Coactivator-Associated Arginine Methyltransferase 1 Regulates Fetal Hematopoiesis and Thymocyte Development

This information is current as Jia Li, Ziqin Zhao, Carla Carter, Lauren I. R. Ehrlich, Mark of September 28, 2021. T. Bedford and Ellen R. Richie J Immunol 2013; 190:597-604; Prepublished online 17 December 2012; doi: 10.4049/jimmunol.1102513

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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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Coactivator-Associated Arginine Methyltransferase 1 Regulates Fetal Hematopoiesis and Thymocyte Development

Jia Li,*,† Ziqin Zhao,† Carla Carter,* Lauren I. R. Ehrlich,‡,1 Mark T. Bedford,*,1 and Ellen R. Richie*,1

Coactivator-associated arginine methyltransferase 1 (CARM1) is a protein arginine methyltransferase that methylates histones and transcriptional regulators. We previously reported that the absence of CARM1 partially blocks thymocyte differentiation at em- bryonic day 18.5 (E18.5). In this study, we find that reduced thymopoiesis in Carm12/2 mice is due to a defect in the fetal hematopoietic compartment rather than in the thymic stroma. To determine the cellular basis for impaired thymopoiesis, we examined the number and function of fetal liver (FL) and bone marrow cells. Despite markedly reduced cellularity of hemato- poietic progenitors in E18.5 bone marrow, the number of long-term hematopoietic stem cells and downstream subsets was not 2 2 reduced in Carm1 / E14.5 or E18.5 FL. Nevertheless, competitive reconstitution assays revealed a deficit in the ability of Downloaded from Carm12/2 FL cells to contribute to hematopoiesis. Furthermore, impaired differentiation of Carm12/2 FL cells in a CARM1- sufficient host showed that CARM1 is required cell autonomously in hematopoietic cells. Coculture of Carm12/2 FL cells on OP9- DL1 monolayers showed that CARM1 is required for survival of hematopoietic progenitors under conditions that promote differentiation. Taken together, this report demonstrates that CARM1 is a key epigenetic regulator of hematopoiesis that affects multiple lineages at various stages of differentiation. The Journal of Immunology, 2013, 190: 597–604. http://www.jimmunol.org/ ematopoietic stem cells (HSC) can self-renew through- development. Indeed, recently, genome-wide DNA methylation pat- out life and differentiate into all myeloid and lymphoid terns were analyzed at each major stage of hematopoiesis, reveal- H lineages (1). Epigenetic modifications are a driving force ing clear epigenetic signatures for each cell lineage (4). Like DNA of this cellular differentiation. Because the genetic information of methylation, there are a number of reports that arginine methyla- cells pre- and postdifferentiation is identical, epigenetic marks like tion also plays a critical role in lymphocyte development and signal DNA methylation and histone methylation are critical for facilitat- transduction (5). ing the lock-in of a differentiated state (2). Epigenetic mechanisms Arginine methylation is a common posttranslational modifica- function like a ratchet, allowing lineage-specific differentiation, but tion that subtly alters the function of its substrates (6). It does this in generally not dedifferentiation. There is an element of plasticity to a number ways: 1) Arginine methylation provides a docking site by guest on September 28, 2021 cellular differentiation that can be forcibly reversed by small mol- for Tudor domain–containing effector molecules (7–10); 2) it can ecule epigenetic regulators and/or the overexpression of a few also block protein–protein interactions, as in the case of certain SH3 specific to generate an induced pluripotent stem cell state domain–driven interactions (11); 3) arginine methylation can neg- (3). Perhaps one of the best biological systems to study epigenetic atively regulate AKT-mediated phosphorylation, because the AKT changes that correlate with differentiation is hematopoietic cell consensus motif contains key arginine residues (12, 13); and sim- ilarly, 4) lysine methylation can also be blocked by adjacent argi- nine methylation events (14, 15). The substrates for protein arginine *Department of Molecular Carcinogenesis, The University of Texas MD Anderson Center, Smithville, TX 78957; †Department of Forensic Medicine, Shanghai methyltransferases (PRMTs) are both nuclear and cytoplasmic, and Medical College of Fudan University, Shanghai 200032, China; and ‡Section of in the nucleus, histones are a major target of these . Histone Molecular Genetics and Microbiology, Institute for Cellular and Molecular Biology, methylation allows the PRMTs to feed into the epigenetic code and The University of Texas at Austin, Austin, TX 78712 1 contribute to key molecular switches that dictate cell fate. L.I.R.E., M.T.B., and E.R.R. contributed equally to this work. The mammalian PRMT family of enzymes consists of nine Received for publication August 31, 2011. Accepted for publication November 7, members, PRMT1–9; the majority of these enzymes target the 2012. N-terminal tails for histones H3, H4, and H2A for methylation This work was supported by National Institutes of Health Grant DK62248 (to M.T.B. and E.R.R.) and through MD Anderson’s Cancer Center Support Grant CA016672. (16). Coactivator-associated arginine methyltransferase 1 (CARM1)/ Facility core support for flow cytometry was provided by National Institute of En- PRMT4 was the first family member to be identified as a transcrip- vironmental Health Sciences Grant P30ES007784 to the Center for Research on tional coactivator, which methylates the H3R17 and H3R26 sites, Environmental Disease. as well as other transcriptional regulators (6, 17). CARM1-null Address correspondence and reprint requests to Dr. Ellen R. Richie, The University of Texas MD Anderson Cancer Center, Science Park-Research Division, Smithville, embryos display no overt developmental defects, although they TX 78957. E-mail address: [email protected] are smaller than their wild-type counterparts, and once born, the The online version of this article contains supplemental material. nulls die without taking their first breath (18). In-depth analysis of Abbreviations used in this article: BM, bone marrow; CARM1, coactivator-associ- these CARM1-null embryos has revealed a number of clear phe- ated arginine methyltransferase 1; CLP, common lymphoid progenitor; DN, double- notypes, many of them associated with cell differentiation defects. negative; DP, double-positive; E, embryonic day; FL, fetal liver; GMP, - progenitor; HSC, ; KLS, Lineageloc-Kit+Sca-1+; CARM1-null lethality at birth is likely due to the fact that lungs LT-HSC, long-term HSC; MEP, megakaryocyte/erythroid progenitor; MPP, multipo- from mice lacking CARM1 are inundated with immature alveolar tent progenitor; PRMT, protein arginine methyltransferase; SP, single-positive; ST- type II cells, which do not develop into more mature alveolar type I HSC, short-term HSC. cells; thus, CARM1 is required for the proper differentiation of Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 alveolar cells (19). In addition, CARM1-null embryos lack brown www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102513 598 CARM1 REGULATES FETAL HEMATOPOIESIS fat, and cells that do not express CARM1 are not able to differ- to allophycocyanin, anti–c- (clone 2B8) conjugated to allophycocyanin- entiate into mature adipocytes (20). CARM1 is also required for Cy7, and anti-FcgR (clone 93) conjugated to PE were purchased from chondrogenesis (21) and skeletal muscle development (22). Fi- BioLegend. Abs to CD8a (clone 53-6.7) conjugated to FITC or PE-Cy7, gdTCR (clone eBioGL3) conjugated to PE, CD3ε (145-2C11), and CD127 nally, we have also observed that functional CARM1 is required (clone A7R34) biotinylated, CD4 (GK1.5) and TER-119 conjugated to for normal cellularity and differentiation (23, 24). Thus, allophycocyanin, c-Kit (clone 2B8) conjugated to allophycocyanin-eFluor there is genetic evidence that CARM1 activity impacts the differ- 780, CD45 (clone 30-F11) conjugated to eFluor 450, CD25 (PC61.5) entiation of lung, fat, muscle, cartilage, and thymocytes. conjugated to Alexa Fluor 488, and CD34 (RAM34) conjugated to Alexa Fluor 700 were purchased from eBioscience. A lineage mixture containing We previously reported that CARM1 null embryos have a 5-fold Abs to CD3ε (145-2C11) CD4 (GK1.5), CD8a (clone 53-6.7) CD11c (clone reduction in thymocyte cellularity and a partial block in early N418), CD11b (clone M1/70), B220 (clone RA3-6B2), TER-119, and Gr-1 T cell development (24). We now confirm a role for CARM1 in conjugated to PE-Cy5 was purchased from eBioscience. Anti-CD4 conju- thymocyte development at the transition between the double- gated to Pacific Orange was purchased from Invitrogen. Biotinylated Abs negative 1 (DN1) and DN2 stages. We investigated the cellular were detected using streptavidin conjugated to Qdot 655 or allophycocyanin (Invitrogen). Cells were analyzed on an FACS Aria II (BD Science), and data basis for impaired thymopoiesis in this mouse model. In this ar- were analyzed using FlowJo software (Tree Star). The lineage mixture in- ticle, we report that CARM1 functions cell intrinsically to regulate cluded Abs to B220, Ter119, Gr-1, Mac-1, NK1.1, and CD11c to exclude hematopoietic progenitor cell activity and cellularity in the fetal lineage-positive cells in thymocyte analyses. The lineage mixture included liver (FL) and bone marrow (BM), respectively. Furthermore, Abs to CD4, CD8, B220, Ter119, Gr-1, Mac-1, NK1.1, and CD11c to ex- clude lineage-positive cells in BM and FL analyses. when FL progenitors were cocultured on OP9-DL1 stroma, CARM1 was dispensable for T lineage differentiation in response Kidney capsule transplantation to Notch ligands but was essential for survival. Taken together, 2 2 Downloaded from Fetal thymic lobes from E15.5 Carm1 / or littermate controls were placed these results demonstrate that CARM1 regulates fetal hematopoiesis on Millicell 30-mm round 0.4-mm culture plate inserts (Millipore) such and thymocyte development, bolstering the notion that epigenetic that they were floating at the liquid–air interface in RPMI 1640 medium regulation is critical for proper differentiation and survival of mul- (Invitrogen) containing 10% FBS (Atlanta Biologicals), 1% penicillin– tiple hematopoietic lineages. streptomycin (Invitrogen), 2 mM L-glutamine (Invitrogen), 1 mM sodium pyruvate (Invitrogen), and 1.35 mM 29-deoxyguanosine for 5 d. The thymocyte-depleted lobes were transplanted under the kidney capsule of

nu/nu http://www.jimmunol.org/ Materials and Methods athymic recipient NCr mice. Mice Competitive repopulation assay 2/2 Carm1 embryos were described previously (18) and generated by 3 6 2/2 + 2/+ FL cells (2.5 10 ) from Carm1 EGFP E14.5 embryos or controls crossing Carm1 breeders. Timed pregnancies were established, and the (Carm1+/2 EGFP+ or Carm1+/+ littermates) were mixed 1:1 with com- day of vaginal plug was designated embryonic day 0.5 (E0.5). Embryos were petitor EGFP2 FL cells from wild-type C57Bl6/J E18.5 embryos and genotyped using the primers 59-CCCACTTCTGTTACCTCCTTTG-39 and transplanted into sublethally irradiated (4.5 Gy) Rag22/2gc2/2 mice by 59-TAACTAAAAGAAAATGGAATGG-39. Mice transgenic for EFGP under retroorbital injection. The recipient mice were analyzed 8 or 16 wk after control of the b-actin promoter were kindly provided by Dr. Irving Weiss- BM transplantation. man (25). C57BL/6J and RAG22/2gc2/2 mice were purchased from The nu/nu Jackson Laboratory (Bar Harbor, ME). Athymic NCr mice were ob- OP9-DL1 cocultures, proliferation, and apoptosis assays by guest on September 28, 2021 tained from NCI (Frederick, MD). Mice were maintained in the Vivarium at the MD Anderson Cancer Center Science Park in accordance with guidelines Five hundred FACS-purified Lineageloc-Kit+Sca-1+ (KLS) FL progenitors established by the Association for the Accreditation of Laboratory Animal obtained from individual E14.5 Carm12/2 or control littermates were di- Care. All protocols using these mice were reviewed and accepted by the MD rectly sorted into triplicate wells of 24-well plates containing a monolayer Anderson Animal Care and Use Committee. of OP9-DL1 cells in a-MEM + 10% FCS with 5 ng/ml IL-7 and 5 ng/ml Flt3L (PeproTech). After 6 d in culture (37˚C, 5% CO2), hematopoietic Immunofluorescence microscopy cells were recovered and analyzed by flow cytometry for T cell differen- tiation markers. For DNA content analysis, live cells were stained with Serial sections (5 mm) from OCT-embedded frozen tissue were air dried Vybrant Dye Cycle Violet (Life Sciences), according to manufacturer’s and fixed in cold acetone for 5 min at room temperature. After washing in instructions. FlowJo software (Tree Star) was used to analyze the fre- TNT (0.1 M Tris pH 7.5, 0.15 M NaCl, 0.05% Tween 20), sections were quency of proliferating cells (S + G2/M), as determined by the Dean–Jett– blocked for 15 min in TNB (0.1 M Tris-HCL pH 7.5, 0.15 M NaCl, 0.5% Fox algorithm. Apoptosis was assessed by staining with Pacific Blue–con- blocking reagent from TSA Biotin System [NEN Life Science Products, jugated Annexin V (BioLegend), according to manufacturer’s instructions, in Boston, MA]). The slides were incubated at room temperature with pri- conjunction with propidium iodide to detect dead cells. Forward and side mary Abs including rat anti-mouse CD45 (clone 30-F11; BD), polyclonal scatter gates were set to tightly enclose live lymphocytes, thus excluding rabbit anti-cytokeratin (Dako), rat anti-mouse CD25 (clone PC61; BD), debris and small dead cells from our analyses. rabbit anti-mouse K5 (Covance), and rat anti-mouse cKit (clone ACK45; BD) for 1 h. Secondary reagents included streptavidin-FITC (Vector Statistical analysis Laboratories), Texas Red–conjugated donkey anti-rabbit IgG, and Texas Red–conjugated donkey anti-mouse IgG (Jackson Immunoresearch). In Prism 5.0 (GraphPad version 5.0c; GraphPad, San Diego, CA) and some cases, tyramide amplification was performed. Microsoft Excel were used for all statistical analyses. The Kolmogorov– Smirnov test was first used to assess normality of the data in each ex- Flow cytometry periment. To analyze the mean cell number in different hematopoietic Single-cell suspensions were prepared from E18.5 thymi, spleen, and FL by subsets, we performed a Student t test on data distributed normally, dissociation of tissues through a 70-mm strainer (Fisher). Fetal BM cells whereas the two-tailed Mann–Whitney U test was performed on data that were not distributed normally. For comparison of the ratio of GFP+:GFP2 were obtained by finely mincing E18.5 femur, tibia, humerus, radius, and , ulna with a single-edge razor blade to release cells into FACS buffer. The cells, the one-tailed Mann–Whitney U test was used. A p value 0.05 was dissociated cells and fragments were filtered through a 70-mm strainer considered statistically significant. (Fisher). BM and spleen cells were treated with RBC lysis buffer (17 mM Tris, 160 mM NH4Cl, pH 7.3) to remove RBCs, then resuspended in FACS Results buffer (PBS pH 7.2, 0.005 M EDTA, 2% FBS). Cells were stained with fluorochrome-conjugated Abs in FACS buffer for 30 min on ice and Carm1 deficiency results in a block at the DN1-DN2 transition washed with FACS buffer. Propidium iodide (Invitrogen) was added (0.5 in thymopoiesis mg/ml) to the samples immediately before data acquisition for dead cell exclusion. Anti-B220 (clone RA3-6B2) and anti-CD45 (clone 30-F11) We previously observed that CARM1 deficiency results in a re- conjugated to Pacific Blue, anti-CD27 (clone LG.3A10) conjugated to duction in thymic cellularity, associated with an accumulation of 2 2 2 PerCP-Cy5.5, anti-CD44 (IM7) and anti-CD135 (clone A2F10) conjugated CD44+CD25 CD4 CD8 thymocytes at E18.5 (24). This pop- The Journal of Immunology 599 ulation is heterogeneous, containing both T and non-T lineage might be expected from a reduction in their progenitors, DN2 cells progenitors. c-Kit is expressed on the most immature T cell pro- (Fig. 1B). Together, these data indicate that CARM1 is required for genitors, DN1 cells, within this population (DN1: c-Kit+ CD44+ continued maturation of thymocytes beyond the DN1 stage. 2 2 2 CD25 CD4 CD8 ) (26, 27). DN1 thymocytes give rise to DN2 2 2 Reduced thymopoiesis in Carm1 / mice is not due to thymic progenitors (c-Kit+CD44+CD25+CD42CD82) that subsequently stromal defect downregulate CD25 to become DN3 cells (c-Kit2CD442CD25+ CD42CD82) that are committed to the T cell lineage. To further Thymopoiesis requires input from stromal cells in the thymic characterize the block in thymocyte development observed in microenvironment. Thus, the block in thymocyte development 2/2 Carm12/2embryos (24), we analyzed c-Kit expression within the observed in Carm1 embryos could be an indirect consequence CD44+CD252CD42CD82 compartment to distinguish the earliest of a defect in the thymic stromal compartment. To determine T cell progenitor population. Consistent with our previous report, whether Carm1 is required for proper thymic stromal function, we 2/2 there was an increased frequency of CD44+CD252 thymocytes in transplanted E15.5 2-deoxyguanosine–treated Carm1 versus the CD42CD82 compartment in Carm12/2 E18.5 embryos (Fig. control fetal thymic lobes under the kidney capsule of athymic 1A). Furthermore, analysis of c-Kit expression revealed an increase nude recipients. Eight to 12 wk after transplantation, thymic grafts in the frequency of DN1 versus DN2 progenitors in Carm12/2 were recovered and analyzed by flow cytometry. In contrast with 2/2 E18.5 embryos, consistent with a block in differentiation of the the developmental block observed in Carm1 E18.5 thymi (Fig. 1), earliest thymocyte subset. thymopoiesis was not impaired when CARM1 deficiency was re- The absolute number of DN1 thymocytes was not significantly stricted to thymic stromal cells (Fig. 2). The cellularity was com- 2 2 different in Carm1 / E18.5 versus control embryos. However, the parable for all thymocyte subsets regardless of whether they Downloaded from 2/2 DN2 subset was significantly reduced, consistent with a block in developed in a control or Carm1 stromal environment. Engrafted the DN1-DN2 transition (Fig. 1B). CARM1 deficiency resulted in CARM1-deficient lobes were smaller than controls at the outset of a .90% reduction in the number of DN2, DN3, and DN4 (c-Kit2 the transplantation experiment; this initial difference in thymic size CD42CD82CD442CD252) thymocytes. DN4 cells are the im- could account for the slight decreases in thymocyte numbers ob- 2/2 mediate precursors of double-positive (DP) thymocytes (CD4+ served in transplanted Carm1 lobes (Fig. 2). In addition, com- + CD8 ), which then give rise to mature CD4 single-positive (SP; parable numbers of CD4SP and CD8SP T cells were recovered from http://www.jimmunol.org/ 2/2 CD4+CD82) and CD8SP (CD42CD8+) subsets. These latter sub- the spleens of athymic recipients transplanted with Carm1 or sets were reduced by ∼75% in the absence of CARM1 (Fig. 1B). control fetal thymi (Supplemental Fig. 1). Altogether, the normal We also observed a significant reduction of 85% in gd T cells, as differentiation of thymocyte progenitors in a CARM1-deficient microenvironment, in contrast with the severely reduced cellular- ity in Carm12/2 E18.5 thymi, indicates that loss of CARM1 pre- dominantly affects thymocyte progenitors as opposed to the thymic stromal microenvironment. CARM1 deficiency affects thymocyte cellularity in the E12.5 by guest on September 28, 2021 thymic rudiment Given the defect in thymic cellularity at E18.5, we examined cryosections from E12.5–E18.5 from Carm12/2 embryos to de- termine whether the reduction in thymocyte cellularity was ap- parent earlier in ontogeny. At E12.5, there was a striking paucity in the number of CD45+ hematopoietic progenitors in the Carm12/2 thymic rudiment (Fig. 3A). This deficiency was also apparent E13.5–E17.5 in sections stained for c-Kit and CD25, markers of

FIGURE 1. CARM1 deficiency results in an early thymopoiesis block in E18.5 embryos. (A) Representative flow cytometric analysis of CD45+Lin2 thymocytes from E18.5 Carm12/2 and Carm1+/2 or Carm1+/+ littermate FIGURE 2. CARM1 deficiency in the thymic stromal compartment does controls. Arrows indicate gating strategy. DN thymocyte subsets are spec- not impair thymocyte development. E15.5 Carm12/2 or littermate control ified. Dot plots were depicted at low resolution when subset cellularity was thymic lobes were incubated in 2-deoxyguanosine for 5 d and transplanted low. (B) Cellularity of thymocyte subsets from E18.5 Carm12/2 embryos under the kidney capsule of athymic recipients. After 8 wk, thymocytes and littermate controls. Each point represents an individual embryo. Mean recovered from the grafts were analyzed by flow cytometry. Data shown values are indicated by the horizontal bars. Results are combined from four are from five individual experiments with two or more animals per ex- independent experiments with two or more mice per experiment. **p , periment. Mean values are indicated by the horizontal bars. *p , 0.05, 0.01, ***p , 0.001. **p , 0.01, ***p , 0.001. 600 CARM1 REGULATES FETAL HEMATOPOIESIS

DN1-DN3 thymocytes (Fig. 3B, 3C). By E13.5, Carm12/2 thymic of thymic organogenesis (Fig. 3), we hypothesized that the absence lobes were markedly smaller than wild-type and remain hypo- of Carm1 might affect prethymic hematopoietic progenitors. Be- plastic, containing fewer thymocyte progenitors throughout em- cause hematopoietic progenitors are present in the fetal BM bryogenesis (Fig. 3B, 3C). Thus, CARM1 plays a significant role in by E18.5 (28), we compared E18.5 BM from Carm12/2 and thymocyte development before and after thymic vascularization, control littermates for hematopoietic progenitor subset composi- which occurs at ∼E14.5. tion. Cellularity was reduced in Carm12/2 BM compared with 2 2 littermate controls (data not shown). Interestingly, the proportion Early hematopoietic defect in Carm1 / BM and number of c-Kit+ cells within the lineagelo fraction was greatly Given the profound reduction in DN subsets at E18.5 (Fig. 1), reduced in CARM1-deficient embryos (Fig. 4A, left panels,4B). together with the decrease in thymocyte progenitors at early stages The KLS progenitors, which contain the most undifferentiated hematopoietic progenitors, are reduced in both frequency and ab- solute number in the E18.5 Carm12/2 BM (Fig. 4). The KLS population consists of several progenitor subsets: long-term HSCs (LT-HSCs: Slamf1+Flk22KLS), short-term HSCs (ST-HSCs: Slamf12Flk22KLS), and lymphoid-biased multipotent progenitors (Flk2+MPP: Flk2+Slamf12KLS). All three of these early hemato- poietic progenitors are significantly reduced in the E18.5 Carm12/2 BM. Flk2+MPP are reduced by 95%, whereas LT-HSCs and ST-

HSCs were reduced by ∼80% (Fig. 4). Downloaded from In addition to the decrease in oligopotent hematopoietic pro- genitors in E18.5 Carm12/2 BM, downstream lineage-restricted progenitors were also diminished. The lineageloCD27+Flk2+ BM subset contains all progenitors with thymocyte differentiation http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. Carm12/2 fetal thymi contain fewer thymocyte progenitors than Carm1+/+ littermates. Transverse cryosections of Carm12/2 and Carm1+/+ fetal thymi were costained for thymocyte and thymic eptihelial cell (TEC) markers as indicated. (A) Distribution of CD45+ progenitors in FIGURE 4. CARM1 deficiency results in reduced cellularity of hema- E12.5 thymus rudiment. Scale bar, 100 mm. (B) Distribution of c-kit+ topoietic progenitors in E18.5 BM. (A) Representative flow cytometric (DN1 or DN2) thymocytes in E13.5 thymus rudiment (denoted by dashed analysis of Lin2 BM cells from E18.5 Carm12/2 null mice and littermate line). Scale bar, 200 mm. (C) Distribution of CD25+ (DN2 or DN3) thy- controls. Top row is a representative stain from C57Bl6/J adult BM, which mocytes and K5+ TECs in E13.5 (scale bar, 100 mm), E15.5 (scale bar, was used to define gating strategy for progenitor populations. Arrows in- 500 mm), and E17.5 thymi (scale bar, 1000 mm). Three experiments were dicate gating strategy. Hematopoietic subsets are specified. Dot plots were performed with thymi from two Carm12/2 and one Carm1+/+ embryo per depicted at low resolution when subset cellularity was low. (B) Cellularity experiment. Sections were mounted in VECTASHIELD mounting medium of BM progenitors from E18.5 Carm12/2 embryos and littermate controls. (Vector Laboratories), and staining was analyzed at room temperature Each point represents an individual embryo. Mean values are indicated by using an Olympus ProVis AX70 microscope with UPlanFl 40/0.75, the horizontal bars. Results are combined from five independent experi- UPlanFl 20/0.50, and UPlanFl 10/0.30 objectives, DP Controller and DP ments with two or more animals per experiment. *p , 0.05, **p , 0.01, Manager software. ***p , 0.001. The Journal of Immunology 601 potential (29). Interestingly, this population was severely reduced by 93% in Carm12/2 BM. The CD27+Flk2+ compartment con- tains the lymphoid-restricted common lymphoid progenitor subset (CLP: Lineagelo CD27+Flk2+IL-7R+), which was reduced by 85% (Fig. 4). We also observed a reduction in myeloid-restricted pro- genitors (MP, cKit+ Lineagelo Sca12) in the absence of CARM1 (Fig. 4). These progenitors can be subdivided into common my- eloid progenitors (LineageloCD34+FcgR2MP), further down- stream granulocyte-macrophage progenitors (GMP: LinloCD34+ FcgR+MP), and megakaryocyte/erythroid progenitors (MEP: Linlo CD342FcgR2MP). Common myeloid progenitors and MEPs were significantly reduced by 92 and 66%, respectively, whereas neither BM GMPs nor splenic were significantly diminished (Fig. 4, Supplemental Fig. 2). The reduction in LT-HSCs and subsequent progenitors through CLPs, together with our obser- vations that E18.5 thymocytes and splenic B cells are reduced in Carm12/2 embryos (Fig. 1, Supplemental Fig. 2), suggest that CARM1 is required for early stages of hematopoiesis and con- tinued lymphoid differentiation. Downloaded from

Cellularity of hematopoietic progenitors is not decreased in Carm12/2 FL Although hematopoiesis is shifting to the BM by E18.5, hema- topoietic progenitors are still present in the FL (28). Therefore, we analyzed the hematopoietic progenitor compartments from E18.5 http://www.jimmunol.org/ FL in CARM1-deficient mice and littermate controls. In contrast with E18.5 BM, the number of FL cells in Carm12/2 mice is comparable with controls (data not shown). Interestingly, the number of LT-HSCs was slightly increased in Carm12/2 FL, resulting in an increase in overall KLS cells (Fig. 5). This indi- FIGURE 5. CARM1 is not necessary for maintenance of hematopoietic A 2 cates that CARM1 is not required to maintain HSC cellularity progenitors in FL. ( ) Representative flow cytometric analysis of Lin FL cells from E18.5 Carm12/2 mice and littermate controls. Top row is du- in the FL. All other hematopoietic progenitors, from ST-HSCs plicated from Fig. 4 to show the gating used to identify hematopoietic through lymphoid committed CLPs and myeloid committed progenitor subsets. Arrows indicate gating strategy. Hematopoietic subsets by guest on September 28, 2021 GMPs, as well as MEPs, were present in similar numbers to are specified. (B) Cellularity of FL progenitors from E18.5 Carm12/2 controls (Fig. 5). Although the lack of CARM1 did not reduce embryos and littermate controls. Each point represents an individual em- cellularity of LT-HSCs and subsequent hematopoietic progenitors bryo. Mean values are indicated by the horizontal bars. Results are com- (Fig. 5), we observed a striking decrease in hematopoiesis in the bined from five independent experiments using the same animals as were fetal BM, thymus, and spleen at the same time point. Therefore, used in Fig. 4. *p , 0.05, **p , 0.01. we questioned whether the hematopoietic potential of Carm12/2 FL progenitors was impaired. matopoiesis of all lineages. Given that the recipient mice were CARM1 sufficient, these data also demonstrate that CARM1 is Carm12/2 FL cells have impaired hematopoietic potential required cell autonomously in hematopoietic progenitors. in vivo 2/2 To assess the functional potential of E18.5 FL progenitors, we Carm1 FL cells are capable of responding to Notch-driven performed a competitive reconstitution experiment. We first crossed T cell differentiation signals, but display reduced viability Carm12/2 mice with an actin-driven EGFP transgenic line (25). in vitro Similar to E18.5 FL, we did not observe a decrease in cellularity or The impaired ability of Carm12/2 FL cells to contribute to he- in hematopoietic progenitor subsets in E14.5 FL (data not shown). matopoietic lineages, including T cells, suggests that they are Equal numbers of E14.5 FL cells from GFP+ Carm12/2 embryos compromised either in their differentiation potential or in their or GFP+ littermate controls were mixed with GFP2 wild-type FL response to survival cues. Although there were insufficient num- cells from E14.5 C57BL/6 embryos. This mixture was injected into bers of Carm12/2 fetal BM progenitors to compare the frequency sublethally irradiated RAG22/2 gc2/2 mice. A schematic of the of apoptotic cells relative to controls, we observed a slight in- experiment is shown in Fig. 6A. Recipient thymi and BM were crease in apoptosis in ex vivo Carm12/2 FL KLS progenitors analyzed for donor chimerism in all hematopoietic subsets 8–12 (data not shown). Therefore, to functionally assess the ability of wk after transfer. Carm12/2 FL cells were dramatically impaired Carm12/2 FL progenitors to respond to differentiation and sur- in their ability to give rise to all thymocyte subsets, from DN1 vival cues, we used the OP9-DL1 coculture system, which robustly through CD4SP and CD8SP, when in competition with control FL promotes T cell differentiation from hematopoietic progenitors, cells (Fig. 6B). Furthermore, in this competitive setting, Carm12/2 largely through activation of the Notch1 signaling pathway (30, 31). FL cells failed to contribute efficiently in establishing hemato- Equal numbers of KLS progenitors from control or Carm12/2 E14.5 poietic progenitor chimerism in the BM, with the exception of FL were sorted into triplicate wells containing monolayers of OP9- MEP (Fig. 6C). Thus, despite the fact that hematopoietic progen- DL1 stroma. After 6 d of culture in the presence of IL-7 and Flt3L, itors were present at near-normal numbers in E18.5 Carm12/2 FL, the cells were harvested and analyzed by flow cytometry to deter- they were severely impaired in their ability to contribute to he- mine their ability to commit to the T cell lineage. Both control and 602 CARM1 REGULATES FETAL HEMATOPOIESIS Downloaded from http://www.jimmunol.org/

FIGURE 6. Competitive reconstitution reveals a cell-autonomous re- quirement for CARM1 in hematopoiesis. (A) Schematic of competitive 2 reconstitution assay. Equal numbers of EGFP C57Bl6/J FL cells were by guest on September 28, 2021 mixed with EGFP+ FL cells from Carm12/2 or control E14.5 embryos. The cell mixture was injected i.v. into sublethally irradiated (4.5 Gy) RAG- 22/2 gc2/2 mice. Eight to 12 wk postinjection, recipient thymocytes and BM cells were analyzed for the relative EGFP+ versus EGFP2 chimerism. (B) The ratio of EGFP+ to EGFP2 chimerism within each thymocyte subset in competitive reconstitution recipients was determined by flow cytometric analysis. Results are from four independent experiments with two or more animals per experiment. (C) The ratio of EGFP+ to EGFP2 chimerism within each BM progenitor subset in competitive reconstitution FIGURE 7. CARM1 deficiency impairs survival of E14.5 FL progenitors recipients was determined by flow cytometric analysis. Results are from during in vitro T cell differentiation. Five hundred sorted KLS from E14.5 three independent experiments with two or more animals per experiment. Carm12/2 or control FL were cultured on OP9-DL1 stroma in the presence 6 , , Error bars show SEM. *p 0.05, **p 0.01. of 5 ng/ml IL-7 and Flt3L for 6 d. (A) Cellularity of DN1, DN2, DN3, and DN4 subsets was determined by flow cytometry. Each point represents an Carm12/2 progenitors were capable of T cell commitment, as average of three replicate wells plated from a single embryo. Mean values are indicated by horizontal bars. Data are combined from six independent evidenced by differentiation to the DN3 developmental stage. experiments. (B) Percentage of cycling cells was determined using flow However, we found a significant reduction in the number of DN1, cytometric analysis of DNA content in recovered cells. Each point repre- DN2, DN3, and DN4 cells recovered from wells plated with sents an average of three replicate wells plated from a single embryo. (C) 2/2 Carm1 progenitors compared with controls (Fig. 7A). Neither Percentage of Annexin V+ cells was determined by flow cytometric analysis 2/2 control nor Carm1 cells progressed to the DP stage in this of recovered cells. Each point represents an average of three replicate wells timeframe. Although we consistently observed a block at the DN1 plated from a single embryo. **p , 0.01, ***p , 0.001. (D) Representative to DN2 transition in vivo (Fig. 1) (23, 24), this was not observed in flow cytometric histograms of IL-7R expression on E18.5 FL and thymo- + the in vitro OP9-DL1 coculture system. Instead, there was a con- cyte progenitors. IL-7R expression is comparable on FL Flk2 MPP, CLP, 2/2 sistent decrease of ∼4-fold for all subsets derived from Carm12/2 and on thymocyte DN3 subsets in Carm1 versus control embryos. IL-7R expression is reduced on the Carm12/2 CD44+CD252 thymocyte progen- relative to control progenitors. To clarify the basis for the reduction + 2 2 itor population, including the c-Kit DN1 subset. FL data are representative in cellularity, we assessed whether Carm1 / progenitors were of three separate experiments, including a total of six control and five defective in survival or proliferation in culture. We analyzed the Carm12/2 embryos. Thymocyte data are representative of four separate frequency of cycling cells in the OP9-DL1 cultures, using Vybrant experiments, including a total of five control and six Carm12/2 embryos. Dye Cycle to assess DNA content. We did not find a significant difference in the percentage of cycling cells between Carm12/2 Carm12/2 progenitors (Fig. 7C). Because IL-7 is a critical survival and control cells (Fig. 7B). However, there was a significant in- factor during T cell differentiation, we considered the possibility crease in the frequency of apoptotic cells in wells seeded with that reduced IL-7R expression could account for the survival defect The Journal of Immunology 603 of Carm12/2 progenitors. IL-7R expression was not reduced in Reduced functionality of fetal hematopoietic progenitors could E18.5 Carm12/2 CLPs in the FL. However, IL-7R expression was be sufficient to explain the reduction in thymic cellularity; how- diminished on the entire CD44+CD252 thymocyte subset, includ- ever, our data suggest that CARM1 plays an additional role in reg- ing the c-Kit+ DN1 progenitors in Carm12/2 compared with wild- ulating thymocyte differentiation and/or survival. At E18.5, DN1 type controls (Fig. 7D). After T lineage commitment and pro- thymocyte progenitors are not significantly reduced in Carm12/2 gression to the DN3 stage, there was little or no difference in IL-7R embryos. However, DN2 and subsequent stages of thymocyte dif- expression. Taken together, these data suggest that CARM1 is re- ferentiation are severely impaired (Fig. 1). These data indicate that quired for survival of hematopoietic progenitors, particularly at the CARM1 is required for efficient transition between DN1 and DN2 earliest stages of T cell differentiation. stages in vivo, in keeping with our previous report (24). Interest- ingly, whereas CARM1 deficiency results in a marked deficit in Discussion E18.5 BM progenitors with the potential to seed the thymus (note Protein arginine methylation is a posttranslational modification the reduction in Flk2+CD27+ progenitors in Fig. 4) (29), there is involved in various cellular functions, including signal transduc- a not a significant reduction in DN1 numbers. Thus, DN1 niches tion, subcellular protein localization, transcriptional regulation, may be extremely limiting, so that the reduced number of CLPs in protein–protein interactions, and DNA repair (6). We previously the BM would still provide an adequate number of thymic seeding reported that the absence of CARM1 results in impaired fetal cells to saturate this niche. In addition, homeostatic mechanisms thymopoiesis (24). In this study, we demonstrate that this defect is may be in place to maintain DN1 cellularity. Absence of such due not only to a requirement for CARM1 in T cell development, homeostatic factors in the OP9-DL1 culture system could ac- 2 2 but also to a much earlier requirement for CARM1 in oligopotent count for the reduced DN1 cellularity in the Carm1 / versus Downloaded from fetal hematopoietic progenitors. Although the number of LT-HSCs control cultures (Fig. 7A). Furthermore, increased apoptosis of and downstream KLS subsets was not reduced in Carm12/2 E14.5 Carm12/2 progenitors in vitro supports a role for CARM1 in or E18.5 FL, competitive reconstitution assays revealed a deficit in maintaining survival during T cell differentiation (Fig. 7C). Be- the ability of Carm12/2 FL cells to contribute to hematopoiesis. cause IL-7 is known to be such a potent survival cue during T cell CARM1 is required cell autonomously in hematopoietic cells, as differentiation, the reduction in IL-7R expression on ex vivo 2/2 2/2 revealed by impaired differentiation of Carm1 FL cells in Carm1 DN1 thymocytes could account for the impaired sur- http://www.jimmunol.org/ a CARM1-sufficient host, as well as by unimpeded differentiation vival and reduced cellularity in conditions promoting T cell dif- of wild-type thymocyte progenitors in a Carm12/2 thymic stromal ferentiation (Fig. 7D). We note that CARM1 is not required for IL- microenvironment. 7R expression in all hematopoietic progenitors because IL-7R Our previous observation of reduced thymic cellularity in E18.5 expression is not impaired in FL CLP or lineage committed DN3 Carm12/2 mice (24), together with our current finding of fewer thymocytes. In contrast with its influence on survival, CARM1 is thymocyte progenitors in thymic rudiments as early as E12.5 (Fig. not essential for Notch1-driven T cell commitment, as evidenced 3), suggested that there might be a block in prethymic hemato- by the ability of surviving DN1 progenitors to progress through poiesis. During fetal development, hematopoiesis transitions from subsequent DN2 and DN3 maturation stages (Fig. 7B). Taken to- the liver to the BM, such that at E18.5, hematopoiesis could occur gether, our findings indicate that CARM1 influences hematopoiesis by guest on September 28, 2021 in both compartments (28, 32, and this study). Therefore, we both in BM and FL progenitors, as well as in thymocyte progen- analyzed the frequency and number of hematopoietic progenitors itors, consistent with a role for CARM1 in differentiation or sur- in both the E18.5 FL and BM. Interestingly, although a severe vival, or both, of multiple cell types (6). reduction in all c-Kit+ progenitors was observed in the BM, the FL As a member of the PRMT family of arginine methyltrans- did not recapitulate this phenotype. Indeed, there was a small, but ferases, CARM1 contributes to epigenetic regulation of differen- significant, increase in LT-HSCs in the E18.5 Carm12/2 FL. In tiation in many cell types (6). The enzymatic activity of CARM1 addition, at E14.5, when the FL is the major site of hematopoiesis, is required for thymocyte development, as well as for embryonic lack of CARM1 did not result in altered FL progenitor numbers or survival, adipocyte differentiation, and transcriptional coactivator frequencies (data not shown). However, the competitive FL re- activity (23). Thus, arginine methylation is a critical epigenetic constitution assays did reveal a functional defect in these E14.5 modification that contributes to proper differentiation of hemato- FL progenitors (Fig. 6). There are at least three possible explan- poietic lineages. Indeed, epigenetic regulation of HSC self- ations that could reconcile normal numbers of FL hematopoietic renewal and differentiation has been demonstrated by dynamic progenitors with their impaired function and the reduction in BM changes in the methylation status of both DNA and histones progenitor cellularity in Carm12/2embryos. First, CARM1 defi- during hematopoietic lineage progression (35–37). In this study, ciency could impair the ability of hematopoietic progenitors to we demonstrate a novel function for the epigenetic modifier emigrate from the FL, thus resulting in an accumulation of these CARM1 in the regulation of fetal hematopoiesis in BM and FL, as progenitors in the FL and a reduction in the BM. However, this well as in thymopoiesis. Given this impact of CARM1 on early possibility is unlikely because on FL transplantation, a functional hematopoiesis and thymocyte development, CARM1-mediated defect in these progenitors is revealed despite their manual release epigenetic regulation may contribute to lymphoid and myeloid from the FL. Although this does not rule out a possible emigration leukemogenesis. In this light, small molecule inhibitors of CARM1 defect, this mechanism is not sufficient to account for functional may have therapeutic potential. The feasibility of this approach defects in hematopoiesis. Second, the ability of FL hematopoietic is suggested by the finding that treatment of Th cells with broad- progenitors to home to the BM could be impaired. Chemo- spectrum PRMT small molecule inhibitors partially blocks attractants and are known to regulate cellular trafficking production (38). In conclusion, CARM1 is required at multiple and localization of hematopoietic cells, including LT-HSCs. stages of hematopoietic differentiation, identifying it as a key epi- CARM1 could control expression of these molecules, impacting genetic regulator of a cellular differentiation process that occurs the ability of Carm12/2 FL HSCs to migrate to the fetal BM (33). throughout life. Future investigations will further elucidate the role Finally, CARM1 deficiency could alter the ability of HSCs in the of CARM1 at specific stages of hematopoiesis and determine fetal BM to respond to molecular cues in the HSC BM niche, the potential for therapeutic modulation of CARM1, which could be which regulate self-renewal, survival, and/or differentiation (34). beneficial for hematopoietic disorders. 604 CARM1 REGULATES FETAL HEMATOPOIESIS

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