CARM1Regulates Fetal Hematopoiesis And Thymocyte Development

IntroductionHematopoietic 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 (7-10);2) It can also block protein-protein interactions, as in the case of certain SH3domain-driven interactions (11);3) Arginine methylation can negatively regulate AKT-mediated phosphorylation, because the AKT consensus motif contains key arginine residues (12’13); and similarly4) 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, H4and H2A for methylation (16). CARM1/PRMT4was the first family member to be identified as a transcriptional coactivator, which methylates the H3R17and 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 CARM1are inundated with immature alveolar type II cells, which do not develop into more mature alveolar type I cells, thus CARM1is required for the proper differentiation of alveolar cells (19). In addition, CARM1-null embryos lack brown fat, and cells that do not express CARM1are not able to differentiate into mature adipocytes (20). CARM1is also required for chondrogenesis (21) and skeletal muscle development (22). Finally, we have also observed that functional CARM1is required for normal T cell cellularity and differentiation (23,24). Thus, there is genetic evidence that CARM1activity 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 further investigated the cellular basis for impaired thymopoiesis in this mouse model. Here we report that CARM1functions cell intrinsically to regulate long-term hematopoietic stem cell activity and cellularity in the fetal liver and bone marrow, respectively. Furthermore, CARM1deficiency has a greater impact on lymphoid versus myeloid differentiation. We confirm a role for CARM1in thymocyte development at the transition between the DN1and DN2stages. Taken together, these results demonstrate that CARM1regulates fetal hematopoiesis and thymocyte development, bolstering the notion that epigenetic regulation is critical for proper differentiation of multiple hematopoietic lineages.Part I Carm1deficiency results in a block at the DN1-DN2transition in thymopoiesisObjectives To elucidate the effect of CARM1deficiency in lymphocytes, including T cell and B cell developmentMethodsWe inbreed CARM1+/-mice, genotyping with Southern blot and PCR. Run FACS to test the subsets of thymocytes and splenic cells.ResultsCARM1was knocked out according to Southern blot and PCR; CARM1deficient embryos are smaller, as big as60%of WT. The thymi are smaller as well.There is a DN1to DN2block, and the percentage of DNs are reduced.In the peripheral lymphocytes, the cellularity is diminished, especially B cells.CoclusionsCARM1was successfully knocked out in vivo. There is a DN1to DN2block, all the downsream subsets are effected. Analysis of c-Kit expression revealed an increase in the frequency of DN1versus DN2progenitors in Carm1-/-E18.5embryos, consistent with a block in differentiation of the earliest thymocyte subset. Together these data indicate that CARM1is required for continued maturation of thymocytes beyond the DN1stage.Part II Reduced thymopoiesis in Carm1-/-mice is not due to thymic stromal defectObjectivesThe cross talk between thymocytes and TEC is important to the development. To identify the possibility that the defect of thymocytes in the absence of CARM1dues to the deficiency of TECs.MethodsWe run IF and FACS analysis on the CARM1null TECs; we transplanted E15.52-deoxyguanosine treated Carm1-/-versus control fetal thymic lobes under the kidney capsule of athymic nude recipients.8-12weeks after transplantation, thymic grafts were recovered and analyzed by flow cytometry.ResultsThymopoiesis was not impaired when CARM1deficiency was restricted to thymic stromal cells. The cellularity was comparable for all thymocyte subsets regardless of whether they developed in a control or Carm1-/-stromal environment. Engrafted CARM1deficient lobes were smaller than controls at the outset of the transplantation experimen.ConclusionsThe normal differentiation of thymocyte progenitors in a CARM1deficient microenvironment, in contrast to the severely reduced cellularity in Carm1-/-E18.5thymi, indicates that loss of CARM1predominantly affects thymocyte progenitors as opposed to the thymic stromal microenvironment.Part III Early hematopoietic defect in CARM1-/-mice bone marrow and Caarm^fetal liver cells have impaired hematopoietic potentialObjectivesHypothesized that the absence of Carm1might affect prethymic hematopoietic progenitors, assess the functional potential of E18.5fetal liver progenitorsMethodsWe examined cryosections from E12.5-E18.5from Carm1-/-embryos to determine if the reduction in thymocyte cellularity was apparent earlier in ontogeny. We compared E18.5bone marrow and fetal liver from Carm1-/-and control littermates for hematopoietic progenitor subset composition. To assess the functional potential of E18.5fetal liver progenitors, we performed a competitive reconstitution experiment. Equal numbers of E14.5fetal liver cells from GFP+Carm1-/-embryos or GFP+littermate controls were mixed with GFP-wild-type fetal liver cells from E14.5C57BL/6embryos. This mixture was injected into sublethally irradiated RAG2-/-γc-/-mice. Recipient thymi and bone marrow were analyzed for donor chimerism in all hematopoietic subsets8-12weeks after transfer. ResultsAt E12.5there was a striking paucity in the number of CD45+hematopoietic progenitors in the Carm1-/-thymic rudiment. This deficiency was also apparent E13.5-E17.5, Cellularity was reduced in Carm1-/-bone marrow compared with littermate controls. Flk2+MPP are reduced by95%, while LT-HSC and ST-HSC were reduced by approximately80%.There is a decrease in oligopotent hematopoietic progenitors in E18.5Carm1-/-bone marrow, downstream lineage-restricted progenitors were also diminished. CD27+Flk2+compartment was reduced by85%, CMP and MEP were significantly reduced by92%and66%, respectively, GMP were not significantly diminished.The number of fetal liver cells in Carm1-/-mice is comparable to, LT-HSCs subset was slightly increased in Carm1-/-" fetal liver, resulting in an increase in overall KLS cells. All other hematopoietic progenitors, were present in similar numbers to controls.Carm1-/-fetal liver cells from DN1through CD4SP and CD8SP, when in competition with control fetal liver cells. 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.ConclusionsCARM1plays a significant role in thymocyte development prior to and after thymic vascularization, which occurs at-E14.5Early hematopoietic defect in CARM1-/-mice bone marrow suggests that CARM1is required for early stages of hematopoiesis and continued lymphoid differentiation. However, CARM1is less essential for later stages of myeloid differentiation, as observed by normal numbers of GMP and splenic granulocytes.Cellularity of hematopoietic progenitors is not decreased in Carm1-/-fetal liver, indicates CARM1is not required to maintain HSC cellularity in the fetal liver.Carm1-/-fetal liver cells have impaired hematopoietic potentialdespite the fact that hematopoietic progenitors were present at near normal numbers in E18.5Carm1-/fetal liver, they were severely impaired in their ability to contribute to hematopoiesis of all lineages. Given that the recipient mice were CARM1sufficient, these data also demonstrate that CARM1is required cell-autonomously in hematopoietic progenitors. ConclusionsIn conclusion, CARM1is required at multiple stages of hematopoietic differentiation, identifying it as a key epigenetic regulator of a cellular differentiation process that occurs throughout life.