFZ) Im Neuenheimer Feld associated factor complex (PCAF; mammalian equivalent of 280, Heidelberg, Germany yeast Spt-Ada-Gcn5-acetyl-transferase, SAGA), HIV Tat-inter- Received for publication June 22, 2009. Accepted for publication September acting 60 KDa protein complex (Tip60; mammalian equivalent 15, 2009. of yeast nucleosomal acetyl-transferase of histone H4, NuA4), The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance TATA-binding protein-free TAFII-containing complex, novel with 18 U.S.C. Section 1734 solely to indicate this fact. SAGA-related complex and SAGA-like complexes (8, 11). 1 J.I.L., G.O., V.S., C.S., and R.M. performed all the experiments. Z.H. and J.I.L. TRRAP was found in association with two major HAT families: coordinated the project and wrote the manuscript. Z.Q.W. and A.T. gave conceptual the general control nonderepressible-related (GCN5) acetyl- assistance and contributed with planning the experiments. All authors discussed the results and manuscript text. transferase HAT family and the MOZ, Ybf2/Sas3, Sas2, Tip60- 2 Address correspondence and reprint requests to Dr. Zdenko Herceg, Epigenetics related HAT family. These include SAGA/GCN5 (general con- Group, International Agency for Research on Cancer (IARC), 150 Cours Albert- trol nonderepressible), PCAF, Tip60, NuA4, TAFII-containing Thomas, Lyon, France. E-mail address: [email protected] complex (TATAbinding protein-free TAF(II) complex), STAGA 3 Abbreviations used in this paper: HSC, hematopoietic stem cell; HAT, histone (SPT3-TAFII 31-GCN5L acetylase), SAGA-like, and SALSA acetyltransferase; TRRAP, TRansformation/tRanscription domain-Associated Pro- tein; PCAF, p300/CBP-associated factor complex; pIpC, poly(inosinic)-poly(cyti- (SAGA altered, Spt8 absent). Both SAGA/GCN5/PCAF (a complex dylic) acid; WT, wild type; BM, bone marrow; CKO, conditional knockout; FISH, that preferentially acetylates histone H3) and NuA4/Tip60 (a complex fluorescence in situ hybridization. that targets histone H4) contact several transcription activators Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 through TRRAP/Tra1, the interactions that were shown to mediate www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901969 The Journal of Immunology 6423

chromatin-based processes including transcription and DNA repair (8, Western blot analysis 12, 13). Several transcription factors have been shown to interact with Total protein lysates were prepared from lin-BM cells, resolved by elec- Trrap/HAT complexes and are potential regulators or mediators of trophoresis on 10% SDS-PAGE gel, and blotted on nitrocellulose mem- Trrap/HAT chromatin modifying activities (14–19). Among these, brane (Bio-Rad). The following Abs were used: rabbit anti-c-Myc (1/500; the c-Myc oncoprotein has been shown to directly interact with Trrap/ Santa Cruz Biotechnology), rabbit anti-N-myc (1/200; Santa Cruz Biotech- HAT complexes and this interaction is required for its transcriptional nology), and mouse anti-actin (1/10000; Santa Cruz Biotechnology). Pro- teins were visualized with HRP-conjugated anti-rabbit IgGs (1/5000; Dako- and transforming activities (17, 20, 21). c-Myc and N-Myc regulate a Cytomation) and anti-mouse IgGs (1/2000; DakoCytomation), followed by variety of genes involved in the control of cell proliferation and cell use of the ECL chemiluminiscence system (Amersham). death (22) and their disruption lead to compromised maintenance of Blood analysis HSCs and hematopoietic function (23, 24), although the precise molecular mechanism remains to be elucidated. Furthermore, a recent For analysis of peripheral blood elements, ϳ200 ␮l of blood was collected ϳ ␮ large-scale RNAi screen in mouse embryonic stem cells revealed that retro-orbitally into a glass Pasteur pipette containing 30 l heparin. Anal- ysis was performed by a commercial company. Tip60-p400 HAT is necessary for the maintenance of embryonic stem cell identity (25), although the role of HATs and histone acetylation Histological analysis of BM and liver in establishing and maintaining distinct chromatin signature required Femur and liver samples were fixed in 4% buffered formaldehyde over- for unique stem cell properties remains largely unknown (26). night, and then in 70% ethanol overnight before embedding in paraffin. In the present study, we generated conditional knockout Femurs were additionally decalcified before performing histological anal- ␮ (CKO) mice that allow for inactivation of the Trrap gene in a yses on 3 m sections stained with H&E.

spatialtemporal manner in vivo after expression of Cre-recom- Assessment of BM, spleen, and thymus cellularity Downloaded from binase, and studied the function Trrap/HAT and histone acety- BM cells were flushed from the long bones of hindlegs and prepared by lation in vivo in adult mice and in adult tissue stem cells. This standard procedures. Cell suspensions of BM, spleen and thymus were study has revealed an essential function of Trrap in the main- filtered through a nylon mesh filter (40 ␮m), stained with trypan blue, and tenance of hematopoietic stem/progenitor cells and homeostasis counted under a light microscope. of the hematopoietic system. CFU assay http://www.jimmunol.org/ Materials and Methods BM was cultured in M3434 Methocult (Stem Cell Technologies) containing insulin, transferrin, SCF, IL-3, IL-6, and erythropoietin for 7 to 10 days Generation of Trrap conditional knockout mice at 37°C in an atmosphere of 5% CO2 in air. Resulting colonies were scored Embryonic stem cells carrying the Trrap “conditional” (“floxed”; according to morphology as directed by the manufacturer using an inverted Trrapwild-type/floxed, Trrapϩ/f) allele, generated using the Cre-loxP system light microscope. (27), were injected into blastocysts for the production of chimeric and Trrap Ϫ ϩ ϩ ϩ ⌬ Purification of lin BM cells “floxed” (Trrap /f) mice. Trrap /f mice were intercrossed with Trrap / mice ⌬ (27) to obtain Trrapf/ and Trrapf/f mice. To inactivate the Trrap “conditional” To purify lineage cells, the BM from several mice was pooled and linϪ ⌬ allele in tissues and organs in an inducible manner, Trrapf/ and Trrapf/f mice cells for CD3␧, CD11b, CD45/B220, Ly-6G and Ly-6C (Gr-1), and were crossed to transgenic mice expressing Cre recombinase under the control Ter119/erythroid cells (Ly-76) were recovered using a biotin-conjugated by guest on September 26, 2021 of the Mx-1 promoter, a poly(inosinic)-poly(cytidylic) acid (pIpC)-inducible mouse lineage panel of Abs (BD Biosciences), as recommended by the ⌬ ϩ ϩ promoter (28), to produce Trrapf/ Cre and Trrapf/fCre mice. To delete manufacturer. Dynabeads (Invitrogen) were then used to exclude biotin Trrap in vivo, mice harboring the Trrap “floxed” allele and Cre transgene bound cells as directed by the manufacturer. The enrichment of linϪ cells acquired an inactivating mutation of the gene upon treatment with three intra- was confirmed by FACS using a c-kit Ab labeled with FITC. peritoneal injections of 250 ␮g pIpC (InvivoGen), each 2 days apart, unless otherwise stated. To generate Trrap conditional knockout mice in a p53 null Analysis of apoptosis, cell cycle profile, and proliferation Ϫ/Ϫ f/⌬ ϩ background Trp53 mice and Trrap Cre mice were interbred to generate Ϫ ⌬ ϩ ϩ Ϫ ⌬ For cell cycle profiling lin BM cells were stained with propidium iodide Trrapf/ Cre Ϫ Trp53 / mice which were intercrossed to generate Trrapf/ ϩ ϩ ϩ ⌬ ϩ Ϫ Ϫ using the CycleTEST PLUS kit according to the recommendations of the Cre Ϫ Trp53 / or Trrapf/ Cre Ϫ Trp53 / mice. To maintain this colony manufacturer (BD Biosciences). To detect apoptotic BM cells we stained mice heterozygous for Trp53 were crossed. All animal experiments were ap- Ϫ lin cells with Annexin-V-Fluos that detects phosphatidylserine on cell proved by and performed in accordance with the guidelines of the International membranes as recommended by the manufacturer (Roche). All FACS anal- Agency for Research on Cancer’s Animal Care and Use Committee. yses were performed using a FACScalibur apparatus equipped with Genotyping of tissue and mice by PCR and Southern blot CellQuest software (BD Biosciences). The Trrap, Trp53, and Mx1-Cre genotypes of mice were determined by DNA fluorescence in situ hybridization (FISH) performing PCR on DNA extracted from tail tissue. For Trp53, the primers Ϫ Lin cells were spun using a cytospin and slides were fixed and hybridized used for PCR were Neo19, X6.5, and X7.0. For Trrap, the primers used with a 5Ј-biotinylated DNA probe (Invitrogen) recognizing the mouse ma- were I3.0 and I3.1 to detect the floxed and wild-type (WT) alleles and G7.0 jor satellite repeats as described previously (5). and I2.1 to detect WT/floxed and ␦ alleles. For detection of the Mx1-Cre transgene the primers B2.1, B2.2, Cre1, and Cre2 were used. Primer se- Abs and FACS analysis of hematopoietic compartments quences are listed in supplementary Table I.4 To detect the Trrap floxed, WT, and ␦ alleles by Southern blotting, DNA was digested overnight with Abs against Gr-1, Ter 119, B220, CD11b, CD4, CD8␣, CD11a, and CD18 KpnI, resolved on 1% agarose gel, transferred to nitrocellulose, and probed were purified and conjugated in the laboratory following standard proto- with a 32P labeled probe as described previously (27). cols. Abs to CD34, CD135, CD117, ScaI, CD43, CD41, IgM, CD19, CD71, CD93, CD49d, and CD29 were purchased from eBioscience. Pro- RT-PCR tocols used for FACS analysis of hematopoietic cells were described previously (23). Total RNA from bone marrow (BM), spleen, Lin- cells, thymus, and liver was extracted using the RNeasy mini kit (Qiagen). Between 70 ng and 300 BM transplantation ng, total RNA was used for reverse transcription using 200 U of M-MLV Reverse Transcriptase (Invitrogen). Two microliters of the reverse tran- In the case, where mice carrying at least one wild-type allele were used as scriptase reaction was subsequently subjected to PCR amplification. Primer the recipients, mice were lethally irradiated with 10 Gy of whole-body 6 sequences are shown in supplementary Table I. PCR products were re- irradiation and then reconstituted with 3 ϫ 10 BM cells by retro-orbital solved on 2% agarose gels. Hprt or Gapdh mRNA abundance was used as injection. Trrap-CKO BM cells were extracted from the femurs of mice 6 control. days post deletion. In the case where mice amenable to deletion of Trrap were used as the recipients, ϳ4-wk-old mice were transplanted with 3 ϫ 106 BM cells by retro-orbital injection and after 2 wk administered with 4 The online version of this article contains supplementary material. three i.p. injections of 250 ␮g pIpC, each 2 days apart. 6424 TRRAP MAINTAINS HEMATOPOIETIC STEM CELLS

FIGURE 1. Trrap is required for viability of adult mice. A, Trrapf/f and Trrapf/⌬ mice were crossed with transgenic mice expressing Cre recombinase under the control of the Mx 1 promoter. To delete Trrap in vivo expression of Cre was induced with three intraperitoneal (IP) injections of 250 ␮g polyI-polyC (pIpC), each two days apart. B, Cre mediated deletion of Trrap was monitored by Southern blotting on DNA extracted from tail (T), thymus (Th) and liver (L) samples taken from Trrapf/f MxϪCreϩ mice treated either with PBS (Co) or pIpC (Trrap-CKO). As an additional control Trrapf/f MxϪCreϪ mice were treated with pIpC, along side Trrapf/f MxϪCreϩ litter mates. DNA was extracted from the spleen and thymus and subjected to Southern blotting to detect Trrap ␦ allele. C, Depletion of Trrap mRNA in spleen (S), liver (L) and bone marrow (BM) was monitored by performing RT-PCR from control mice treated with pIpC and Trrap-CKO mice treated with pIpC. The hprt levels were used as an internal load- Downloaded from ing control. D, Within 5 wk of Trrap deletion only ϳ30% of Trrap conditional knock-out (CKO) maintain viability in comparison to control (Co) mice that maintain 100% viability. E, Trrap-CKO mice appear moribund in comparison to litter-mate control mice. http://www.jimmunol.org/

Results gene could be induced by treatment of Trrapf/⌬Creϩ and Trrapf/f ϩ Generation of mice with a floxed allele of Trrap and Cre mice with three injections of pIpC at 2-day intervals. We inactivation of Trrap in adult mice found that 2 days after the third injection of pIpC, the deletion of Trrap was highly efficient in BM and liver (nearly 100%), slightly We have previously shown that homozygous mutation of Trrap in less efficient in spleen (80%), whereas deletion in thymus was mice induced embryonic lethality (27). To circumvent problems of

significantly less efficient (25% deletion) (Fig. 1B, and data not by guest on September 26, 2021 embryonic lethality and to study the function of Trrap and Trrap/ shown). The similar efficiency of deletion of Trrap ‘floxed’ allele HAT-mediated histone acetylation in vivo in adult mice, we have was observed from homozygous (Trrapf/fCreϩ) and hemizygous generated Trrap conditional knockout mice that allow inducible (Trrapf/⌬Creϩ) mice (data not shown). In agreement with these deletion of the gene in a spatiotemporal manner. Mouse embryonic findings, the Trrap mRNA was 90% less abundant in BM and liver stem cells carrying the Trrap conditional (floxed; Trrapwild-type/floxed, ϩ of Trrap conditional knockout mice 2 days after the 3rd injection Trrap /f) allele generated using the Cre-loxP system (27) were of pIpC (Fig. 1C). These data indicate that pIpC-mediated condi- injected into blastocysts for the production of chimeric and Trrap ϩ/f ϩ/f tional deletion of the Trrap floxed allele effectively inactivated the floxed (Trrap ) mice. Trrap mice were either intercrossed or ϩ/⌬ f/⌬ f/f Trrap gene in the hematopoietic system, including BM and spleen. crossed with Trrap mice (27) to obtain Trrap and Trrap f/⌬ ϩ f/f ϩ mice (Fig. 1A). Although Trrap⌬/⌬ embryos did not survive (data Hereafter, Trrap Cre and Trrap Cre mice treated with pIpC f/⌬ are designated as Trrap-CKO mice. The control group (Trrap-Co) not shown), consistent with our previous report (27), Trrap and ϩ/ϩ Ϫ f/⌬ f/f was composed of several genotypes (Trrap Cre ; Trrap Trrap mice were healthy and phenotypically normal, suggesting Ϫ ϩ/⌬ ϩ ϩ/f ϩ f/f Ϫ Trrap Cre ; Trrap Cre ; Trrap Cre ; and Trrap Cre injected that the floxed allele is functional and that one functional f/⌬ ϩ f/f ϩ allele of Trrap is sufficient to support normal development and with pIpC; and Trrap Cre Trrap Cre injected with PBS), all viability. of which carried at least one intact Trrap allele and produced con- To inactivate the Trrap conditional allele in tissues in an induc- sistent results in all experiments. Within 5 to 6 wk after pIpC ible manner, Trrapf/⌬ and Trrapf/f mice were crossed to transgenic treatment, the majority of Trrap-CKO mice, but not control mice mice expressing Cre recombinase under the control of the Mx including several genotypes, appeared moribund and either died or promoter (Mx-Cre) (28) to produce Trrapf/⌬Creϩ and Trrapf/f were killed (Fig. 1, D and E). Creϩ mice (Fig. 1A). For different requirements of the experiments, these mice were then either intercrossed or crossed with ϩ ⌬ Ϫ ⌬ Ϫ Ϫ Fatal failure of BM after inactivation of Trrap Trrap / Cre , Trrapf/ Cre ,orTrrapf/fCre to produce different genotypes, all of which were healthy and phenotypically normal. Analysis of Trrap-CKO mice revealed a dramatic decrease in cel- To delete Trrap in vivo, mice harboring the Trrap floxed allele and lularity of the femur and spleen at days 6 and 12 post deletion (Fig. Cre transgene acquired an inactivating mutation of the gene upon 2, A and B). Furthermore, analysis of peripheral blood indicated a treatment with pIpC (28) (Fig. 1A). The Mx1 promoter was chosen reduction in platelets, white blood cells, and lymphocytes in to drive Cre expression as it allows deletion of the gene in IFN- Trrap-CKO mice in comparison to control mice (Fig. 2, C–E). responsive tissues and hence generates a high level of deletion in Indeed, blood analysis of severely moribund mice revealed a four- hematopoietic tissues, including hematopietic stem cells (28). Cre- fold reduction in RBC, 36-fold reduction in platelets and 21-fold mediated deletion of Trrap was monitored by Southern blotting reduction in white blood cells (data not shown). Hence, Trrap- and PCR analysis of genomic DNA. Efficient deletion of the Trrap CKO died from severe aneamia and cytopenia. A comprehensive The Journal of Immunology 6425 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. Trrap is required to maintain homeostasis of the hematopoietic system in adult mice. Trrap-CKO mice, both at 6 and 12 days, display about a 60% reduction in cells from the BM (A) and about a 30% reduction in cells from the spleen (B) in comparison to control (Co) mice. Analysis of peripheral blood indicates a reduction in white blood cells (C), platelets (D), and lymphocytes (E)inTrrap-CKO mice at 2, 7, and 20 days, in comparison to control (Co) mice. F, Histological analysis by H&E staining reveals that Trrap-CKO mice have reduced nucleated cells in BM but not liver at 2 and 7 days. G, FACS analysis on cells from BM and spleen at 6 and 12 days postadministration of pIpC reveals a dramatic reduction in both the relative numbers of granulocytes (Gr1ϩ CD111bϩ) (FACS scan) and the total number of granulocytes (bar chart) in Trrap-CKO in comparison to Co mice. H, Additionally, the relative numbers of reticulocytes (Ter119ϩ CD71ϩ) and erythrocytes (Ter119ϩ CD71Ϫ) are down-regulated in the spleen (FACS scan) of Trrap-CKO mice (day 12 postdeletion) as compared with control mice. Intriguingly, analysis of the total number of reticulocytes and erythrocytes (bar chart) in the BM of Trrap-CKO mice (day 12) indicates the erythrocytes to be increased and the reticulocytes to be moderately decreased in comparison to control mice. I, Megakaryocytes were drastically decreased in both the spleen and BM of Trrap-CKO mice both at day 6 and 12 as indicated in the bar chart and FACS scan. J, The relative numbers (FACS scan) and total numbers (bar charts) of mature B cells (CD19ϩ IgMϩ) and T cells from the spleen of Trrap-CKO mice were not signiﬁcantly changed both at 6 and 12 days postdeletion of Trrap as compared with control mice. All FACS scans indicate analyses performed at 6 and 12 days postdeletion. The data shown are representative of two independent experiments. 6426 TRRAP MAINTAINS HEMATOPOIETIC STEM CELLS

FIGURE 3. Lethality of Trrap deletion is BM speciﬁc. A, BM from Trrap- CKO mice (6 days postdeletion) and control mice was extracted from the long bones and injected retro-orbitally into lethally irradiated recipient mice (that carry at least one WT Trrap allele to distinguish from the donor) following which survival was followed for at least 35 days. B, All recipient mice transplanted with control BM survived whereas only ϳ20% of recipient mice transplanted with Trrap-CKO BM survived. All mice that were lethally irradiated and transplanted with PBS died. C, PCR analysis was performed from tail and blood DNA from the surviving 20% of mice transplanted with Trrap- CKO BM. These mice were found to

carry the Trrap ﬂoxed allele in blood Downloaded from DNA, indicating that the transplanted BM cells were not efﬁciently deleted for Trrap.

histological analysis revealed no apparent abnormalities in any tis- Loss of BM progenitor populations and HSCs after Trrap http://www.jimmunol.org/ sues of Trrap-CKO mice except in BM (Fig. 2F; and data not inactivation shown) that conﬁrmed the severe decrease in cellularity at 2 and 7 Loss of Trrap results in a reduction of several mature blood ele- days post deletion (Fig. 2A). As a control, histological analysis of ments including granulocytes, megakaryocytes, platelets, and the liver, another target tissue of Mx-Cre, displayed no difference white blood cells, therefore we reasoned that the progenitors of between Trrap-CKO mice and control mice (Fig. 2F). To further these cells may be affected upon deletion of Trrap. To test this investigate the phenotype of these mice, we performed FACS anal- possibility, we have cultured BM cells under conditions that allow ysis of blood elements on BM and splenocyte cells. We found a for the progenitors to differentiate into varying lineages which can vast reduction in the number of granulocytes in the spleen and BM

then be identified based on their morphology in vitro. BM from by guest on September 26, 2021 at both 6 and 12 days post deletion in Trrap-CKO mice (Fig. 2G). Trrap-containing and Trrap-CKO mice were cultured for 10 days Similarly, the number of reticulocytes and megakaryocytes was following which colonies with different morphology were scored. decreased post deletion in the BM and spleen (Fig. 2, H and I), This assay revealed that Trrap-CKO BM generated dramatically whereas the relative number of mature B/T cells and erythrocytes Trrap was unaffected and increased, respectively (Fig. 2, H and J), sug- fewer colonies than did control BM and most progenitors gesting that erythroid and lymphoid progenitors might be less de- tested were markedly down-regulated (Fig. 4, A and B). To deter- pendent on HSCs, at least in the short term. These results indicate mine the status of hematopoietic progenitors using a more quan- that Trrap depletion affected a wide range of mature hematopoietic titative assay, phenotypic FACS analysis was performed on cells cells in BM and spleen. taken from BM, spleen and thymus. Strikingly, we found that the common myeloid progenitor, common lymphoid progenitor, gran- Trrap maintains the hematopoietic system in a cell-autonomous ulocyte-monocyte progenitor, and megakaryocyte-erythrocyte pro- manner genitor were all dramatically reduced in Trrap-CKO mice (Fig. 4, To examine whether the reduced survival of the mice and deple- C and D and data not shown). Additionally, Trrap deletion resulted in a loss of the immature PreproB, ProB, and PreB cells (Fig. 4, C tion of hematopoietic cells associated with Trrap deletion was a Ϫ result of hematopoietic failure, we performed BM reconstitution and D; and data not shown) as well as lineage negative (lin ) experiments (Fig. 3A). BM cells from Trrap-CKO mice or control stem/progenitor cells (Fig. 4E). This decrease in progenitors, as mice were transferred into lethally irradiated Trrapϩ/ϩ or Trrapf/ϩ determined both by FACS and colony formation assay, could be recipients and engraftment was allowed to occur. As expected, the due to an inability of the progenitors to proliferate or an absence of mice reconstituted with control BM cells retained 100% survival, the progenitors. Considering that in adult BM these progenitors ϩ Ϫ however only 30% of those reconstituted with Trrap-CKO BM arise from the rare (Ͻ0.1%) HSCs (lineage marker c-Kit Lin ϩ cells survived (Fig. 3B). Importantly, analysis of the DNA isolated Sca-1 , KLS), we next investigated the number of these cells in from the blood of those 30% surviving mice revealed only con- the BM by FACS. As early as 2 days postdeletion of Trrap, and tributing DNA from the Trrapf allele but not the host origin (i.e., even more drastically at 12 days postdeletion, we found the num- the Trrapϩ allele) (Fig. 3C), indicating that these mice are “es- ber of both long-term and short-term HSCs to be vastly reduced in capers” of deletion. All mice lethally irradiated and mock trans- comparison to control mice (Fig. 4, F–H). Quantitative analysis planted (administered with PBS) died within 2 wk postirradiation revealed that both absolute and relative numbers of either long- (Fig. 3B). These results demonstrate that Trrap-deficient hemato- term-HSCs, short-term-HSCs, and multipotent progenitor cells poietic stem/precursor cells lost the capacity to repopulate lethally were severely reduced in Trrap-deficient BM, whereas relative irradiated BM, suggesting that the defect in this compartment may number of multipotent progenitor at later time-point (day 12) account for the hematopoietic failure and reduced survival of seems to be more strongly affected (Fig. 4H, and data not shown). Trrap-CKO mice. Furthermore, expression of adhesion molecules CD29, CD49d, The Journal of Immunology 6427 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. Loss of hematopoietic progenitors and hematopoietic stem cells after Trrap deletion. A and B, Trrap-CKO BM was harvested 2 days (A) and 7 days (B) after three intraperitoneal dose of pIpC and cultured in medium that allows for the formation of hematopoietic progenitor colonies. Data are presented as mean number of colonies from triplicate performed assays Ϯ SEM counted 10 days after plating of 10,000 BM cells. C and D, B cell progenitors (common lymphoid progenitor, pre-pro B, proB, and preB) in BM deleted for Trrap were found to be down-regulated in BM as assessed by FACS using CD43, CD93, CD19 and IgM Abs, indicated by FACS (C) scan plot and bar chart (D). E, Lin- cells in BM were down-regulated in Trrap-CKO mice both at 6 and 12 days postdeletion. F–H, HSCs were dramatically reduced in Trrap-CKO BM. F, Gating strategy for quantitative analysis of HSCs in BM. BM cells were stained for lineage markers, gated on the linϪ (linnegative) subset, and further stained for c-Kit (CD117) and Sca-1. Numbers on the plot are the frequency of cells in the indicated regions. Hematopoietic stem cells are contained within the c-Kitϩ linϪ Sca-1ϩ (KLS cells) population. G and H, HSC (ScaIϩ CD117ϩ) in BM from Trrap proficient (Co) and Trrap-CKO mice was assessed by FACS using specific Abs both at 2 days and 12 days postdeletion. Boxes indicate KLS-HSC populations. H, Quantification of hematopoietic stem/progenitor cells as in G. Data are representative of two independent experiments. 6428 TRRAP MAINTAINS HEMATOPOIETIC STEM CELLS and CD49e on KLS immature cells were unaffected in Trrap that p53Ϫ/Ϫ Trrap-CKO mice displayed high mortality rates depleted BM at 2 days after Trrap deletion, and only slightly that were similar to Trrap-CKO mice (data not shown), these down-regulated 12 days post deletion (data not shown), arguing data indicate that Trrap may prevent apoptosis of hematopoietic that changes in the expression of adhesion molecules are un- stem/progenitor cells through a p53-independent mechanism. likely to induce the migration of HSCs out of the niche and Deregulation of c-Myc, a transcription factor which directly subsequently cause their loss in Trrap-CKO mice. These results interacts with TRRAP (17), was shown to alter hematopoietic show that hematopoietic failure in Trrap deficient mice is most stem cell pools (23, 24). Therefore, to test the expression and likely due to the loss of HSCs or cell autonomous defects in function of the Myc transcription factors in hematopietic stem/ HSCs, indicating that Trrap plays an essential role in the main- progenitor cells lacking Trrap, we measured quantitatively by tenance of HSCs and the repopulating capacity of hematopoi- RT-PCR the expression of c-Myc and N-Myc and the sets of etic stem/progenitor cells. genes known to be the targets of these transcription factors in Trrap-CKO linϪ cells. Loss of Trrap resulted in up-regulation Apoptosis of BM progenitor populations after Trrap deletion of c-Myc and N-Myc expression in linϪ cells, whereas expres- BM failure and the reduction in hematopoietic cells could be sion of the anti-apoptotic gene Mcl1 and housekeeping gene due to a blockage of cell cycle progression or induction of hprt remains unchanged (Fig. 5J, and data not shown). Surpris- apoptosis in hematopoietic stem/progenitor populations. To ad- ingly, Western blot analysis revealed that c-Myc protein levels dress what contributes to this cellular reduction, linϪ cells were were dramatically reduced in Trrap-CKO linϪ cells, whereas purified from total BM and used for further analysis (Fig. 5A). N-Myc protein showed a moderate decrease (Fig. 5J), indicat- Ϫ The purity of the lin population was verified by performing ing that the protein Trrap is required for the stability of Myc Downloaded from FACS analysis using an anti c-kit Ab (Fig. 5B), and the effi- proteins. Furthermore, we found that Trrap deficient linϪ cells ciency of Trrap deletion in these cells following pIpC injection exhibit a significant reduction in cyclin D2, Mcm7, and Id2 and was analyzed by PCR (Fig. 5C). To examine the proliferation concomitant up-regulation of cyclin D1 (Fig. 5, J and K). These dynamics and apoptotic fraction of linϪ BM cells lacking Trrap, results show that loss of Trrap in linϪ cells, despite down- Trrap was deleted in BM and 48 h thereafter the cell cycle regulation of c-Myc and N-Myc proteins, compromises both Ϫ profile and cell death of lin BM cells were analyzed ex vivo by transcriptional activation and repression function of Myc tran- http://www.jimmunol.org/ flow cytometry after staining cells with PI or FITC-conjugated scription factors on their respective targets. Because deregula- Annexin V, respectively. Although cell cycle profiles were vir- tion of c-Myc and N-Myc was shown to alter hematopoietic tually indistinguishable between Trrap-deficient and Trrap-con- stem cell pools (23, 24), these results suggest that deregulation taining BM cells (Fig. 5D), linϪ cells lacking Trrap exhibited a of c-Myc and N-Myc function in the absence of Trrap leads to marked increase (over 2-fold) in apoptotic fraction in compar- apoptosis and/or defect in the maintenance of hematopoietic ison to Trrap-proficient controls (Fig. 5E; and data not shown). stem/progenitor cells. In addition, a fraction of cells with nuclear features of apoptosis (chromatin condensation visualized by FISH staining of major satellite repeats MSR) was significantly increased in Trrap-de- Discussion by guest on September 26, 2021 pleted linϪ cells as compared with Trrap-proficient controls Recent studies have indicated a role of Tip60-p400 HAT com- (Fig. 5F, and data not shown). These results indicate that in- plex in the maintenance of stem cell identity (25), however the creased apoptosis, and not cell cycle block, in the stem/progen- underlying mechanism remains poorly understood. In the itor compartment is likely to be the cause of BM failure and loss present study, we conditionally deleted Trrap in bone marrow of hematopoietic progenitors in Trrap-deficient mice. We there- stem/progenitor cells as a model system to further identify fore asked whether Trrap controls viability of hematopoietic Trrap as a part of the mechanism that promotes the maintenance stem/progenitor cells via a p53-dependent mechanism. For this of stem cell pools. Our study reveals that deletion of Trrap purpose, we crossed mice harboring floxed alleles of Trrap and leads to a loss of hematopoietic stem and progenitor cells via a the inducible Mx-Cre transgene with p53-deficient mice cell autonomous manner without affecting the HSC niche hence ( p53Ϫ/Ϫ mice) (29) to obtain p53Ϫ/ϪTrrapf/fCreϩ double-mu- Trrap is essential for maintaining the integrity of adult BM. tant mice. BM from these mice isolated 2 and 7 days after the Furthermore, Trrap-deficient hematopoietic stem/progenitor treatment with pIpC ( p53Ϫ/ϪTrrap-CKO mice) or PBS ( p53Ϫ/Ϫ cells exhibited a marked increase in p53 independent apoptosis, Trrap-Co mice) was cultured and colonies with different mor- arguing that loss of hematopoietic stem/progenitor pool results phology were scored. As shown in Fig. 5, G and H, most stem/ in BM failure leading to the severe phenotype and frequent progenitors tested were markedly down-regulated in BM from death of Trrap-CKO mice. Similar phenotypes have been ob- p53Ϫ/ϪTrrap-CKO compared with that from p53Ϫ/ϪTrrap-Co served after deletion of several transcriptional regulators and mice, showing that the absence of p53 could not rescue the molecular players involved in chromatin modifications includ- hematopoietic stem/progenitor cell defect in Trrap-deficient ing Zfx (30), Foxo1/3/4 (31), Foxo3a (32), Bmi-1 (33), and mice. To further determine whether the increased apoptosis of Mi2␤. These studies suggest that Trrap belongs to a group of hematopoietic stem/progenitor cells in the absence of Trrap is molecules involved in transcriptional reprogramming and chro- p53-dependent, Trrap was deleted in BM of p53Ϫ/Ϫ mice and matin reconfiguration that play critical roles in the maintenance apoptotic fraction of linϪ BM cells from p53Ϫ/ϪTrrap-deficient of hematopoietic stem/progenitor cell pool. The severe pheno- double mutant mice was analyzed by flow cytometry after An- type of TRRAP deficient mice and bone marrow progenitor/ nexin V staining. As shown in Fig. 5I, Trrap-deficient linϪ cells stem cells raises the question on the contribution of different on p53Ϫ/Ϫ background exhibited a significantly higher apopto- TRRAP-dependent processes to the TRRAP-deficient pheno- tic fraction compared with linϪ cells from p53Ϫ/ϪTrrap-Co type. TRRAP is a component of several HAT complexes (8) and mice. This increase in apoptotic fraction was comparable to that interacts with transcription factors c-Myc and E2F (17). How- observed in Trrap-deficient mice on p53ϩ/ϩ background (Fig. ever, deletion of any single TRRAP-interacting player (such as 5E), indicating that p53 status does not affect cell death induc- individual HAT enzyme, c-Myc or E2F) leads to embryonic tion associated with loss of Trrap. Together with the findings lethality at only later embryonic stage (c-Myc at E10.5; Gcn5 at The Journal of Immunology 6429 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 5. Loss of Trrap leads to increased apoptosis and deregulation of the Myc transcription factors in BM stem/progenitor cells. A, Trrapf/⌬ Creϩ and Trrapf/⌬ CreϪ mice were injected with pIpC and linϪ cells were purified from BM 2 days later. Cells were then used for Annexin V staining (to detect apoptotic cells), cell cycle profiling, immunofluorescent staining, and RT-PCR analysis. B, The purity of linage negative cells was assessed by FACS using ac-kit (CD117) Ab. C, RT-PCR analysis of Trrap depletion efficiency by pIpC-induced Cre recombinase in hematopoietic linϪ cells. Total RNA was isolated from linϪ cells purified from control (Co) and Trrap-CKO mice and Trrap mRNA levels were analyzed by RT-PCR. HPRT levels were used as internal loading control. D–F, Analysis of cell cycle and apoptosis in Trrap-deficient BM cells. D, BM cells isolated from Trrap-CKO and control mice were stained with propidium iodide and analyzed by FACS. E, Flow cytometry analysis of BM cells isolated from Trrap-CKO and control after staining with FITC-Annexin V. The numbers indicate the percentage of Annexin V-positive (apoptotic) cells. F, DNA-FISH analysis of the mouse major satellite repeats in linϪ cells isolated from Trrap-containing (Co) and Trrap-depleted (CKO) BM. Cells were counterstained by DAPI (DNA). G and H, Apoptosis of hematopoietic stem/progenitor cells after Trrap deletion is p53 independent. BM cells from Trrap-CKO in Trp53Ϫ/Ϫ (p53Ϫ/Ϫ) background were harvested 2 days (G) and 7 days (H) after three intraperitoneal dose of pIpC and cultured in medium that allows for the formation of hematopoietic progenitor colonies. Data are presented as mean number of colonies from triplicate performed assays Ϯ SEM counted 10 days after plating of 10,000 BM cells. I, Analysis of apoptosis in linϪ BM cells from Trrap deficient mice on p53Ϫ/Ϫ background. LinϪ BM cells from p53Ϫ/ϪTrrap-CKO and p53Ϫ/Ϫ Trrap-Co (after intraperitoneal injection of pIpC) were analyzed by flow cytometry after Annexin V staining. The numbers indicate the percentage of Annexin V-positive cells. J and K, RT-PCR and Western blot analysis comparing expression of Myc transcription factors and their targets in Trrap deficient stem/progenitor cells. J, Total RNA was isolated from linϪ cells purified from control (Co) and Trrap-CKO mice and mRNA levels of indicated genes were analyzed by RT-PCR and Western blotting. All data was normalized against the expression of hprt (RT-PCR) and actin (Western blot). K, Quantification of gene expression after normalization as in J. mRNA levels of indicated genes were analyzed densitometrically and normalized to hprt. 6430 TRRAP MAINTAINS HEMATOPOIETIC STEM CELLS

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