ALBERT

Description: Abstract 3440 Background: How components of the cytoskeleton regulate complex cellular responses is fundamental to understanding cellular function. Megakaryocyte Leukemia 1 (MKL1), an activator of serum response factor (SRF) transcriptional activity, plays critical roles in muscle, neuron, and megakaryocyte differentiation. Regulation of MKL1 subcellular localization is one mechanism by which a cell can control SRF activity with MKL1 localization to the nucleus being critical for its function as a transcriptional activator. MKL1 subcellular localization is cell-type specific; MKL1 is predominantly cytoplasmic in unstimulated fibroblasts and some muscle cell types until it is sequestered in the nucleus following actin polymerization. In contrast, MKL1 is constitutively localized to the nucleus in neuronal cells. Objective: We tested the hypothesis that MKL1 subcellular localization is tightly regulated in megakaryocytic cells during induction of maturation. Methods and Results: Using a human erythroleukemia (HEL) cell line, we systematically dissected the events that occur after 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced megakaryocytic differentiation to assess the relationships between RhoA activation, actin polymerization, MKL1 subcellular localization, and upregulation of SRF-target genes essential for megakaryocyte differentiation. In response to treatment with TPA, the percentage of HEL cells with predominantly nuclear localization went from

Description: Abstract 2502 Megakaryopoiesis involves the differentiation of progenitor cells into diploid megakaryocytes, which then undergo endomitosis and cytoplasmic maturation resulting in platelet release. Acute Megakaryoblastic Leukemia (AMKL) results when this process goes awry. Nearly one third of pediatric AMKL patients are infants who have the t (1;22) chromosomal translocation (t (1;22)-AMKL) resulting in fusion of the RNA Binding Motif 15 gene (RBM15) on chromosome 1 upstream of the transcriptional cofactor Megakaryoblastic Leukemia 1 gene (MKL1) on chromosome 22. Given that t (1;22)-AMKL primarily affects infants, the leukemia likely originates in utero when the hematopoietic system is in its embryonic stage.Our goal was to establish in vitro methods to study embryonic megakaryopoiesis and leukemogenesis using human and murine embryonic stem cells (hESCs and mESCs). Using the Invitrogen Gateway System, human MKL1 (hMKL1) and human RBM15-MKL1 (hRM) constructs were designed. H9-rtTA, a hESC line constitutively expressing the reverse transcriptional activator (rtTA) under the control of the Ubiquitin C promoter, were transduced with lentivirus harboring either hMKL1 or hRM with an IRES-GFP promoter under the control of the tetracycline responsive element (TRE) to derive doxycycline (dox) inducible cell lines. In the presence of dox, the rtTA binds to the TRE, causing transcriptional activation of the downstream transgene. hESC were co-cultured on murine OP-9 stromal cell layers with thrombopoietin to promote megakaryocytic differentiation. For construction of the inducible mESC lines, mESCs already containing the rtTA at the ROSA26 locus were electroporated with either murine MKL1 (mMKL1) or murine RBM15-MKL1 (mRM). Site-specific insertion of the transgenes using a cre recombinase and modified loxP sites placed mMKL1 or mRM under the control of a TRE. The inducible mMKL1 mESCs were differentiated into hematopoietic cells using embryoid body formation and subsequently co-cultured on OP-9s. The human and murine MKL1 and RM constructs were transiently transfected into 293FT cells and Western blotting was used to confirm their functionality. In the hESC studies, GFP expression was detected by flow cytometry 24 hrs after dox induction in both cell lines, with 54.7% (SD+3.1) GFP+ cells in hMKL1 transduced cells and 47.5% (SD+16.2) GFP+ cells in hRM transduced cells. hMKL1 protein was detected 24 hrs after dox induction; however, the hRM fusion protein was not detected in undifferentiated hESCs by Western blot or IP/IB after 6, 24, 48, 72, 96 hrs of dox exposure. Nearly one fourth of the cells transduced with hRM were GFP+ after only 6 hrs of doxycycline exposure. Interestingly, with continued dox exposure over six days, GFP expression in the undifferentiated hESCs declined, with a mean decrease in GFP expression of 14.5% in both cell lines. Successful incorporation of the vector carrying the hRM was confirmed by genomic PCR and resulted in dox-inducible expression of hRM mRNA detected by RT-PCR. Upon megakaryocytic differentiation, GFP and CD41 expression, detected by flow cytometry, occur in a mutually exclusive fashion in both MKL and RBM15-MKL1 transduced hESCs. In the mESC experiements, protein expression of mMKL1 in the undifferentiated cells was confirmed by Western blot 24, 48, and 72 hrs after dox addition. Expression of mRM protein in the undifferentiated mESCs was undetectable 6, 14, 24, 30, 48, or 72 hrs after dox addition. However, mRM mRNA was detected by qRT-PCR at 0, 6, 14, and 30 hours after dox treatment in the undifferentiated mESCs. Of note, transient transfection of undifferentiated mESCs with mRM resulted in protein expression by Western blot as early as 6 hrs after transfection. The fusion protein continued to be detected at 12 and 18 hrs after transfection, but by 24 hrs, it was no longer detectable. When differentiated, the mMKL1 mESC line no longer demonstrated inducible mMKL1 expression in the CD41+ (hematopoietic) population as determined by qRT-PCR. These findings confirm ongoing activity of the promoters in the undifferentiated hESC and mESC lines and indicate that the promoters may be silenced upon induction of hematopoietic differentiation of both hESC and mESC lines. In addition, the transient detection of RM after transfection into the mESCs suggests post-translational modification of the fusion protein likely results in its rapid degradation. Disclosures: No relevant conflicts of interest to declare.

Description: Serum response factor and its transcriptional cofactor MKL1 are critical for megakaryocyte maturation and platelet formation. We show that MKL2, a homologue of MKL1, is expressed in megakaryocytes and plays a role in megakaryocyte maturation. Using a megakaryocyte-specific Mkl2 knockout (KO) mouse on the conventional Mkl1 KO background to produce double KO (DKO) megakaryocytes and platelets, a critical role for MKL2 is revealed. The decrease in megakaryocyte ploidy and platelet counts of DKO mice is more severe than in Mkl1 KO mice. Platelet dysfunction in DKO mice is revealed by prolonged bleeding times and ineffective platelet activation in vitro in response to adenosine 5′-diphosphate. Electron microscopy and immunofluorescence of DKO megakaryocytes and platelets indicate abnormal cytoskeletal and membrane organization with decreased granule complexity. Surprisingly, the DKO mice have a more extreme thrombocytopenia than mice lacking serum response factor (SRF) expression in the megakaryocyte compartment. Comparison of gene expression reveals approximately 4400 genes whose expression is differentially affected in DKO compared with megakaryocytes deficient in SRF, strongly suggesting that MKL1 and MKL2 have both SRF-dependent and SRF-independent activity in megakaryocytopoiesis.

Description: Key Points Deletion of the erythroid enhancer of Bcl11a from the mouse genome does not affect viability or Bcl11a expression in nonerythroid lineages. Elevated levels of γ-globin in Bcl11a enhancer–deleted mice are comparable to those in erythroid-specific Bcl11a gene knockout mice.

Description: Abstract 2336 Background: The transcriptional cofactors, MKL1 and MKL2, are members of the myocardin related factor family, which bind and activate the transcription factor Serum Response Factor (SRF) (Cen et al J Cell Biochem, 2004). Megakaryocytes, large, highly polyploid myeloid cells give rise to platelets, which promote blood clotting in response to tissue damage. The Krause lab has reported a significant decrease in Mk ploidy with a 50% decrease in mature platelets in Mkl1 knockout (KO) mice, indicating that MKL1 may contribute to megakaryocyte maturation (Cheng et al Blood, 2009). The phenotype of the Mk specific (PF4-Cre) Srf conditional KO (SRF PF4-cKO) further supports the importance for the MKL1/SRF pathway in Mk differentiation (Halene et al Blood, 2010); although similar to the Mkl1 KO mouse, the Srf PF4-cKO mouse has a more severe phenotype with a 90% decrease in platelet counts suggesting the presence of compensatory proteins or redundant pathways in Mkl1 KO mice. Objective: This study addresses the hypothesis that MKL2 is critical for normal megakaryocytopoiesis particularly in the absence of MKL1. Methods and Results: Using bone marrow (BM) transplantation, we determined that the Mkl1 KO phenotype (decreased megakaryocyte ploidy and decreased platelets) is cell autonomous. Lethally irradiated WT recipients of Mkl1 KO BM cells had a 50% decrease in platelet count, as well as significantly decreased ploidy indicated by the percentage of Mk with 2N (60% and 40% in the recipients of Mkl1 KO vs WT BM, respectively, p

Description: Introduction Genome-wide association studies (GWAS) have ascertained numerous trait-associated common genetic variants localized to regulatory DNA. The hypothesis that regulatory variation accounts for substantial heritability has undergone scarce experimental evaluation. Common variation at BCL11A is estimated to explain ∼15% of the trait variance in fetal hemoglobin (HbF) level but the functional variants remain unknown. Materials and Methods We use chromatin immunoprecipitation (ChIP), DNase I sensitivity and chromosome conformation capture to evaluate the BCL11A locus in mouse and human primary erythroblasts. We extensively genotype 1,263 samples from the Collaborative Study of Sickle Cell Disease within three HbF-associated erythroid DNase I hypersensitive sites (DHSs) at BCL11A. We pyrosequence heterozygous erythroblasts to assess allele-specific transcription factor binding and gene expression. We conduct transgenic analysis by mouse zygotic microinjection and genome editing with transcription activator-like effector nucleases (TALENs) and clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) RNA-guided nucleases. Results Common genetic variation at BCL11A associated with HbF level lies in noncoding sequences decorated by an erythroid enhancer chromatin signature. Fine-mapping this putative regulatory DNA uncovers a motif-disrupting common variant associated with reduced GATA1 and TAL1 transcription factor binding, modestly diminished BCL11A expression and elevated HbF. This variant, rs1427407, accounts for the HbF association of the previously reported sentinel SNPs. The composite element functions in vivo as a developmental stage-specific lineage-restricted enhancer. Genome editing reveals that the enhancer is required in erythroid but dispensable in B-lymphoid cells for expression of BCL11A. We demonstrate species-specific functional components of the composite enhancer in mouse as compared to human erythroid precursor cells. The mouse sequences homologous to the human DHS sufficient to drive reporter activity are dispensable from the mouse composite element, whereas the adjacent DHS, whose human homolog does not direct reporter activity, is absolutely required for BCL11A expression. Conclusions We describe a comprehensive and widely applicable approach, including chromatin mapping followed by fine-mapping, allele-specific ChIP and gene expression studies, and functional analyses, to reveal causal variants and critical elements. We assert that functional validation of regulatory DNA ought to include perturbation of the endogenous genomic context by genome editing and not solely rely on in vitro or ectopic surrogate assays. These results validate the hypothesis that common variation modulates cell type-specific regulatory elements, and reveal that although functional variants themselves may be of modest impact, their harboring elements may be critical for appropriate gene expression. We speculate that species-level functional differences in components of the composite enhancer might partially account for differences in timing of globin gene expression among animals. We suggest that the GWAS-marked BCL11A enhancer represents a highly attractive target for therapeutic genome editing for the major b-hemoglobin disorders. Disclosures: No relevant conflicts of interest to declare.

Description: Common genetic variation associated with fetal hemoglobin (HbF) level and β-hemoglobin disorder clinical severity marks an erythroid enhancer within the BCL11A gene. The 12 kb intronic enhancer contains three ~1 kb erythroid DNase I hypersensitive sites (DHSs), termed +55, +58, and +62. Here we utilized a human adult-stage erythroid cell line to show by CRISPR-Cas9 mediated targeted deletion that the composite enhancer is required both for BCL11A expression and HbF repression. Because deletion of the entire enhancer is currently too inefficient to consider for a gene editing approach to hemoglobin disorders, we sought to define the critical features of the enhancer in its natural genomic context. We designed and synthesized a tiling pooled guide RNA (gRNA) library to conduct saturating mutagenesis of the enhancer sequences in situ using the CRISPR-Cas9 gene editing platform. The gRNAs direct Cas9 cleavage and non-homologous end-joining repair at discrete sites throughout the enhancer. By comparing the representation of lentiviral gRNA integrants in high and low HbF pools of the adult erythroid cells, we generated a functional map approaching nucleotide resolution of sequences within the enhancer influencing BCL11A regulation. We observed several discrete enhancer regions required for maximal expression. The largest effect was observed by producing mutations within a narrow functional core of the +58 DHS. These sequences include a GATA1 motif conserved among vertebrates located within a primate-specific context. This region constitutes an Achilles Heel for functional inactivation of the enhancer. We also identified rare genetic variants within the +58 DHS core in individuals with sickle cell disease that are associated with HbF level, independent of all known associations of common genetic variants. In parallel, we performed a similar saturating CRISPR mutagenesis screen of the corresponding murine Bcl11a enhancer. To our surprise, despite low-resolution evidence of conservation by primary sequence homology, syntenic genomic position, and shared chromatin signature, the mouse enhancer sequence determinants of BCL11A expression showed substantial functional divergence. The +58 orthologous sequences were dispensable whereas the +62 orthologous sequences were critically required in murine adult erythroid cells. These results were validated by producing targeted deletions in mouse and human adult erythroid cell lines. Furthermore we subjected cells to individual gRNAs to correlate individual nucleotide disruptions with loss of BCL11A expression. To substantiate the tissue-restricted effect of the enhancer mutations, we generated transgenic mice with deletion of the Bcl11a enhancer and found these sequences were dispensable for expression in developing neurons and B-lymphocytes (unlike conventional Bcl11a knockout) but essential for appropriate hemoglobin switching in vivo. We showed that in primary CD34+ hematopoietic stem and progenitor derived human erythroid precursors that delivery of an individual gRNA and Cas9 is sufficient to produce robust reinduction of HbF. These results validate the BCL11A erythroid enhancer as a promising therapeutic target. Our findings define the most favorable regions for generation of indel mutations in the BCL11A erythroid enhancer as a therapeutic genome editing strategy for HbF reinduction for the β-hemoglobin disorders. Disclosures Bauer: Biogen: Research Funding; Editas Medicine: Consultancy. Zhang:Editas Medicine: Membership on an entity's Board of Directors or advisory committees; Horizon Discovery: Membership on an entity's Board of Directors or advisory committees. Orkin:Editas Medicine: Membership on an entity's Board of Directors or advisory committees; Biogen: Research Funding; Pfizer: Research Funding; Sangamo Biosciences: Consultancy.

Description: Reactivation of fetal hemoglobin (HbF, α2γ2) expression in adults ameliorates the clinical symptoms in patients with the major β-hemoglobin disorders, sickle cell disease (SCD) and β-thalassemias. The zinc-finger protein BCL11A is a major modulator of hemoglobin switching and HbF silencing. BCL11A was initially identified by genome-wide association studies (GWAS) as a new HbF-associated gene. Down-regulation of BCL11A in primary human erythroid cells induces HbF expression. Knockout of BCL11A in mice impairs HbF silencing in adult erythroid cells. Most importantly, inactivation of BCL11A alone in humanized SCD mice corrects the hematologic and pathologic defects through high-level HbF induction. These studies established BCL11A as a genetically and functionally validated transcriptional regulator of HbF switching and silencing. In human and mouse erythroid cells, BCL11A is expressed as several isoforms, yet their individual roles in globin gene expression remain unexplored. Furthermore, the functional domains within the BCL11A protein responsible for its activity in HbF repression are largely unknown. To further understand the mechanistic roles of BCL11A in globin expression, we established a functional assay based on a BCL11A-null erythroid cell line generated by transcription activator-like effector nucleases (TALENs)-mediated deletion of an obligate erythroid-specific enhancer of BCL11A in murine erythroleukemia (MEL) cells. In the BCL11A-null cells, the expression of β-like embryonic globin genes is markedly induced (〉200-fold), consistent with the role of BCL11A in repression of murine embryonic globin genes. To examine the activity of known BCL11A isoforms in HbF silencing, we expressed various BCL11A isoforms in these engineered BCL11A-null cells. Ectopic expression of full-length BCL11A-XL isoform, but not the alternatively spliced, C-terminally truncated L isoform, restored the full repression of β-like embryonic globins in BCL11A-null cells. Since XL and L differ only by 91 amino acids containing three tandem C2H2-type zinc finger motifs, these results indicate that the C-terminal zinc finger motifs are indispensable for BCL11A-mediated transcriptional repression. To systemically define BCL11A functional domains for globin gene repression, we next generated a panel of BCL11A mutant cDNAs, including deletion of the N-terminal NuRD-interacting motif and one or more C2H2-type zinc finger domains. Analysis of various BCL11A mutants in the functional rescue assay identified several functional domains, including the N-terminal NuRD-interacting motif and five out of the six C2H2 zinc fingers, that are required for BCL11A-mediated repression. These findings provide the foundation for further molecular analysis of BCL11A functional domains in globin gene repression. BCL11A is known to interact with several transcriptional co-repressor complexes including Mi-2β/NuRD/HDAC1/HDAC2, LSD1/CoREST and SWI/SNF complexes, occupy discrete regions within the human β-globin cluster, and promote long-range chromosomal interactions. Our results suggest that BCL11A functional domains may be involved in protein-protein interactions, protein homo-/heterodimerization, and/or chromatin/DNA association that are required for its activity in HbF silencing. In summary, we demonstrate that several functional domains on BCL11A protein are indispensable for its transcriptional activity in HbF silencing. Further focused studies of BCL11A structure-function domains in HbF silencing not only will advance our understanding of the molecular mechanisms by which BCL11A controls the clinically important fetal-to-adult globin switch, but may identify novel cellular targets for therapeutic HbF induction in β-hemoglobinopathies. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points RhoA-induced actin polymerization promotes nuclear accumulation of MKL1 and transcriptional activation. Thrombopoietin activates nuclear accumulation of MKL1 and transcriptional activation in primary megakarocytes.