Abstract 549
RUNX1 (also known as AML1) is the DNA binding component of the Core Binding Factor (CBF)-transcriptional regulatory complex, which plays an important role in hematopoiesis. Upon binding ...to the common binding sequence -PyGpyGGTPy (Py = pyrimidine) in the regulatory regions of promoters and enhancers of its target genes, RUNX1 acts either as an activator or a repressor, depending on promoter context and its interacting partners. Thus, modulation of the network of RUNX1 interactions can influence hematopoiesis. However, how RUNX1 selects one set of partners over another to assemble a functional complex is largely unknown. Posttranslational modifications, including ubiquitination, phosphorylation, acetylation and methylation, present a viable mean to fine-tune its functions.
Here we shown that RUNX1 is arginine methylated at a specific residue, R223, by PRMT4, a type I arginine methyltransferase generally thought of as a co-activator molecule. We hypothesized that arginine methylation of RUNX1 by PRMT4 affects its protein-protein interactions, therefore, to identify proteins that specifically interact with unmethylated and/or methylated-R223 RUNX1, in an unbiased manner, we performed a peptide pull-down experiment, using a methyl-R223 RUNX1 peptide and an unmodified RUNX1 peptide as bait, following by mass spectrometry analysis. We identified several proteins that preferentially interacted with the R223 methyl peptide, but focused on a novel interacting protein, DPF2 (double PhD Finger 2), which is a widely expressed member of the d4 protein family, characterized by the presence of a tandem plant-homodomain (PHD domain). We confirmed the specific interaction between methylated-RUNX1 with DPF2 in vivo by immunoprecipitation. We generated an antibody specific for the R223 methylated-RUNX1 protein, and found that RUNX1 methylation decreases during the myeloid differentiation of human CD34+ haematopoietic stem/progenitor cells (HSPCs), without a change in the total level of RUNX1 protein, and this occurred co-incident with a downregulation of PRMT4 protein expression. Having determined that PRMT4 expression declines during myeloid differentiation, we examined the role of PRMT4 in this process, using short hairpin RNAs to knockdown PRMT4 expression in CD34+ cells. Knockdown of PRMT4 accelerates the myeloid differentiation of the cells, whereas overexpression of PRMT4 in human CD34+ cells blocked their myeloid differentiation. When analyzing the expression of several “master” regulators of myeloid differentiation, we identified microRNA-223, a myeloid specific microRNA, as a common target gene of PRMT4 and RUNX1. Furthermore, we have found that by promoting the assembly of a functional complex containing R223 methylRUNX1 and DPF2 at the transcriptional regulatory region of the microRNA-223 promoter, PRMT4 can control miR-223 expression and myeloid differentiation. We have verified the role of DPF2 in this process, as DPF2 represses miR-223 expression and loss of DPF2 promotes myeloid differentiation. Thus, DPF2 acts in a common pathway with PRMT4 to regulate myeloid differentiation.
In conclusion, our study elucidates a novel mechanism, where the arginine methylation of RUNX1 regulates its recruitment of interacting partner(s). In addition to demonstrating that PRMT4 can trigger repression of gene expression, we have identified a novel role for PRMT4 (aka CARM1) in myeloid differentiation.
No relevant conflicts of interest to declare.
Abstract 3632
Protein arginine methyltransferase 4 is a Type I member of PRMT family, that catalyses the addition of a methyl-group to arginine residues of a wide range of proteins, including ...histones, transcription factors, and RNA binding proteins. PRMT4 has been shown to regulate gene expression through its interaction with various transcription factors and via methylation of numerous substrates. Although PRMT4 has been reported to play an important role in T cell development, lung development and adipocyte differentiation in mouse models, the function of PRMT4 during hematopoiesis has not been studied.
To investigate the function of PRMT4 in the hematopoietic system, we utilized human CD34+ haematopoietic stem/progenitor cells (HSPCs). We observed that PRMT4 protein level is markedly downregulated during the myeloid differentiation of CD34+ cells without a significant change in the mRNA level. We then utilized a loss of function approach, using short hairpin RNAs, and found that knockdown of PRMT4 leads to an acceleration of myeloid differentiation, with a concomitant loss of the clonogenic potential of the cells. Interestingly, knocking down PRMT4 results in upregulation of miR-223, a myeloid specific microRNA. We also found that, during the myeloid differentiation of CD34+ cells, miR-223 expression steadily increased. Using a microRNA target prediction program, we identified a binding site for miR-223 in the 3′-UTR region of PRMT4 and found that when we over-expressed miR-223 in CD34+ cells, PRMT4 protein expression decreased. To determine the importance of PRMT4 downregulation in myeloid differentiation, we expressed the PRMT4-ORF (that should not be regulated by microRNAs) in CD34+ cells. The forced expression of PRMT4, that lacks the 3′-UTR region, leads to a block in myeloid differentiation and the inability of cells to up-regulate miR-223 during differentiation. Taken together, these data indicate a regulatory loop between PRMT4 and miR-223 that controls the differentiation of CD34+ toward the myeloid lineage.
To examine how PRMT4 regulates transcription of miR-223, we examined the miR-223 locus and found a RUNX1 binding site in the promoter of pri-miR-223. We discovered that PRMT4 interacts with RUNX1 and methylates RUNX1 at a specific arginine residue. This results in the recruitment of several novel interacting partners, which appear to control the expression of miR-223. Thus our results indicate that PRMT4 regulate the transcription of miR-223 transcription via its effects on RUNX1. Our study demonstrates a novel function of PRMT4 in myeloid differentiation, through regulation of RUNX1 function and miR-223 expression.
No relevant conflicts of interest to declare.
RNA binding proteins (RBPs) tightly control mRNA abundance, stability and translation while mutations or altered expression of specific factors can drive malignancy. Yet, the identity of the RBPs ...that govern myeloid stem cells remains poorly characterized. We and others have recently demonstrated that MUSASHI-2 (MSI2) is a central regulator of the cancer stem cell program in myeloid leukemia. Therefore, we curated a list of 127 MSI2 direct protein interactors and associated genes to perform an in vivo shRNA screen using MLL-AF9 leukemia cells. We identified shRNAs corresponding to 24 genes that were significantly depleted in vivo after sequencing and comparing their representation from day 16 to day 0. We confirmed knockdown and demonstrated marked reduction in myeloid colony formation in vitro after depleting 7 hits identified in our screen. Additionally, we tested these genes in normal bone marrow c-Kit positive cells and found that the most differentially required gene in leukemia cells compared to normal cells was SYNCRIP (Synaptotagmin-binding, cytoplasmic RNA-interacting protein). SYNCRIP is an RNA binding protein that has been implicated in various RNA regulatory processes but its role in the hematopoietic system is virtually unknown. Depletion of SYNCRIP with shRNAs in murine MLL-AF9 leukemia cells resulted in an increase in myeloid differentiation, apoptosis and delayed leukemogenesis in vivo (median survival of 35 days; control versus 61 days shRNA#1 knockdown was selected against, and "not reached" shRNA#2). To further assess SYNCRIP function in vivo, we developed a germline Syncrip knockout (KO) by injecting Cas9-DNA and Syncrip - guides RNAs into embryos and harvested E13 fetal liver cells. After Syncrip deletion was verified by immunoblotting, we observed normal numbers of HSCs and equivalent engraftment in lethally irradiated animals in both primary and secondary transplants. In contrast, we observed a delay in leukemeogenesis (median survival of 87.5 days; WT versus 118 days KO) in recipient mice after transplantation of MLL-AF9 transformed LSKs. Notably, non-deleted leukemia cells outcompeted the SYNCRIP deleted cells based on a reemergence of SYNCRIP expression. These data suggest that SYNCRIP is differentially required in myeloid leukemia cells compared to normal cells. Furthermore, we found that SYNCRIP was highly expressed in wide variety of human AML cell lines and in primary AML patients (n=4/5). SYNCRIP depletion with shRNAs resulted in reduced cell proliferation and the induction of apoptosis in human AML cell lines (MOLM13, NOMO-1, KASUMI-1 and NB4) and a marked decrease in engraftment of primary AML patient cells. To gain insights into SYNCRIP function, we performed RNA-sequencing of leukemia cells depleted for SYNCRIP. Gene set enrichment analysis (GSEA) negatively enriched for the MLL-AF9, HOXA9 and stem cell programs in SYNCRIP-KD cells and positively enriched for MSI2's direct mRNA binding targets and a MSI2 deficient LSC signature. Reciprocal immunoblotting in the presence or absence of RNAse demonstrated that SYNCRIP and MSI2 interaction is RNA dependent. We validated their shared targets by performing SYNCRIP RNA-immunoprecipitation (RIP) for previously identified MSI2's direct mRNAs targets (HOXA9 and c-MYC). SYNCRIP depletion resulted in reduced protein abundance of HOXA9 and c-MYC. Forced MSI2 expression partially rescued the colony formation and HOXA9 expression in SYNCRIP-KD cells. To assess the functional downstream targets of SYNCRIP in leukemia, we overexpressed HOXA9 and c-MYC in SYNCRIP-KD cells and observed that HOXA9 expression but not c-MYC partially rescues the effect of SYNCRIP depletion on myeloid colony formation. Mechanistically, we showed that SYNCRIP regulates translation of HOXA9 without affecting HoxA9 mRNA stability. Overall, we provide a strategy for interrogating the functional RNA binding network in leukemia using shRNA screening. Additionally, we validated SYNCRIP as a novel RBP that controls the leukemia stem cell program and propose that targeting these functional complexes might provide a novel therapeutic strategy in myeloid leukemia.
Melnick:Janssen: Research Funding. Levine:Novartis: Consultancy; Qiagen: Membership on an entity’s Board of Directors or advisory committees. Järås:Cantargia AB: Equity Ownership.
Abstract 3403
RUNX1 is a transcription factor that is required for definitive hematopoietic development, and helps regulate long term hematopoietic stem cell self-renewal, platelet production, and ...lymphocyte development during adult hematopoiesis. RUNX1 is known to be modified via phosphorylation, acetylation, ubiquitination and methylation, for example on R208 and R210 by PRMT1, which activates its activating function. We continue to investigate how the methylation of RUNX1 by other protein arginine methyl transferases (PRMTs) regulates its function. Loop 9 of the DNA binding domain (the Runt domain) of RUNX1 contains an SGRGK sequence that is also present on the tails of histone H2A and H4. The histone tails of H4 and H2A can be methylated by a purified PRMT5 complex in vitro. An enzymatically active in vitro PRMT5 complex capable of methylating histones and SM proteins requires two subunits: both PRMT5 and MEP50, a WD 40 repeat domain protein. Nevertheless, this purified PRMT5/MEP50 complex cannot methylate the DNA binding domain of the RUNX1 protein in vitro. We show that RUNX1 also can be symmetrically methylated at R142 within the SGRGK motif in vitro by a nuclear PRMT5/MEP50 complex which also contains COPR5. We show after RUNX1 is methylated on R142 within the nucleus of HEL cells, RUNX1 is exported to the cytoplasm in a CRM1 dependent manner, as the export of methylated RUNX1 is blocked by lemptomycin B. CRM1 interacts with PRMT5, supporting that PRMT5 mediated arginine methylation tags protein for nuclear export. Therefore, PRMT5 not only involves in epigenetic regulation by methylation of histones but also it can directly controls the level of transcription factor proteins within the nucleus. Polycytocemia Vera patients who express the Jak2V617F mutation have low PRMT5 activity due to JAK2V617F mediated PRMT5 phosphorylation (Liu et al 2011). How Jak2 signaling affects RUNX1 methylation and RUNX1 localization within the nucleus is still under investigation. By controlling the amount of RUNX1 available within the cell nucleus, PRMT5 may regulate lineage differentiation potential and growth potential of hematopoietic stem and progenitor cells. The nuclear localization of RUNX1 can be changed through post translational modification such as arginine methylation in addition to point mutations and translocations involving RUNX1 found patients with leukemia and pre-leukemic diseases.
No relevant conflicts of interest to declare.
Abstract 352
L3MBTL1 is a Polycomb group protein, commonly deleted in patients with myeloid disorders associated with the 20q- chromosomal abnormality. After crystallizing the MBT repeat domain, we ...demonstrated that L3MBTL1 compacts chromatin by binding mono- and di-methylated lysine residues in histones H1 (H1K26) and H4 (H4K20), ultimately leading to gene repression. Despite its role in affecting the chromatin structure, the role of L3MBTL1 in hematopoiesis has remained largely unknown.
We recently demonstrated that lack of L3MBTL1 accelerates the erythroid differentiation of human hematopoietic stem cells and here we reveal that L3MBTL1 represses the expression of the fetal gamma globin gene. We lentivirally expressed shRNAs targeting L3MBTL1 in human cord blood (CB) CD34+ cells and in K562 erythroleukemia cells, and consistently observed upregulation of gamma globin gene expression, while beta globin gene expression decreased. Remarkably, we observed similar findings in human embryonic stem (hES) cells, where knock-down of L3MBTL1 triggered a BMP4-like spontaneous differentiation. Given the potential impact of therapeutically increasing fetal hemoglobin expression in patients with hemoglobinopathies, we targeted L3MBTL1 in induced pluripotent stem (iPS) cells derived from patients with β-thalassemia. The gene expression profile of L3MBTL1-KD normal and thalassemic iPS cells indicated clear activation of fetal hemoglobin (HbF) expression, activation of BMP4 signaling and upregulation of specific smad5 target genes (e.g. EKLF, HHEX, ID2/3). We generated and utilized a model of “stress erythropoiesis” in L3MBTL1 KO mice and observed in vivo BMP4-mediated expansion of spleen immature erythroid progenitors, as indicated by increased spleen weight and splenic BFU-E colonies in KO mice compared to controls.
We also examined K562 cells, human CB CD34+ cells and hES cells, using chromatin immunoprecipitation assays, and found that L3MBTL1 directly associates with the human β-globin locus, occupying discrete regions within the human β-globin cluster. Furthermore, L3MBTL1 colocalized with H4K20me within the Locus Control Region (LCR), a primary attachment site for chromatin modifiers. We observed clearance of L3MBTL1 and its associated histone marks (H4K20me1/2) from the LCR upon treatment with hemin, erythropoietin or TGFβ, three agents that potently induce erythroid differentiation. This suggests that this polycomb repressor complex responds to cytokine signaling.
In summary, we have identified a novel epigenetic regulatory mechanism to control fetal globin gene expression; the Polycomb protein L3MBTL1 regulates BMP4 signaling and the chromatin structure of globin genes. Targeting this regulatory system represents a means to efficiently increase HbF in a human model of β-thalassemia (i.e. with the use of patient-derived iPS cells) and to potentially ameliorate hematological and clinical symptoms of patients with red cell disorders.
No relevant conflicts of interest to declare.
Leukemia stem cells (LSCs) are found in most aggressive myeloid diseases and contribute to therapeutic resistance. LSCs are characterized by their gain of a self-renewal program that is normally ...associated with hematopoietic stem cells (HSCs). Previously we have shown that the RNA binding protein, Msi2 contributes to both HSC and myeloid leukemia function. Elevated MSI2 expression predicts a poor prognosis in a variety of leukemias and shRNA-mediated depletion in human AML cell lines reduces proliferation, increases differentiation and induces apoptosis. Despite these in vitroand correlative studies, MSI2’s molecular mechanism is not known and its role in LSC function has not been assessed.
To elucidate MSI2’s role in LSC function, we utilized the MLL-AF9 leukemia mouse model. Initially we found MSI2 was elevated in the LSC enriched compartment (c-KitHigh cells) compared to non-LSCs (c-KitLow cells) based on flow cytometric intracellular staining. Therefore, to establish a model to study Msi2 and its contribution to myeloid LSCs, we have utilized the Msi2 conditional knockout mice that we previously crossed (Msi2f/f) into an Mx1-Cre background to generate the Msi2Δ/Δallele (injection of polyinositol-polycytosine; pIpC). In order to test if Msi2 is critical for MLL-AF9 mediated initiation, we transduced control Msi2f/f and Msi2Δ/ΔLin- Sca1+ c-Kit+cells (LSKs) with MLL-AF9 expressing retroviruses co-expressing GFP.
Msi2 deleted LSKs or granulocyte-monocyte progenitors (GMPs) transduced with MLL-AF9 demonstrated delayed leukemogenesis with dramatically reduced diseased burden. Msi2 deficient leukemias were found to have a 4-fold reduced phenotypic LSC population and were more differentiated based on cellular morphology. Msi2 deficient leukemias failed to transplant into secondary recipients demonstrating that Msi2 is required for maintaining LSCs. Deletion of Msi2after leukemia engraftment led to a delay in leukemogenesis indicating that Msi2 is also important for leukemia maintenance.
Gene expression profiling of the Msi2 ablated LSCs resulted in a loss of the HSC/LSC program and an increase in differentiation gene sets. The gene signature from the Msi2 deleted murine LSCs (121 genes) was overlapped and subjected to unsupervised clustering with the gene expression profiles from 336 AML patients (ECOG1900 dataset). This analysis resulted in distinct clusters that had differential MSI2 expression and the MSI2“high” cluster predicted a worse clinical outcome when compared to the other clusters.
Overlapping of the differential transcriptional analysis of the Msi2 deleted murine LSCs with our global MSI2 direct mRNA targets (HITS-CLIP) led us to identify that MSI2 binds to transcripts that are associated with the downstream MLL self-renewal program, including Myc and Ikzf2. Ikzf2 is a member of the Ikaros transcription factor family and is known to regulate lymphocyte development by controlling regulatory T-cell function and chromatin remodeling. Ikzf2 shRNA mediated depletion resulted in reduced colony formation, decreased proliferation and increased apoptosis. The MLL associated targets were also reduced, which included Bcl-2 and Hoxa9. In contrast to its tumor suppressor role in hypodiploid B-ALL, these results suggest that Ikzf2 contributes to MLL leukemia cell maintenance. Thus, we provide evidence that MSI2 maintains the oncogenic LSC epigenetic program and the rationale for clinically targeting MSI2 in myeloid leukemia.
No relevant conflicts of interest to declare.
Stem cells balance cellular fates through asymmetric and symmetric divisions in order to self-renew or to generate downstream progenitors. Symmetric commitment divisions in stem cells are required ...for rapid regeneration during tissue damage and stress. The control of symmetric commitment remains poorly defined. Using single-cell RNA sequencing (scRNA-seq) in combination with transcriptomic profiling of HSPCs (hematopoietic stem and progenitor cells) from control and m
A methyltransferase Mettl3 conditional knockout mice, we found that m
A-deficient hematopoietic stem cells (HSCs) fail to symmetrically differentiate. Dividing HSCs are expanded and are blocked in an intermediate state that molecularly and functionally resembles multipotent progenitors. Mechanistically, RNA methylation controls Myc mRNA abundance in differentiating HSCs. We identified MYC as a marker for HSC asymmetric and symmetric commitment. Overall, our results indicate that RNA methylation controls symmetric commitment and cell identity of HSCs and may provide a general mechanism for how stem cells regulate differentiation fate choice.
Abstract 2296
The Hippo signaling pathway, first discovered in Drosophila, is emerging as an important regulator of stem cell behavior. Upon still-unclear upstream stimuli, the hippo pathway kinase ...cascade phosphorylates and inhibits the function of YAP, a transcription coactivator, by inducing its cytoplasmic retention. While recent evidences indicate that inhibition of YAP affects cell fate decisions, and proliferation, in many tissues, little is known about the relevance of this pathway in hematopoiesis. However, the interaction of YAP with Smad1, identified in flies and human cells (Alarcon C. et al. Cell 2009), prevents smurf-mediated Smad1 degradation, potentially enhancing BMP signaling.
Our ongoing studies have indentified crosstalk between the BMP4 and the Hippo pathways in hematopoietic cells, and in induced-pluripotent stem (iPS) cells that we differentiated towards the erythroid lineage. This crosstalk involves the chromatin-binding, Polycomb protein L3MBTL1, which clearly regulate the effects of BMP on the erythroid differentiation of hematopoietic stem/progenitor cells and on fetal globin gene expression.
We find that the Lats2 kinase, a core component of the Hippo pathway, physically interacts with L3MBTL1 and that treatment with BMP4 or Erythropoietin decreases the expression of both proteins in various hematopoietic cells, including primary human cord blood-derived CD34+ cells. By altering L3MBTL1 levels in K562 cells, we were able to show that the L3MBTL1-Lats2 interaction enhances Lats-mediated phosphorylation and the cytoplasmic retention of YAP. Furthermore, L3MBTL1-depleted iPS cells have an enhanced smad-mediated transcriptional response; by analyzing the gene expression profile of these cells, we found increased expression of several BMP target genes (such as HHEX and ID genes), suggesting that L3MBTL1 negatively titrates the BMP4 signaling pathway at least in part by affecting YAP phosphorylation and localization. Gene Set Enrichment Analysis confirmed enrichment of many smad-related genes, and yet, these cells presented enhanced smad1/5/8 phosphorylation by WB analysis, indicating that BMP4 signaling is triggered by L3MBTL1 depletion. We also found that hematopoietic differentiation of L3MBTL1-KD iPS cells generates high-fetal globin gene expressing erythroid progeny, suggesting a role for the BMP4 signaling pathway and the targeting of L3MBTL1 in the treatment of hemoglobinopathies.
To further evaluate the effect of BMP4 signaling on hematopoietic cells that lack L3MBTL1, we analyzed the stress erythroid response of L3MBTL1 KO mice: while no difference was observed at baseline in the null mice compared to wt littermates, the L3mbtl1 null mice had a more severe anemia, with increased leukocytosis, and thrombocytosis post-hydrazine (PHZ) or Epo. We found a significant increase in the colony-forming ability of the l3mbtl1 null spleen and bone marrow cells, compared to controls, as well as increased spleen size and an expansion of the spleen erythroid compartment. Thus, l3mbtl1 null hematopoietic stem cells are more sensitive to the PHZ-mediated cytokine storm, which includes BMP4. Interestingly, the L3mbtl1 null BM and spleen cells showed diminished expression of Lats2 and phospho-YAP, consistent with our in vitro findings.
In conclusion, these investigations have shown that L3MBTL1 not only negatively titrates the BMP4 signaling pathway, but also provides a nodal point for crosstalk between the BMP4 and Hippo signaling pathways in erythropoiesis. Thus, these data provide insights into possible novel treatments for genetic red cell disorders (such as β-thalassemia) and for acquired bone marrow failure syndromes such as Epo-resistant anemia.
Levine:Agios Pharmaceuticals: Research Funding.
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