PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Immunol. Author manuscript; available in PMC 2014 January 15.
Published in final edited form as:
PMCID: PMC3538901
NIHMSID: NIHMS420743

CARM1 regulates fetal hematopoiesis and thymocyte development

Abstract

CARM13 (coactivator-associated arginine methyltransferase 1) is a protein arginine methyltransferase that methylates histones and transcriptional regulators. We previously reported that the absence of CARM1 partially blocks thymocyte differentiation at embryonic day 18.5 (E18.5). Here we find that reduced thymopoiesis in Carm1−/− 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 and bone marrow cells. Despite markedly reduced cellularity of hematopoietic progenitors in E18.5 bone marrow, the number of long-term hematopoietic stem cells and downstream subsets was not reduced in Carm1−/− E14.5 or E18.5 fetal liver. Nevertheless, competitive reconstitution assays revealed a deficit in the ability of Carm1−/− fetal liver cells to contribute to hematopoiesis. Furthermore, impaired differentiation of Carm1−/− fetal liver cells in a CARM1 sufficient host showed that CARM1 is required cell-autonomously in hematopoietic cells. Co-culture of Carm1−/− fetal liver 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.

Introduction

Hematopoietic stem cells (HSC) can self renew throughout life and differentiate into all myeloid and lymphoid lineages (1). Epigenetic modifications are a driving force of this cellular differentiation. Because the genetic information of cells pre- and post-differentiation is identical, epigenetic marks like DNA methylation and histone methylation are critical for facilitating the lock-in of a differentiated state (2). Epigenetic mechanisms function like a ratchet, allowing lineage-specific differentiation, but generally not de-differentiation. There is an element of plasticity to cellular differentiation, which can be forcibly reversed by small molecule epigenetic regulators and/or the overexpression of a few specific genes to generate an induced pluripotent stem (iPS) cell state (3). Perhaps one of the best biological systems to study epigenetic changes that correlate with differentiation is hematopoietic cell development. Indeed, recently genome-wide DNA methylation patterns were analyzed at each major stage of hematopoiesis, revealing clear epigenetic signatures for each cell lineage (4). Like DNA methylation, there are a number of reports that arginine methylation also plays a critical role in lymphocyte development and signal transduction (5).

Arginine methylation is a common posttranslational modification that subtly alters the function of its substrates (6). It does this in a number ways: 1) Arginine methylation provides a docking site for Tudor domain-containing effector molecules (710); 2) It can also block protein-protein interactions, as in the case of certain SH3 domain-driven interactions (11); 3) Arginine methylation can negatively regulate AKT-mediated phosphorylation, because the AKT consensus motif contains key arginine residues (12, 13); and similarly 4) Lysine methylation can also be blocked by adjacent arginine methylation events (14, 15). The substrates for arginine methyltransferases (PRMTs) are both nuclear and cytoplasmic, and in the nucleus, histones are a major target of these enzymes. Histone methylation allows the PRMTs to feed into the epigenetic code and contribute to key molecular switches that dictate cell fate.

The mammalian PRMT family of enzymes consists of nine members – PRMT1–9, the majority of these enzymes target the N-terminal tails for histones H3, H4 and H2A for methylation (16). CARM1/PRMT4 was the first family member to be identified as a transcriptional coactivator, which methylates the H3R17 and H3R26, sites as well as other transcriptional regulators (6,17). CARM1-null embryos display no overt developmental defects, although they are smaller than their wild-type counterparts, and once born, the nulls die without taking their first breath (18). In-depth analysis of these CARM1-null embryos has revealed a number of clear phenotypes, many of them associated with cell differentiation defects. CARM1-null lethality at birth is likely due to the fact that lungs from mice lacking CARM1 are inundated with immature alveolar type II cells, which do not develop into more mature alveolar type I cells, thus CARM1 is required for the proper differentiation of alveolar cells (19). In addition, CARM1-null embryos lack brown fat, and cells that do not express CARM1 are not able to differentiate into mature adipocytes (20). CARM1 is also required for chondrogenesis (21) and skeletal muscle development (22). Finally, we have also observed that functional CARM1 is required for normal T cell cellularity and differentiation (23, 24). Thus, there is genetic evidence that CARM1 activity impacts the differentiation of lung, fat, muscle, cartilage and thymocytes.

We previously reported that CARM1 null embryos have a five-fold reduction in thymocyte cellularity and a partial block in early T cell development (24). We now confirm a role for CARM1 in thymocyte development at the transition between the DN1 and DN2 stages. We investigated the cellular basis for impaired thymopoiesis in this mouse model. Here we report that CARM1 functions cell intrinsically to regulate hematopoietic progenitor cell activity and cellularity in the fetal liver and bone marrow, respectively. Furthermore, when fetal liver progenitors were co-cultured on OP9-DL1 stroma, CARM1 was dispensable for T lineage differentiation in response to Notch ligands, but was essential for survival. Taken together, these results demonstrate that CARM1 regulates fetal hematopoiesis and thymocyte development, bolstering the notion that epigenetic regulation is critical for proper differentiation and survival of multiple hematopoietic lineages.

Materials and Methods

Mice

Carm1−/− embryos were described previously (18) and generated by crossing Carm1−/+ breeders. Timed pregnancies were established, and the day of vaginal plug was designated E0.5. Embryos were genotyped using the primers 5’-CCCACTTCTGTTACCTCCTTTG-3’ and 5’-TAACTAAAAGAAAATGGAATGG-3’. Mice transgenic for EFGP under control of the b-actin promoter were kindly provided by Dr. Irving Weissman (25). C57BL/6J and RAG2−/−γc−/− mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Athymic NCrnu/nu mice were obtained from NCI (Frederick, MD) Mice were maintained in the Vivarium at the M.D. Anderson Cancer Center Science Park in accordance with guidelines established by the Association for the Accreditation of Laboratory Animal Care. All protocols using these mice were reviewed and accepted by the M.D. Anderson Animal Care and Use Committee.

Immunofluorescence microscopy

Serial sections (5 µm) from OCT-embedded frozen tissue were air dried and fixed in cold acetone for 5 min at room temperature. After washing in TNT (1M Tris pH7.5, 15M NaCl, 0.05% Tween 20), sections were blocked for 15 min in TNB (0.1M Tris-HCL pH7.5, 0.15NaCl, 0.5% blocking reagent from TSA Biotin System, NEN Life Science Products, Boston, MA). The slides were incubated at room temperature with primary antibodies including rat anti-mouse CD45 (clone 30-F11, BD), polyclonal rabbit anti-Cytokeratin (Dako), rat anti-mouse CD25 (clone PC61, BD), rabbit anti-mouse K5 (Covance) and rat anti-mouse cKit (clone ACK45, BD) for 1 hour. Secondary reagents included streptavidin-FITC (Vector Laboratories), Texas Red conjugated donkey anti-rabbit IgG and Texas Red conjugated donkey anti-mouse IgG (Jackson Immunoresearch). In some cases, tyramide amplification was performed.

Flow cytometry

Single cell suspensions were prepared from E18.5 thymi, spleen and fetal liver by dissociation of tissues through a 70-µm strainer (Fisher). Fetal bone marrow cells 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 dissociated cells and fragments were filtered through a 70-µm strainer (Fisher). Bone marrow and spleen cells were treated with RBC lysis buffer (17mM Tris, 160mM NH4Cl, pH7.3) to remove red blood cells then resuspended in FACS buffer (PBS pH7.2, 0.005M EDTA, 2% FBS). Cells were stained with fluorochrome-conjugated antibodies in FACS buffer for 30 min on ice and washed with FACS buffer. Propidium iodide (Invitrogen) was added (0.5 µg/ml) to the samples immediately before data acquisition for dead cell exclusion. Anti-B220 (clone RA3-6B2) and anti-CD45 (clone 30-F11) conjugated to Pacific Blue, anti-CD27 (clone LG.3A10) conjugated to PerCP-Cy5.5, anti-CD44 (IM7) and anti-CD135 (clone A2F10) conjugated to APC, anti-c-kit (clone 2B8) conjugated to APC-Cy7, anti-FcγR (clone 93) conjugated to PE were purchased from Biolegend. Antibodies to CD8a (clone 53-6.7) conjugated to FITC or PE-Cy7, γδTCR (clone eBioGL3) conjugated to PE, CD3e (145–2C11), and CD127 (clone A7R34) biotinylated, CD4 (GK1.5) and TER-119 conjugated to APC, c-Kit (clone 2B8) conjugated to APC-eFluor® 780, CD45 (clone 30-F11) conjugated to eFluor® 450, CD25 (PC61.5) conjugated to Alexa Fluor® 488, CD34 (RAM34) conjugated to Alexa Fluor® 700 were purchased from eBioscience. A lineage cocktail containing antibodies to CD3e (145–2C11) CD4 (GK1.5), CD8a (clone 53-6.7) CD11c (clone N418), CD11b (clone M1/70), B220 (clone RA3-6B2), TER-119, Gr-1 were conjugated to PE-Cy5 was purchased from eBioscience. Anti-CD4 conjugated to Pacific Orange was purchased from Invitrogen. Biotinylated Abs were detected using streptavidin conjugated to Qdot 655 or APC (Invitrogen). Cells were analyzed on a FACS Aria II (BD Science) and data was analyzed using FlowJo software (Tree Star). To exclude lineage positive cells in thymocyte analyses, the lineage cocktail included antibodies to B220, Ter119, Gr1, Mac-1, NK1.1 and CD11c. To exclude lineage positive cells in bone marrow and fetal liver analyses, the lineage cocktail included antibodies to CD4, CD8, B220, Ter119, Gr-1, Mac-1, NK1.1, CD11c.

Kidney capsule transplantation

Fetal thymic lobes from E15.5 Carm1−/− or littermate controls were placed on Millicell 30mm round 0.4µm culture plate inserts (Millipore) such that they were floating at the liquid/air interface in RPMI 1640 medium (Invitrogen) containing 10% Fetal Bovine Serum (Atlanta Biologicals), 1% Penicillin-Streptomycin (Invitrogen), 2mM L-Glutamine (Invitrogen), 1mM Sodium Pyruvate (Invitrogen) and 1.35 mM 2′-deoxyguanosine for 5 days. The thymocyte-depleted lobes were transplanted under the kidney capsule of athymic recipient NCrnu/nu mice.

Competitive repopulation assay

2.5×106 fetal liver cells from Carm1−/− EGFP+ E14.5 embryos or controls (Carm1+/− EGFP+ or Carm1+/+ littermates) were mixed 1:1 with competitor EGFP− fetal liver cells from wildtype C57Bl6/J E18.5 embryos and transplanted into sublethally irradiated (4.5 Gy) Rag2−/−γc−/− mice by retroorbital injection. The recipient mice were analyzed 8 or 16 wk after BM transplantation.

OP9-DL1 co-cultures, proliferation and apoptosis assays

500 FACS purified KLS fetal liver progenitors obtained from individual E14.5 Carm1−/− or control littermates were directly sorted into triplicate wells of 24-well plates containing a monolayer of OP9-DL1 cells in α-MEM + 10% FCS with 5 ng/ml IL-7 and 5ng/ml Flt3L (Peprotech). After 6 days in culture (37°C, 5% CO2), hematopoietic cells were recovered and analyzed by flow cytometry for T cell differentiation markers. For DNA content analysis, live cells were stained with Vybrant Dye Cycle Violet (Life Sciences), according to manufacturer’s instructions. FlowJo software (Tree Star) was used to analyze the frequency of proliferating cells (S + G2/M), as determined by the Dean-Jett-Fox algorithm. Apoptosis was assessed by staining with Pacific Blue-conjugated Annexin V (Biolegend) according to manufacturer’s instructions, in conjunction with PI to detect dead cells. Forward and side scatter gates were set to tightly enclose live lymphocytes, thus excluding debris and small dead cells from our analyses.

Statistical analysis

Prism 5.0 (GraphPad version 5.0c San Diego, CA) and Microsoft Excel were used for all statistical analyses. The Kolmogorov–Smirnov test was first used to assess normality of the data in each experiment. To analyze the mean cell number in different hematopoietic subsets, a student t test was performed on data distributed normally, whereas the two-tailed Mann-Whitney U test was on for data that were not distributed normally. For comparison of the ratio of GFP+:GFP− cells, the one-tailed Mann-Whitney U test was used. A p value <0.05 was considered statistically significant.

Results

Carm1 deficiency results in a block at the DN1-DN2 transition in thymopoiesis

We previously observed that CARM1 deficiency results in a reduction in thymic cellularity, associated with an accumulation of CD44+CD25CD4CD8 thymocytes at E18.5 (24). This population is heterogeneous, containing both T and non-T lineage progenitors. c-Kit is expressed on the most immature T cell progenitors, DN1 cells, within this population (DN1: c-Kit+ CD44+CD25CD4CD8) (26, 27). DN1 thymocytes give rise to DN2 progenitors (c-Kit+CD44+CD25+CD4CD8) that subsequently down-regulate CD25 to become DN3 cells (c-KitCD44CD25+CD4CD8) that are committed to the T cell lineage. To further characterize the block in thymocyte development observed in Carm1−/−embryos (24), we analyzed c-Kit expression within the CD44+CD25CD4CD8 compartment to distinguish the earliest T cell progenitor population. Consistent with our previous report, there was an increased frequency of CD44+CD25 thymocytes in the CD4CD8 compartment in Carm1−/− E18.5 embryos (Fig. 1 A). Furthermore, analysis of c-Kit expression revealed an increase in the frequency of DN1 versus DN2 progenitors in Carm1−/− E18.5 embryos, consistent with a block in differentiation of the earliest thymocyte subset.

Figure 1
CARM1 deficiency results in an early thymopoiesis block in E18.5 embryos

The absolute number of DN1 thymocytes was not significantly different in Carm1−/− E18.5 versus control embryos. However, the DN2 subset was significantly reduced, consistent with a block in the DN1-DN2 transition (Fig. 1 B). CARM1 deficiency resulted in a greater than 90% reduction in the number of DN2, DN3 and DN4 (c-KitCD4CD8CD44CD25) thymocytes. DN4 cells are the immediate precursors of DP thymocytes (CD4+CD8+), which then give rise to mature CD4SP (CD4+CD8) and CD8SP (CD4CD8+) subsets. These latter subsets were reduced by ~75% in the absence of CARM1 (Fig. 1 B). We also observed a significant reduction of 85% in γδ T cells, as might be expected from a reduction in their progenitors, DN2 cells (Fig. 1 B). Together these data indicate that CARM1 is required for continued maturation of thymocytes beyond the DN1 stage.

Reduced thymopoiesis in Carm1−/− mice is not due to thymic stromal defect

Thymopoiesis requires input from stromal cells in the thymic microenvironment. Thus, the block in thymocyte development observed in Carm1−/− embryos could be an indirect consequence of a defect in the thymic stromal compartment. To determine whether Carm1 is required for proper thymic stromal function, we transplanted E15.5 2-deoxyguanosine treated Carm1−/− versus control fetal thymic lobes under the kidney capsule of athymic nude recipients. 8–12 weeks after transplantation, thymic grafts were recovered and analyzed by flow cytometry. In contrast to the developmental block observed in Carm1−/− E18.5 thymi (Fig. 1), thymopoiesis was not impaired when CARM1 deficiency was restricted to thymic stromal cells (Fig. 2). The cellularity was comparable for all thymocyte subsets regardless of whether they developed in a control or Carm1−/− stromal environment. Engrafted CARM1 deficient lobes were smaller than controls at the outset of the transplantation experiment; this initial difference in thymic size could account for the slight decreases in thymocyte numbers observed in transplanted Carm1−/− lobes (Fig. 2). In addition, comparable numbers of CD4SP and CD8SP T cells were recovered from the spleens of athymic recipients transplanted with Carm1−/− or control fetal thymi (Fig. S1). Altogether, the normal differentiation of thymocyte progenitors in a CARM1 deficient microenvironment, in contrast to the severely reduced cellularity in Carm1−/− E18.5 thymi, indicates that loss of CARM1 predominantly affects thymocyte progenitors as opposed to the thymic stromal microenvironment.

Figure 2
CARM1 deficiency in the thymic stromal compartment does not impair thymocyte development

CARM1 deficiency affects thymocyte cellularity in the E12.5 thymic rudiment

Given the defect in thymic cellularity at E18.5, we examined cryosections from E12.5 – E18.5 from Carm1−/− embryos to determine if the reduction in thymocyte cellularity was apparent earlier in ontogeny. At E12.5 there was a striking paucity in the number of CD45+ hematopoietic progenitors in the Carm1−/− thymic rudiment (Fig. 3 A). This deficiency was also apparent E13.5 – E 17.5 in sections stained for c-Kit and CD25, markers of DN1-DN3 thymocytes (Fig. 3 B–C). By E13.5, Carm1−/− thymic lobes were markedly smaller than wildtype and remain hypoplastic, containing fewer thymocyte progenitors throughout embryogenesis (Figure 1 B–C). Thus, CARM1 plays a significant role in thymocyte development prior to and after thymic vascularization, which occurs at ~E14.5.

Figure 3
Carm1−/− fetal thymi contain fewer thymocyte progenitors than Carm1+/+ littermates

Early hematopoietic defect in Carm1−/− bone marrow

Given the profound reduction in DN subsets at E18.5 (Fig. 1), along with the decrease in thymocyte progenitors at early stages of thymic organogenesis (Fig. 3), we hypothesized that the absence of Carm1 might affect prethymic hematopoietic progenitors. Since hematopoietic progenitors are present in the fetal bone marrow by E18.5 (28), we compared E18.5 bone marrow from Carm1−/− and control littermates for hematopoietic progenitor subset composition. Cellularity was reduced in Carm1−/− bone marrow compared with littermate controls (data not shown). Interestingly, the proportion and number of c-Kit+ cells within the lineagelo fraction was greatly reduced in CARM1 deficient embryos (Fig. 4 A, far left panels, and Fig. 4 B). The Lineageloc-Kit+Sca-1+ (KLS) progenitors, which contain the most undifferentiated hematopoietic progenitors, are reduced in both frequency and absolute number in the E18.5 Carm1−/− bone marrow (Fig. 4). The KLS population consists of several progenitor subsets: long-term hematopoietic stem cells (LT-HSC: Slamf1+Flk2KLS), short-term hematopoietic stem cells (ST-HSC: Slamf1Flk2KLS), and lymphoid-biased multipotent progenitors (Flk2+MPP: Flk2+Slamf1KLS). All three of these early hematopoietic progenitors are significantly reduced in the E18.5 Carm1−/− bone marrow. Flk2+MPP are reduced by 95%, while LT-HSC and ST-HSC were reduced by approximately 80% (Fig. 4).

Figure 4
CARM1 deficiency results in reduced cellularity of hematopoietic progenitors in E18.5 bone marrow

In addition to the decrease in oligopotent hematopoietic progenitors in E18.5 Carm1−/− bone marrow, downstream lineage-restricted progenitors were also diminished. The lineageloCD27+Flk2+ bone marrow subset contains all progenitors with thymocyte differentiation potential (29). Interestingly, this population was severely reduced by 93% in Carm1−/− bone marrow. The CD27+Flk2+ compartment contains 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 progenitors (MP, cKit+ Lineagelo Sca1) in the absence of CARM1 (Fig. 4). These progenitors can be subdivided into common myeloid progenitors (CMP: LineageloCD34+FcgRMP), further downstream granulocyte-macrophage progenitors (GMP: LinloCD34+FcgR+MP), and megakaryocyte/erythroid progenitors (MEP: LinloCD34FcgRMP). CMP and MEP were significantly reduced by 92% and 66%, respectively, whereas neither bone marrow GMP nor splenic granulocytes were significantly diminished (Fig. 4 and Fig. S2). The reduction in LT-HSC and subsequent progenitors through CLP, along with our observations that E18.5 thymocytes and splenic B cells are reduced in Carm1−/− embryos (Fig. 1 and Fig. S2), suggest that CARM1 is required for early stages of hematopoiesis and continued lymphoid differentiation.

Cellularity of hematopoietic progenitors is not decreased in Carm1−/− fetal liver

Although hematopoiesis is shifting to the bone marrow by E18.5, hematopoietic progenitors are still present in the fetal liver (28). Therefore, we analyzed the hematopoietic progenitor compartments from E18.5 fetal liver in CARM1 deficient mice and littermate controls. In contrast to E18.5 bone marrow, the number of fetal liver cells in Carm1−/− mice is comparable to controls (data not shown). Interestingly, the number of LT-HSCs was slightly increased in Carm1−/− fetal liver, resulting in an increase in overall KLS cells (Fig. 5). This indicates that CARM1 is not required to maintain HSC cellularity in the fetal liver. All other hematopoietic progenitors, from ST-HSC through lymphoid committed CLP and myeloid committed GMP, as well as MEP were present in similar numbers to controls (Fig. 5). While the lack of CARM1 did not reduce cellularity of LT-HSC and subsequent hematopoietic progenitors (Figure 5), we observed a striking decrease in hematopoiesis in the fetal bone marrow, thymus, and spleen at the same time point. Therefore, we questioned whether the hematopoietic potential of Carm1−/− fetal liver progenitors was impaired.

Figure 5
CARM1 is not necessary for maintenance of hematopoietic progenitors in fetal liver

Carm1−/− fetal liver cells have impaired hematopoietic potential in vivo

To assess the functional potential of E18.5 fetal liver progenitors, we performed a competitive reconstitution experiment. We first crossed Carm1−/− mice with an actin-driven EGFP transgenic line (25). Similar to E18.5 fetal liver, we did not observe a decrease in cellularity or in hematopoietic progenitor subsets in E14.5 fetal liver (data not shown). Equal numbers of E14.5 fetal liver cells from GFP+ Carm1−/− embryos or GFP+ littermate controls were mixed with GFP− wild-type fetal liver cells from E14.5 C57BL/6 embryos. This mixture was injected into sublethally irradiated RAG2−/− γc−/− mice. A schematic of the experiment is shown in Figure 6A. Recipient thymi and bone marrow were analyzed for donor chimerism in all hematopoietic subsets 8–12 weeks after transfer. Carm1−/− fetal liver cells were dramatically impaired in their ability to give rise to all thymocyte subsets, from DN1 through CD4SP and CD8SP, when in competition with control fetal liver cells (Fig. 6 B). Furthermore, in this competitive setting Carm1−/− fetal liver cells failed to contribute efficiently in establishing hematopoietic progenitor chimerism in the bone marrow, with the exception of MEP (Figure 6C). Thus, despite the fact that hematopoietic progenitors were present at near normal numbers in E18.5 Carm1−/− fetal liver, they were severely impaired in their ability to contribute to hematopoiesis of all lineages. Given that the recipient mice were CARM1 sufficient, these data also demonstrate that CARM1 is required cell-autonomously in hematopoietic progenitors.

Figure 6
Competitive reconstitution reveals a cell autonomous requirement for CARM1 in hematopoiesis

Carm1−/− fetal liver cells are capable of responding to Notch-driven T cell differentiation signals, but display reduced viability in vitro

The impaired ability of Carm1−/− fetal liver cells to contribute to hematopoietic lineages, including T cells, suggests that they are compromised either in their differentiation potential or in their response to survival cues. While there were insufficient numbers of Carm1−/− fetal bone marrow progenitors to compare the frequency of apoptotic cells relative to controls, we observed a slight increase in apoptosis in ex vivo Carm1−/− fetal liver KLS progenitors (data not shown). Therefore, to functionally assess the ability of Carm1−/− fetal liver progenitors to respond to differentiation and survival cues, we used the OP9-DL1 co-culture system, which robustly promotes T cell differentiation from hematopoietic progenitors, largely through activation of the Notch1 signaling pathway (30, 31). Equal numbers of KLS progenitors from control or Carm1−/− E14.5 fetal livers were sorted into triplicate wells containing monolayers of OP9-DL1 stroma. After six days of culture in the presence of IL-7 and Flt3L, the cells were harvested and analyzed by flow cytometry to determine their ability to commit to the T cell lineage. Both control and Carm1−/− progenitors were capable of T cell commitment, as evidenced by differentiation to the DN3 developmental stage. However, we found a significant reduction in the number of DN1, DN2, DN3 and DN4 cells recovered from wells plated with Carm1−/− progenitors compared to controls (Fig 7A). Neither control nor Carm1−/− cells progressed to the DP stage in this timeframe. While we consistently observed a block at the DN1 to DN2 transition in vivo (Fig 1 and 23,24), this was not observed in the in vitro OP9-DL1 co-culture system. Instead, there was a consistent decrease of approximately 4-fold for all subsets derived from Carm1−/− relative to control progenitors. To clarify the basis for the reduction in cellularity, we assessed whether Carm1−/− progenitors were defective in survival or proliferation in culture. We analyzed the frequency of cycling cells in the OP9-DL1 cultures, using Vybrant® Dye Cycle to assess DNA content. We did not find a significant difference in the percentage of cycling cells between Carm1−/− and control cells (Fig 7B). However, there was a significant increase in the frequency of apoptotic cells in wells seeded with Carm1−/− progenitors (Fig 7C). Because IL-7 is a critical survival factor during T cell differentiation, we considered the possibility that reduced IL-7 receptor expression could account for the survival defect of Carm1−/− progenitors. IL-7 receptor expression was not reduced in E18.5 Carm1−/− CLPs in the fetal liver. However, IL-7 receptor expression was diminished on the entire CD44+CD25 thymocyte subset, including the c-Kit+ DN1 progenitors in Carm1−/− compared to wildtype controls (Fig 7D). Following T lineage commitment and progression to the DN3 stage, there was little or no difference in IL-7 receptor expression. Taken together, these data suggest that CARM1 is required for survival of hematopoietic progenitors, particularly at the earliest stages of T-cell differentiation.

Figure 7
CARM1 deficiency impairs survival of E14.5 fetal liver progenitors during in vitro T cell differentiation

Discussion

Protein arginine methylation is a post-translational modification involved in various cellular functions, including signal transduction, subcellular protein localization, transcriptional regulation, protein–protein interactions and DNA repair (6). We previously reported that the absence of CARM1 results in impaired fetal thymopoiesis (24). Here we demonstrate that this defect is due not only to a requirement for CARM1 in T cell development, but also to a much earlier requirement for CARM1 in oligopotent fetal hematopoietic progenitors. Although the number of LT-HSCs and downstream KLS subsets was not reduced in Carm1−/− E14.5 or E18.5 fetal liver, competitive reconstitution assays revealed a deficit in the ability of Carm1−/− fetal liver cells to contribute to hematopoiesis. CARM1 is required cell-autonomously in hematopoietic cells, as revealed by impaired differentiation of Carm1−/− fetal liver cells in a CARM1 sufficient host, as well as by unimpeded differentiation of wild-type thymocyte progenitors in a Carm1−/− thymic stromal microenvironment.

Our previous observation of reduced thymic cellularity in E18.5 Carm1−/− mice (24), along with our current finding of fewer thymocyte progenitors in thymic rudiments as early as E12.5 (Fig. 3), suggested that there might be a block in pre-thymic hematopoiesis. During fetal development hematopoiesis transitions from the liver to the bone marrow, such that at E18.5, hematopoiesis could occur in both compartments ((28, 32) and current results). Therefore, we analyzed the frequency and number of hematopoietic progenitors in both the E18.5 fetal liver and bone marrow. Interestingly, while a severe reduction in all c-Kit+ progenitors was observed in the bone marrow, the fetal liver did not recapitulate this phenotype. Indeed, there was a small, but significant increase in LT-HSC in the E18.5 Carm1−/− fetal liver. In addition, at E14.5, when the fetal liver is the major site of hematopoiesis, lack of CARM1 did not result in altered fetal liver progenitor numbers or frequencies (data not shown). However, the competitive fetal liver reconstitution assays did reveal a functional defect in these E14.5 FL progenitors (Fig. 6). There are at least three possible explanations that could reconcile normal numbers of fetal liver hematopoietic progenitors with their impaired function and the reduction in bone marrow progenitor cellularity in Carm1−/−embryos. First, CARM1 deficiency could impair the ability of hematopoietic progenitors to emigrate from the fetal liver, thus resulting in an accumulation of these progenitors in the fetal liver and a reduction in the bone marrow. However, this possibility is unlikely because upon FL transplantation, a functional defect in these progenitors is revealed in spite of their manual release from the fetal liver. While this does not rule out a possible emigration defect, this mechanism is not sufficient to account for functional defects in hematopoiesis. Second, the ability of FL hematopoietic progenitors to home to the bone marrow could be impaired. Chemoattractants and integrins are known to regulate cellular trafficking and localization of hematopoietic cells, including LT-HSCs. CARM1 could control expression of these molecules, impacting the ability of Carm1−/− FL HSCs to migrate to the fetal bone marrow (33). Finally, CARM1 deficiency could alter the ability of HSCs in the fetal bone marrow to respond to molecular cues in the HSC bone marrow niche, which regulate self-renewal, survival, and/or differentiation (34).

Reduced functionality of fetal hematopoietic progenitors could be sufficient to explain the reduction in thymic cellularity; however, our data suggest that CARM1 plays an additional role in regulating thymocyte differentiation and/or survival. At E18.5, DN1 thymocyte progenitors are not significantly reduced in Carm1−/− embryos. However, DN2 and subsequent stages of thymocyte differentiation are severely impaired (Fig. 1). These data indicate that CARM1 is required for efficient transition between DN1 and DN2 stages in vivo, in keeping with our previous report (24). Interestingly, whereas CARM1 deficiency results in a marked deficit in E18.5 bone marrow progenitors with the potential to seed the thymus (note the reduction in Flk2+CD27+ progenitors in Figure 4) (29), there is a not a significant reduction in DN1 numbers. Thus, DN1 niches may be extremely limiting, so that the reduced number of CLP in the bone marrow would still provide an adequate number of thymic seeding cells to saturate this niche. Additionally, homeostatic mechanisms may be in place to maintain DN1 cellularity. Absence of such homeostatic factors in the OP9-DL1 culture system could account for the reduced DN1 cellularity in the Carm1−/− versus control cultures (Fig 7A). Furthermore, increased apoptosis of Carm1−/− progenitors in vitro supports a role for CARM1 in maintaining survival during T cell differentiation (Fig 7C). Because IL-7 is known to be such a potent survival cue during T cell differentiation, the reduction in IL-7R expression on ex vivo Carm1−/− DN1 thymocytes could account for the impaired survival and reduced cellularity in conditions promoting T cell differentiation (Fig 7D). We note that CARM1 is not required for IL-7 receptor expression in all hematopoietic progenitors because IL-7 receptor expression is not impaired in fetal liver CLP or lineage committed DN3 thymocytes. In contrast to its influence on survival, CARM1 is not essential for Notch1-driven T cell commitment, as evidenced by the ability of surviving DN1 progenitors to progress through subsequent DN2 and DN3 maturation stages (Fig 7B). Taken together, our findings indicate that CARM1 influences hematopoiesis both in bone marrow and FL progenitors as well as in thymocyte progenitors, consistent with a role for CARM1 in differentiation and/or survival of multiple cell types (6).

As a member of the PRMT family of arginine methyltransferases, CARM1 contributes to epigenetic regulation of differentiation in many cell types (6). The enzymatic activity of CARM1 is required for thymocyte development, as well as for embryonic survival, adipocyte differentiation and transcriptional co-activator activity (23). Thus, arginine methylation is a critical epigenetic modification that contributes to proper differentiation of hematopoietic lineages. Indeed, epigenetic regulation of HSC self-renewal and differentiation has been demonstrated by dynamic changes in the methylation status of both DNA and histones during hematopoietic lineage progression (3537). Here we demonstrate a novel function for the epigenetic modifier CARM1 in the regulation of fetal hematopoiesis in bone marrow and fetal liver, as well as in thymopoiesis. Given this impact of CARM1 on early hematopoiesis and thymocyte development, CARM1-mediated epigenetic regulation may contribute to lymphoid and myeloid leukemogenesis. In this light, small molecule inhibitors of CARM1 may have therapeutic potential. The feasibility of this approach is suggested by the finding that treatment of T helper cells with broad-spectrum PRMT small molecule inhibitors partially blocks cytokine production (38). In conclusion, CARM1 is required at multiple stages of hematopoietic differentiation, identifying it as a key epigenetic regulator of a cellular differentiation process that occurs throughout life. Future investigations will further elucidate the role of CARM1 at specific stages of hematopoiesis and determine the potential for therapeutic modulation of CARM1, which could be beneficial for hematopoietic disorders.

Supplementary Material

Acknowledgments

This work is supported in part by National Institutes of Health grant DK62248 (MTB and ERR). Facility Core support for flow cytometry was funded from P30ES007784 from the National Institute of Environmental Health Sciences to the Center for Research on Environmental Disease. This research was also supported in part by the National Institutes of Health through M.D. Anderson’s Cancer Center Support Grant CA016672.

The authors thank Dr. Irving Weissman for providing the actin-EGFP mice. We also thank Pam Whitney for flow cytometric analysis and Hilary Selden for technical assistance.

Footnotes

3Abbreviations used in this paper: CARM1, coactivator-associated arginine methyltransferase1; DN, double negative; DP, double positive; SP, single positive; BM, bone marrow.

References

1. Chao MP, Seita J, Weissman IL. Establishment of a normal hematopoietic and leukemia stem cell hierarchy. Cold Spring Harb Symp Quant Biol. 2008;73:439–449. [PubMed]
2. Mohn F, Schubeler D. Genetics and epigenetics: stability and plasticity during cellular differentiation. Trends Genet. 2009;25:129–136. [PubMed]
3. Shafa M, Krawetz R, Rancourt DE. Returning to the stem state: epigenetics of recapitulating pre-differentiation chromatin structure. Bioessays. 2010;32:791–799. [PubMed]
4. Ji H, Ehrlich LI, Seita J, Murakami P, Doi A, Lindau P, Lee H, Aryee MJ, Irizarry RA, Kim K, Rossi DJ, Inlay MA, Serwold T, Karsunky H, Ho L, Daley GQ, Weissman IL, Feinberg AP. Comprehensive methylome map of lineage commitment from haematopoietic progenitors. Nature. 2010;467:338–342. [PMC free article] [PubMed]
5. Parry RV, Ward SG. Protein arginine methylation: a new handle on T lymphocytes? Trends Immunol. 2010;31:164–169. [PubMed]
6. Bedford MT, Clarke SG. Protein arginine methylation in mammals: who, what, and why. Mol Cell. 2009;33:1–13. [PMC free article] [PubMed]
7. Cheng D, Cote J, Shaaban S, Bedford MT. The arginine methyltransferase CARM1 regulates the coupling of transcription and mRNA processing. Mol Cell. 2007;25:71–83. [PubMed]
8. Cote J, Richard S. Tudor domains bind symmetrical dimethylated arginines. J Biol Chem. 2005;280:28476–28483. [PubMed]
9. Friesen WJ, Massenet S, Paushkin S, Wyce A, Dreyfuss G. SMN, the product of the spinal muscular atrophy gene, binds preferentially to dimethylarginine-containing protein targets. Mol Cell. 2001;7:1111–1117. [PubMed]
10. Yang Y, Lu Y, Espejo A, Wu J, Xu W, Liang S, Bedford MT. TDRD3 is an effector molecule for arginine-methylated histone marks. Mol Cell. 2010;40:1016–1023. [PMC free article] [PubMed]
11. Bedford MT, Frankel A, Yaffe MB, Clarke S, Leder P, Richard S. Arginine methylation inhibits the binding of proline-rich ligands to Src homology 3, but not WW, domains. J Biol Chem. 2000;275:16030–16036. [PubMed]
12. Sakamaki J, Daitoku H, Ueno K, Hagiwara A, Yamagata K, Fukamizu A. Arginine methylation of BCL-2 antagonist of cell death (BAD) counteracts its phosphorylation and inactivation by Akt. Proc Natl Acad Sci U S A. 2011;108:6085–6090. [PubMed]
13. Yamagata K, Daitoku H, Takahashi Y, Namiki K, Hisatake K, Kako K, Mukai H, Kasuya Y, Fukamizu A. Arginine methylation of FOXO transcription factors inhibits their phosphorylation by Akt. Mol Cell. 2008;32:221–231. [PubMed]
14. Guccione E, Bassi C, Casadio F, Martinato F, Cesaroni M, Schuchlautz H, Luscher B, Amati B. Methylation of histone H3R2 by PRMT6 and H3K4 by an MLL complex are mutually exclusive. Nature. 2007;449:933–937. [PubMed]
15. Hyllus D, Stein C, Schnabel K, Schiltz E, Imhof A, Dou Y, Hsieh J, Bauer UM. PRMT6-mediated methylation of R2 in histone H3 antagonizes H3 K4 trimethylation. Genes Dev. 2007;21:3369–3380. [PubMed]
16. Di Lorenzo A, Bedford MT. Histone arginine methylation. FEBS Lett. 2011;585:2024–2031. [PMC free article] [PubMed]
17. Chen D, Ma H, Hong H, Koh SS, Huang SM, Schurter BT, Aswad DW, Stallcup MR. Regulation of transcription by a protein methyltransferase. Science. 1999;284:2174–2177. [PubMed]
18. Yadav N, Lee J, Kim J, Shen J, Hu MC, Aldaz CM, Bedford MT. Specific protein methylation defects and gene expression perturbations in coactivator-associated arginine methyltransferase 1-deficient mice. Proc Natl Acad Sci U S A. 2003;100:6464–6468. [PubMed]
19. O'Brien KB, Alberich-Jorda M, Yadav N, Kocher O, Diruscio A, Ebralidze A, Levantini E, Sng NJ, Bhasin M, Caron T, Kim D, Steidl U, Huang G, Halmos B, Rodig SJ, Bedford MT, Tenen DG, Kobayashi S. CARM1 is required for proper control of proliferation and differentiation of pulmonary epithelial cells. Development. 2010;137:2147–2156. [PubMed]
20. Yadav N, Cheng D, Richard S, Morel M, Iyer VR, Aldaz CM, Bedford MT. CARM1 promotes adipocyte differentiation by coactivating PPARgamma. EMBO Rep. 2008;9:193–198. [PubMed]
21. Ito T, Yadav N, Lee J, Furumatsu T, Yamashita S, Yoshida K, Taniguchi N, Hashimoto M, Tsuchiya M, Ozaki T, Lotz M, Bedford MT, Asahara H. Arginine methyltransferase CARM1/PRMT4 regulates endochondral ossification. BMC Dev Biol. 2009;9:47. [PMC free article] [PubMed]
22. Dacwag CS, Bedford MT, Sif S, Imbalzano AN. Distinct protein arginine methyltransferases promote ATP-dependent chromatin remodeling function at different stages of skeletal muscle differentiation. Mol Cell Biol. 2009;29:1909–1921. [PMC free article] [PubMed]
23. Kim D, Lee J, Cheng D, Li J, Carter C, Richie E, Bedford MT. Enzymatic activity is required for the in vivo functions of CARM1. J Biol Chem. 2010;285:1147–1152. [PubMed]
24. Kim J, Lee J, Yadav N, Wu Q, Carter C, Richard S, Richie E, Bedford MT. Loss of CARM1 results in hypomethylation of thymocyte cyclic AMP-regulated phosphoprotein and deregulated early T cell development. J Biol Chem. 2004;279:25339–25344. [PubMed]
25. Wright DE, Cheshier SH, Wagers AJ, Randall TD, Christensen JL, Weissman IL. Cyclophosphamide/granulocyte colony-stimulating factor causes selective mobilization of bone marrow hematopoietic stem cells into the blood after M phase of the cell cycle. Blood. 2001;97:2278–2285. [PubMed]
26. Lu M, Tayu R, Ikawa T, Masuda K, Matsumoto I, Mugishima H, Kawamoto H, Katsura Y. The earliest thymic progenitors in adults are restricted to T, NK, and dendritic cell lineage and have a potential to form more diverse TCRbeta chains than fetal progenitors. J Immunol. 2005;175:5848–5856. [PubMed]
27. Porritt HE, Rumfelt LL, Tabrizifard S, Schmitt TM, Zuniga-Pflucker JC, Petrie HT. Heterogeneity among DN1 Prothymocytes Reveals Multiple Progenitors with Different Capacities to Generate T Cell and Non-T Cell Lineages. Immunity. 2004;20:735–745. [PubMed]
28. Christensen JL, Wright DE, Wagers AJ, Weissman IL. Circulation and chemotaxis of fetal hematopoietic stem cells. PLoS Biol. 2004;2:E75. [PMC free article] [PubMed]
29. Serwold T, Ehrlich LI, Weissman IL. Reductive isolation from bone marrow and blood implicates common lymphoid progenitors as the major source of thymopoiesis. Blood. 2009;113:807–815. [PubMed]
30. Schmitt TM, Zúñiga-Pflucker JC. Induction of T cell development from hematopoietic progenitor cells by Delta-like-1 in vitro. Immunity. 2002;17:749–756. [PubMed]
31. Taghon TN, David E-S, Zuniga-Pflucker JC, Rothenberg EV. Delayed, asynchronous, and reversible T-lineage specification induced by Notch/Delta signaling. Genes and Development. 2005;19:965–978. [PubMed]
32. Morrison SJ, Hemmati HD, Wandycz AM, Weissman IL. The purification and characterization of fetal liver hematopoietic stem cells. Proc Natl Acad Sci U S A. 1995;92:10302–10306. [PubMed]
33. Mendez-Ferrer S, Frenette PS. Hematopoietic stem cell trafficking: regulated adhesion and attraction to bone marrow microenvironment. Ann N Y Acad Sci. 2007;1116:392–413. [PubMed]
34. Jones DL, Wagers AJ. No place like home: anatomy and function of the stem cell niche. Nat Rev Mol Cell Biol. 2008;9:11–21. [PubMed]
35. Attema JL, Papathanasiou P, Forsberg EC, Xu J, Smale ST, Weissman IL. Epigenetic characterization of hematopoietic stem cell differentiation using miniChIP and bisulfite sequencing analysis. Proc Natl Acad Sci U S A. 2007;104:12371–12376. [PubMed]
36. Rebel VI, Kung AL, Tanner EA, Yang H, Bronson RT, Livingston DM. Distinct roles for CREB-binding protein and p300 in hematopoietic stem cell self-renewal. Proc Natl Acad Sci U S A. 2002;99:14789–14794. [PubMed]
37. Weishaupt H, Sigvardsson M, Attema JL. Epigenetic chromatin states uniquely define the developmental plasticity of murine hematopoietic stem cells. Blood. 2010;115:247–256. [PubMed]
38. Bonham K, Hemmers S, Lim YH, Hill DM, Finn MG, Mowen KA. Effects of a novel arginine methyltransferase inhibitor on T-helper cell cytokine production. FEBS J. 2010;277:2096–2108. [PMC free article] [PubMed]