The Polycomb Group Protein L3MBTL1 Represses a SMAD5-Mediated Hematopoietic Transcriptional Program in Human Pluripotent Stem Cells

SummaryEpigenetic regulation of key transcriptional programs is a critical mechanism that controls hematopoietic development, and, thus, aberrant expression patterns or mutations in epigenetic regulators occur frequently in hematologic malignancies. We demonstrate that the Polycomb protein L3MBTL1, which is monoallelically deleted in 20q- myeloid malignancies, represses the ability of stem cells to drive hematopoietic-specific transcriptional programs by regulating the expression of SMAD5 and impairing its recruitment to target regulatory regions. Indeed, knockdown of L3MBTL1 promotes the development of hematopoiesis and impairs neural cell fate in human pluripotent stem cells. We also found a role for L3MBTL1 in regulating SMAD5 target gene expression in mature hematopoietic cell populations, thereby affecting erythroid differentiation. Taken together, we have identified epigenetic priming of hematopoietic-specific transcriptional networks, which may assist in the development of therapeutic approaches for patients with anemia

Compound A, a selective glucocorticoid receptor modulator, enhances heat shock protein Hsp70 gene promoter activation

Compound A possesses glucocorticoid receptor (GR)-dependent anti-inflammatory properties. Just like classical GR ligands, Compound A can repress NF-kappa B-mediated gene expression. However, the monomeric Compound A-activated GR is unable to trigger glucocorticoid response element-regulated gene expression. The heat shock response potently activates heat shock factor 1 (HSF1), upregulates Hsp70, a known GR chaperone, and also modulates various aspects of inflammation. We found that the selective GR modulator Compound A and heat shock trigger similar cellular effects in A549 lung epithelial cells. With regard to their anti-inflammatory mechanism, heat shock and Compound A are both able to reduce TNF-stimulated I kappa B alpha degradation and NF-kappa B p65 nuclear translocation. We established an interaction between Compound A-activated GR and Hsp70, but remarkably, although the presence of the Hsp70 chaperone as such appears pivotal for the Compound A-mediated inflammatory gene repression, subsequent novel Hsp70 protein synthesis is uncoupled from an observed CpdA-induced Hsp70 mRNA upregulation and hence obsolete in mediating CpdA's anti-inflammatory effect. The lack of a Compound A-induced increase in Hsp70 protein levels in A549 cells is not mediated by a rapid proteasomal degradation of Hsp70 or by a Compound A-induced general block on translation. Similar to heat shock, Compound A can upregulate transcription of Hsp70 genes in various cell lines and BALB/c mice. Interestingly, whereas Compound A-dependent Hsp70 promoter activation is GR-dependent but HSF1-independent, heat shock-induced Hsp70 expression alternatively occurs in a GR-independent and HSF1-dependent manner in A549 lung epithelial cells

Depletion of L3MBTL1, a PcG Gene Deleted in Patients with 20q-Associated with Hematologic Malignancies, Accelerates Erythroid Differentiation of Hematopoietic Progenitor Cells

Abstract L3MBTL1 is a polycomb gene located in 20q12, within the common deleted region identified in patients with 20q deletion associated with polycythaemia vera (PV), myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML). L3MBTL1 is expressed within CD34+ haematopoietic progenitor cells from which myeloid malignancies arise and its Drosophila homologue encodes a tumour suppressor protein. L3MBTL1 represents then a candidate target gene in 20q deletion patients. To gain insight into the role of L3MBTL1 in hematopoiesis we knocked down the level of L3MBTL1 mRNA through lentiviral expression of short hairpin RNAs in CD34+ hematopoietic stem/progenitor cells isolated from human cord blood (CB) cells. We achieved an approximately 80% drop in endogenous L3MBTL1 mRNA level, as determined using quantitative RT-PCR. Transduced and sorted CD34+ GFP+ CB cells were plated in liquid cultures and induced to cytokine-driven differentiation. The effect of L3MBTL1-knock down was assessed by colony assays and by fluorescence-activated cell sorting (FACS), using lineage–specific cell surface markers. We demonstrate that the knock down (KD) of L3MBTL1 remarkably accelerates the differentiation of hematopoietic CD34+ cells into erythrocytes. The relative percentage of mature erythroid precursors cells, defined as CD71+ and Glycophorin A+ cells, consistently increased in the L3MBTL1-KD population, as the CD34+ cells reached this advanced stage of erythroid differentiation in fewer days than control cells. As confirmation, Giemsa staining after cytospin preparations of L3MBTL1-KD cells showed more mature morphology compared to the control cells, and benzidine staining revealed many more Hb containing-positive cells in the L3MBTL1-depleted cell population compared to the control. Monitoring of globin gene expression demonstrated that L3MBT-Knock down is involved in the regulation of only a subset of these genes, primarily the expression of the epsilon and zeta globin genes, the embryonic globin genes belonging to the beta and alpha globin gene clusters respectively. In addition to these effects a significant slowing of proliferation was seen, which likely reflects the increased differentiation of these cells. We have previously identified a role for L3MBT in binding histones H1 and H4 that contain monomethylated and dimethylated lysine residues H1k2b and H4k20 (Kalakonda et al. 2008) and compacting chromatin (Trojer et al. Cell 2007). Precisely how this feature of L3MBTL1 function in regulating the erythroid differentiation of hematopoietic cells is unknown. We however link lack of L3MBTL1 with the possible pathogenesis of PV associated with 20q deletion. We will present additional data that attempt to define the kinetics of alpha-like and beta-like globin gene expression during the erythroid maturation of L3MBTL1-KD cells in culture and to investigate a supposed synergism with the known molecular pathways of normal and malignant erythroid differentiation

L3MBTL1 Deficiency Directs the Differentiation of Human Embryonic Stem Cells Toward Trophectoderm

Human embryonic stem cells (hESCs) can be used to study the early events in human development and, hopefully, to understand how to differentiate human pluripotent cells for clinical use. To define how L3MBTL1, a chromatin-associated polycomb group protein with transcriptional repressive activities, regulates early events in embryonic cell differentiation, we created hESC lines that constitutively express shRNAs directed against L3MBTL1. The L3MBTL1 knockdown (KD) hESCs maintained normal morphology, proliferation, cell cycle kinetics, cell surface markers, and karyotype after 40 passages. However, under conditions that promote spontaneous differentiation, the L3MBTL1 KD cells differentiated into a relatively homogeneous population of large, flat trophoblast-like cells, unlike the multilineage differentiation seen with the control cells. The differentiated L3MBTL1 KD cells expressed numerous trophoblast markers and secreted placental hormones. Although the L3MBTL1 KD cells could be induced to differentiate into various embryonic lineages, they adopted an exclusive trophoblast fate during spontaneous differentiation. Our data demonstrate that depletion of L3MBTL1 does not affect hESC self-renewal, rather it enhances differentiation toward extra-embryonic trophoblast tissues

Targeting a Novel Epigenetic Silencing Mechanism to Efficiently Upregulate Fetal Globin Gene Expression

Abstract 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. Disclosures: No relevant conflicts of interest to declare

The PcG Protein L3MBTL1 Transcriptionally Represses Human Embryonic and Fetal Globin Genes: a Novel Prospect for HbF Activation

Abstract Abstract 1014 L3MBTL1 is the human homolog of the Drosophila Polycomb Group tumor suppressor gene, lethal(3)malignant brain tumor. We demonstrated that human L3MBTL1 functions as a transcriptional repressor and after crystallizing the MBT repeat domain determined that L3MBTL1 compacts chromatin by binding mono- and di-methylated lysine residues in histones H1 (H1K26) and H4 (H4K20). Despite the known role of L3MBTL1 in affecting chromatin structure, the function of L3MBTL1 in human hematopoiesis has remained largely unknown. We recently demonstrated that L3MBTL1 enforces cell fate decision toward the erythroid lineage and that knockdown of L3MBTL1 accelerates the erythroid differentiation of human hematopoietic stem/progenitor cells, suggesting that its deletion contributes to the pathogenesis of 20q- erythroid malignancies. Consistently with its role in erythropoiesis, here we reveal that L3MBTL1 is a novel transcriptional repressor of fetal globin genes and it may work in concert with BCL11A and EKLF to control globin gene expression. By utilizing RNA interference to reduce L3MBTL1 expression, we have found that knockdown of L3MBTL1 in human cord blood hematopoietic stem/progenitor cells consistently upregulates the expression of the epsilon, gamma, and zeta globin genes, but not the beta globin gene. Similar effects were seen following knockdown of L3MBTL1 in the human erythroleukemia cell line K562, and knockdown of L3MBTL1 in human embryonic stem cells (ESCs) led to the inappropriate expression of fetal and embryonic globin genes (which increases more than 50-fold after the L3MBTL1-KD). These data suggest a role for L3MBTL1 in regulating the globin switch. To investigate the mechanism by which L3MBTL1 silences embryonic and fetal globin gene expression, we used chromatin immunoprecipitation (ChIP) assays to show that L3MBTL1 directly associates with the human β-globin locus. L3MBTL1 occupies several discrete regions within the human β-globin cluster and colocalizes with H4K20me within the Locus Control Region (LCR), a primary attachment site for chromatin modifiers. As confirmation, we found that treatment of K562 cells with hemin, which broadly increases H3K9 acetylation over the β-globin locus and activates the transcription of globin genes, leads to decreases in expression of the repressive H4K20me2 methylmark and L3MBTL1 to the beta-globin cluster. Given the recent identification of the repressor of gamma globin gene expression, BCL11A, we investigated a potential relationship between L3MBTL1 and BCL11A. We found that knockdown of L3MBTL1 led to downregulation of BCL11A mRNA. Accordingly, we have also found that overexpression of L3MBTL1 is associated with an upregulation of BCL11A mRNA, suggesting that L3MBTL1 and BCL11A may function cooperatively to silence globin gene expression. Knockdown of L3MBTL1 also upregulated EKLF mRNA levels which could relate to the decreased BCL11A expression. In summary our data demonstrate that knock-down of L3MBTL1 upregulates embryonic and fetal globin genes in cell contexts where they are usually silenced, indicating the functional importance of this Polycomb protein for repressing the globin gene locus. The clearance of L3MBTL1 and its associated histone mark (H4K20me2) during treatments that induce potent transcriptional activation of globin genes suggest that repression induced by L3MBTL1 is dynamic and may be involved in the fetal-to-adult globin switch. L3MBTL1 therefore emerges as a novel transcriptional repressor of fetal globin genes whose expression may be coordinated with that of BCL11A and EKLF. Understanding the role of L3MBTL1 and the H4K20 methylmark in globin gene switching offers the prospect of the targeted activation of HbF in erythroid cells of patients with hemoglobin disorders. Disclosures: No relevant conflicts of interest to declare

Depletion of L3MBTL1 promotes the erythroid differentiation of human hematopoietic progenitor cells: possible role in 20q− polycythemia vera

L3MBTL1, the human homolog of the Drosophila L(3)MBT polycomb group tumor suppressor gene, is located on chromosome 20q12, within the common deleted region identified in patients with 20q deletion-associated polycythemia vera, myelodysplastic syndrome, and acute myeloid leukemia. L3MBTL1 is expressed within hematopoietic CD34+ cells; thus, it may contribute to the pathogenesis of these disorders. To define its role in hematopoiesis, we knocked down L3MBTL1 expression in primary hematopoietic stem/progenitor (ie, CD34+) cells isolated from human cord blood (using short hairpin RNAs) and observed an enhanced commitment to and acceleration of erythroid differentiation. Consistent with this effect, overexpression of L3MBTL1 in primary hematopoietic CD34+ cells as well as in 20q− cell lines restricted erythroid differentiation. Furthermore, L3MBTL1 levels decrease during hemin-induced erythroid differentiation or erythropoietin exposure, suggesting a specific role for L3MBTL1 down-regulation in enforcing cell fate decisions toward the erythroid lineage. Indeed, L3MBTL1 knockdown enhanced the sensitivity of hematopoietic stem/progenitor cells to erythropoietin (Epo), with increased Epo-induced phosphorylation of STAT5, AKT, and MAPK as well as detectable phosphorylation in the absence of Epo. Our data suggest that haploinsufficiency of L3MBTL1 contributes to some (20q−) myeloproliferative neoplasms, especially polycythemia vera, by promoting erythroid differentiation

L3MBTL1: A Polycomb Protein At the Node of Crosstalk Between the BMP4 and Hippo Signaling Pathways in Erythropoiesis

Abstract 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. Disclosures: Levine: Agios Pharmaceuticals: Research Funding