Transcription Factor BCL11A Research Articles

LIN28B-mediated expression of fetal hemoglobin and production of fetal-like erythrocytes from adult human erythroblasts ex vivo

AbstractReactivation of fetal hemoglobin (HbF) holds therapeutic potential for sickle cell disease and β-thalassemias. In human erythroid cells and hematopoietic organs, LIN28B and its targeted let-7 microRNA family, demonstrate regulated expression during the fetal-to-adult developmental transition. To explore the effects of LIN28B in human erythroid cell development, lentiviral transduction was used to knockdown LIN28B expression in erythroblasts cultured from human umbilical cord CD34+ cells. The subsequent reduction in LIN28B expression caused increased expression of let-7 and significantly reduced HbF expression. Conversely, LIN28B overexpression in cultured adult erythroblasts reduced the expression of let-7 and significantly increased HbF expression. Cellular maturation was maintained including enucleation. LIN28B expression in adult erythroblasts increased the expression of γ-globin, and the HbF content of the cells rose to levels >30% of their hemoglobin. Expression of carbonic anhydrase I, glucosaminyl (N-acetyl) transferase 2, and miR-96 (three additional genes marking the transition from fetal-to-adult erythropoiesis) were reduced by LIN28B expression. The transcription factor BCL11A, a well-characterized repressor of γ-globin expression, was significantly down-regulated. Independent of LIN28B, experimental suppression of let-7 also reduced BCL11A expression and significantly increased HbF expression. LIN28B expression regulates HbF levels and causes adult human erythroblasts to differentiate with a more fetal-like phenotype.

The transcriptional architecture of early human hematopoiesis identifies multilevel control of lymphoid commitment

Understanding how differentiation programs originate from within the gene expression landscape of hematopoietic stem cells (HSC) is crucial to develop new clinical therapies. We mapped the transcriptional dynamics underlying the first steps of commitment by tracking transcriptome changes in human HSC and eight early progenitor populations. Transcriptional programs are extensively shared, extend across lineage-potential boundaries, and are not strictly lineage-affiliated. Elements of stem, lymphoid and myeloid programs are retained in multi-lymphoid progenitors (MLP), reflecting a hybrid transcriptional state. Based on functional single cell analysis, BCL11A, SOX4 and TEAD1 governed transcriptional networks within MLPs, leading to B cell specification. Overall, we show that integrated transcriptome approaches can identify novel regulators of multipotency and uncover additional complexity in lymphoid commitment.

The BCL11A Transcription Factor Directly Activates RAG Gene Expression and V(D)J Recombination

Recombination-activating gene 1 protein (RAG1) and RAG2 are critical enzymes for initiating variable-diversity-joining (VDJ) segment recombination, an essential process for antigen receptor expression and lymphocyte development. The transcription factor BCL11A is required for B cell development, but its molecular function(s) in B cell fate specification and commitment is unknown. We show here that the major B cell isoform, BCL11A-XL, binds the RAG1 promoter and Erag enhancer to activate RAG1 and RAG2 transcription in pre-B cells. We employed BCL11A overexpression with recombination substrates in a cultured pre-B cell line as well as Cre recombinase-mediated Bcl11a(lox/lox) deletion in explanted murine pre-B cells to demonstrate direct consequences of BCL11A/RAG modulation on V(D)J recombination. We conclude that BCL11A is a critical component of a transcriptional network that regulates B cell fate by controlling V(D)J recombination.

The role of BCL11 transcription factors in mouse craniofacial suture development

Craniofacial sutures are the fibrous joints that unite the bones of the skull and are the major sites of bone growth during craniofacial development. These joints normally close postnatally, allowing the brain and the skull to reach the proper size and shape. The premature fusion of one or more sutures leads to a condition called craniosynostosis, which represents a congenital defect affecting approximately one in 2000 live births. Craniosynostosis results in craniofacial morphologic abnormalities and increased intracranial pressure, which can lead to severe neurological and cognitive deficits in children. Surgery, with its inherent risks, remains the only therapeutic modality. Therefore, it is important to understand the molecular mechanisms underlying craniosynostosis in order to develop targeted biologically‐based treatment.The transcription factors BCL11A and BCL11B play essential roles in the development of immune, central nervous, and cutaneous systems. We show that ablation of BCL11 transcription factors in mice leads to craniosynostosis, which results in severe midfacial hypoplasia and malocclusion. We conclude that BCL11A and BCL11B are essential for normal development of craniofacial bones and restrict premature suture fusion. The results suggest the possibility of modulation of BCL11 expression for the treatment of craniosynostosis.Grant Funding Source: DE021879 from NIDCR to ML

Corepressor-dependent silencing of fetal hemoglobin expression by BCL11A

Reactivation of fetal hemoglobin (HbF) in adults ameliorates the severity of the common β-globin disorders. The transcription factor BCL11A is a critical modulator of hemoglobin switching and HbF silencing, yet the molecular mechanism through which BCL11A coordinates the developmental switch is incompletely understood. Particularly, the identities of BCL11A cooperating protein complexes and their roles in HbF expression and erythroid development remain largely unknown. Here we determine the interacting partner proteins of BCL11A in erythroid cells by a proteomic screen. BCL11A is found within multiprotein complexes consisting of erythroid transcription factors, transcriptional corepressors, and chromatin-modifying enzymes. We show that the lysine-specific demethylase 1 and repressor element-1 silencing transcription factor corepressor 1 (LSD1/CoREST) histone demethylase complex interacts with BCL11A and is required for full developmental silencing of mouse embryonic β-like globin genes and human γ-globin genes in adult erythroid cells in vivo. In addition, LSD1 is essential for normal erythroid development. Furthermore, the DNA methyltransferase 1 (DNMT1) is identified as a BCL11A-associated protein in the proteomic screen. DNMT1 is required to maintain HbF silencing in primary human adult erythroid cells. DNMT1 haploinsufficiency combined with BCL11A deficiency further enhances γ-globin expression in adult animals. Our findings provide important insights into the mechanistic roles of BCL11A in HbF silencing and clues for therapeutic targeting of BCL11A in β-hemoglobinopathies.

Hematopoietic SIN Lentiviral Micro RNA-Mediated Silencing of BCL11A: Pre-Clinical Evidence for a Sickle Cell Disease Gene-Therapy Trial

Abstract 753Sickle cell disease (SCD) is caused by a mutation in the β-globin protein, leading to the polymerization of hemoglobin in deoxygenated conditions. The transcription factor BCL11A is a key regulator of developmental silencing of human fetal (γ-) globin, and also critical to repressing γ-globin in adult erythroid cells. A Bcl11a null mouse model carrying a transgenic YAC with a humanized β-globin locus (β-YAC) displays increased levels of fetal hemoglobin (HbF) in adult erythrocytes, and crossing these animals with a SCD murine model abolishes the SCD phenotype. BCL11A therefore constitutes a genetically validated target to induce HbF and reduce erythrocyte “sickling”, which would be predicted to ameliorate the phenotype of SCD patients. However, defective lymphoid development has been observed in Bcl11a genetic null mice, suggesting potential toxicities of BCL11A knockdown. We generated self-inactivating lentiviral vectors (LV) integrating miR-223 microRNA-based inhibitory shRNAs against BCL11A/Bcl11a. Since future clinical applications will need to balance efficacy and potential side effects, LVs were engineered to allow the comparison of effects of high level and ubiquitous versus erythroid lineage-restricted versus inducible expression of miRNA targeting BCL11A. LV backbones therefore included either a strong, viral LTR promoter/enhancer (SFFV-LV), a β-globin locus control region with the endogenous β-globin promoter (LCR-LV), or a tetracycline-inducible promoter (TET-LV). We performed assays to quantify transgenic miRNA expression and demonstrated that the BCL11A knockdown and induction of fetal globin gene output correlated with the expression of targeting miR223-based shRNA. Transduction at low MOI (=2) of murine hematopoietic stem cells (HSC) with LVs carrying the abovementioned regulatory elements leads to long-term engraftment and transgene expression in-vivo. Mice transplanted with SFFV-LV show fluorescent marking up to 70% across myeloid, lymphoid and erythroid lineages. The maximal BCL11A/Bcl11a mRNA and protein knock-down observed in primary hematopoietic cells in-vitro and in-vivo was 70%. This was confirmed in FACS-sorted bone marrow B-lymphoid (B220+) and erythroid progenitors (Terr119+/CD71+) and peripheral blood leukocytes at 4 months post-transplant. BCL11A/Bcl11a knockdown induced fetal globin gene expression depending on the vector backbone and targeting shRNA sequence employed. With SFFV-LV, we observed a 5–20 fold upregulation of fetal globin gene (γ/(ε+γ+β)) output in mice transplanted with HSCs containing the humanized β-YAC transgene. With TET-LV, the induction was dose-dependent and maximally caused a 150-fold increase in murine εγ-globin gene expression in-vitro. Human HSC transduced (MOI=2) with the LCR-LV and differentiated in-vitro resulted in a 3-fold increase of γ-globin mRNA in erythrocytes. SCD patient-derived HSC, which were transduced with LCR-LV (MOI=5) and transplanted into immunodeficient NSG mice, resulted in peripheral human erythrocytes that showed a reversal of the hemoglobin switch with a maximal induction 10% HbF as measured by flow cytometry. In a human ex-vivo B-lymphoid differentiation assay, SFFV-LV transduced (MOI=2) HSC populations with a 70% BCL11A knock-down showed no difference versus control in total cell numbers or in the sequential acquisition of CD43, CD19 and IgM (corresponding to physiological differentiation from common lymphoid progenitor to immature B-lymphocyte), thus showing no evidence for a differentiation block. Since IFN-response gene activation has been described with shRNA silencing and could potentially lead to HSC exhaustion, we quantified ISG20, ISG56 and OAS1 mRNA levels in human HSCs after miR-223-based SFFV-LV transduction (MOI=2). We observed less IFN-response gene activation in miR223-based SFFV-LV transduced HSC than in non-miRNA-based shRNA SFFV-LV transduced controls. In summary, our pre-clinical data demonstrates the potential efficacy of hematopoietic miRNA-mediated BCL11A/Bcl11a silencing to induce the expression of fetal hemoglobin in murine and human model systems, including primary cells. At the levels of BCL11A knock-down obtained, we did not observe any B-lymphoid toxicity. These results support the translation of LV-based miRNA-mediated BCL11A silencing into the clinical setting. Disclosures:No relevant conflicts of interest to declare.

The Oncoprotein BCL11A Binds to Orphan Nuclear Receptor TLX and Potentiates its Transrepressive Function

Nuclear orphan receptor TLX (NR2E1) functions primarily as a transcriptional repressor and its pivotal role in brain development, glioblastoma, mental retardation and retinopathologies make it an attractive drug target. TLX is expressed in the neural stem cells (NSCs) of the subventricular zone and the hippocampus subgranular zone, regions with persistent neurogenesis in the adult brain, and functions as an essential regulator of NSCs maintenance and self-renewal. Little is known about the TLX social network of interactors and only few TLX coregulators are described. To identify and characterize novel TLX-binders and possible coregulators, we performed yeast-two-hybrid (Y2H) screens of a human adult brain cDNA library using different TLX constructs as baits. Our screens identified multiple clones of Atrophin-1 (ATN1), a previously described TLX interactor. In addition, we identified an interaction with the oncoprotein and zinc finger transcription factor BCL11A (CTIP1/Evi9), a key player in the hematopoietic system and in major blood-related malignancies. This interaction was validated by expression and coimmunoprecipitation in human cells. BCL11A potentiated the transrepressive function of TLX in an in vitro reporter gene assay. Our work suggests that BCL11A is a novel TLX coregulator that might be involved in TLX-dependent gene regulation in the brain.

Bcl11a is required for neuronal morphogenesis and sensory circuit formation in dorsal spinal cord development

Dorsal spinal cord neurons receive and integrate somatosensory information provided by neurons located in dorsal root ganglia. Here we demonstrate that dorsal spinal neurons require the Krüppel-C(2)H(2) zinc-finger transcription factor Bcl11a for terminal differentiation and morphogenesis. The disrupted differentiation of dorsal spinal neurons observed in Bcl11a mutant mice interferes with their correct innervation by cutaneous sensory neurons. To understand the mechanism underlying the innervation deficit, we characterized changes in gene expression in the dorsal horn of Bcl11a mutants and identified dysregulated expression of the gene encoding secreted frizzled-related protein 3 (sFRP3, or Frzb). Frzb mutant mice show a deficit in the innervation of the spinal cord, suggesting that the dysregulated expression of Frzb can account in part for the phenotype of Bcl11a mutants. Thus, our genetic analysis of Bcl11a reveals essential functions of this transcription factor in neuronal morphogenesis and sensory wiring of the dorsal spinal cord and identifies Frzb, a component of the Wnt pathway, as a downstream acting molecule involved in this process.

Correction of Murine Sickle Cell Disease Through Interference with Fetal Hemoglobin Silencing

Abstract 351The switch from fetal (HbF, α2γ2) to adult (HbA, α2β2) hemoglobin is critical to the pathogenesis of the major hemoglobin disorders, sickle cell disease (SCD) and β-thalassemias. Increased HbF expression ameliorates the clinical severity of these conditions, forming the basis for the goal of elucidating mechanisms of globin switching and developing target-based therapies to enhance HbF production. The rationale for this therapeutic approach rests on the hypothesis that manipulation of a single target would lead to sufficient HbF reactivation for clinical benefit. Despite recent advances in understanding the complex processes of developmental globin switching and HbF silencing, this hypothesis is untested. The transcription factor BCL11A is a critical mediator of HbF switching and silencing. Previously we showed that BCL11A controls globin switching in mice and silencing of HbF expressed from human β-globin locus transgenes in mouse fetal liver, and contributes to HbF silencing in primary human erythroid cells. Furthermore, we demonstrated that BCL11A is required in vivo for HbF silencing in adult animals, yet dispensable for red cell production. These findings illustrate favorable features of BCL11A as a potential target for reactivation of HbF in the β-hemoglobinopathies.Here, we test whether impairment of BCL11A is sufficient to ameliorate disease symptoms in a relevant preclinical model of a major β-hemoglobin disorder, the humanized SCD mouse model (“Berkeley” SCD model, Paszty et al. Science 278:876–878, 1997). These mice express exclusively human sickle hemoglobin and faithfully recapitulate the principal hematologic and histopathologic phenotypes of patients with SCD. We introduced BCL11A-null alleles into the “Berkeley” SCD model (SCD Bcl11afl/fl EpoR-Cre+, hereafter called SCD/Bcl11a−/− mice). Loss of BCL11A in SCD animals restored the hematologic deficits to normal values. Compared with sickle animals, SCD/Bcl11a−/− mice have marked increases in RBC counts (6.4 ± 0.5 to 9.8 ± 0.4 × 1012/L; non-sickle control 10.1 ± 0.2 × 1012/L ) and hemoglobin level (7.8 ± 0.6 to 13.6 ± 0.7 g/L; non-sickle control 13.1 ± 0.3 g/L), accompanied by a significant reduction in reticulocyte counts (38.2% ± 3.9% to 7.0% ± 0.3%; non-sickle control 3.1% ± 0.6%). Consistent with correction of hematologic parameters, sickled erythrocytes were absent in the peripheral blood of SCD/Bcl11a−/− mice and red cell lifespan was substantially normalized. In addition, tissue damage in multiple organs was prevented in SCD/Bcl11a−/− mice. Urine osmolality of SCD/Bcl11a−/− mice was restored (from 1037 ± 82 mOsm in SCD mice to 2133 ± 333 mOsm in SCD/Bcl11a−/− mice), indicating improved renal function.To assess the extent to which these phenotypic findings were related to HbF induction, we determined the level of γ-globin expression and cellular distribution of HbF in SCD/Bcl11a−/− mice. Compared with SCD mice, expression of γ-globin genes was markedly elevated in adult SCD/Bcl11a−/− mice (1.3% to 28.3% of total β-like human globins; P = 0.00017). Peripheral blood of control and SCD mice contained few F cells (3.6% and 7.2%, respectively). In contrast, peripheral blood of SCD/Bcl11a−/− mice displayed strong pancellular staining of HbF, and F cells contributed to 85.1% of total RBCs (P = 3.9 × 10−5). The level of HbF expression achieved in the absence of BCL11A is well beyond that needed to impede HbS polymerization.In conclusion, we demonstrate that inactivation of a single component involved in HbF regulation, the transcription factor BCL11A, provides phenotypic correction of SCD in a mouse model. Our findings provide a crucial “proof of principle” for targeted reactivation of HbF as a realistic therapeutic goal. Disclosures:No relevant conflicts of interest to declare.

Genetic modifiers of HbF and response to hydroxyurea in sickle cell disease

Fetal hemoglobin (HbF) levels are generally inversely proportional to severity of sickle cell disease (SCD) for given sickle phenotypes. Molecular regulation of HbF occurs through complex interactions cis and trans to the beta globin gene locus. Novel insights made through population-based genetic epidemiologic studies of non-anemic populations were replicated in SCD groups, despite large differences in HbF levels. Identification of the lymphoid transcription factor BCL11A as a key suppressor of HbF expression validates approaches using population genetics to study HbF expression. We review these methods and findings, and speculate on applying pharmaco-genetics to optimize hydroxyurea therapy aimed at increasing HbF.

Transcriptional silencing of γ-globin by BCL11A involves long-range interactions and cooperation with SOX6

The developmental switch from human fetal (gamma) to adult (beta) hemoglobin represents a clinically important example of developmental gene regulation. The transcription factor BCL11A is a central mediator of gamma-globin silencing and hemoglobin switching. Here we determine chromatin occupancy of BCL11A at the human beta-globin locus and other genomic regions in vivo by high-resolution chromatin immunoprecipitation (ChIP)-chip analysis. BCL11A binds the upstream locus control region (LCR), epsilon-globin, and the intergenic regions between gamma-globin and delta-globin genes. A chromosome conformation capture (3C) assay shows that BCL11A reconfigures the beta-globin cluster by modulating chromosomal loop formation. We also show that BCL11A and the HMG-box-containing transcription factor SOX6 interact physically and functionally during erythroid maturation. BCL11A and SOX6 co-occupy the human beta-globin cluster along with GATA1, and cooperate in silencing gamma-globin transcription in adult human erythroid progenitors. These findings collectively demonstrate that transcriptional silencing of gamma-globin genes by BCL11A involves long-range interactions and cooperation with SOX6. Our findings provide insight into the mechanism of BCL11A action and new clues for the developmental gene regulatory programs that function at the beta-globin locus.

During Terminal Differentiation, HbF-Inducing Cytokines Cause Decreased Expression and Reduced Globin Locus Occupancy of BCL11A in Human Erythroblasts.

Abstract 457Genetic association studies and gene regulation studies demonstrate that the transcription factor BCL11A is a regulator of fetal hemoglobin (HbF) expression in humans. Cytokine signal transduction also regulates fetal hemoglobin expression in cultured adult human erythroblasts. To further explore the potential for BCL11A in the cytokine-mediated induction of HbF during adult erythropoiesis, transcript and protein expression levels of BCL11A were measured during erythroblast differentiation. BCL11A expression was detected at all stages of erythroid differentiation with the highest level expression in proerythroblasts during the first week in culture under both low-HbF (%HbF ≤3) and high-HbF (%HbF ≥30) culture conditions. Despite a reduction in BCL11A mRNA expression, Western analyses failed to demonstrate reduced levels of BCL11A nuclear protein expression at the proerythroblast stage of differentiation. However, BCL11A protein expression in the high-HbF producing cells was reduced relative to the low-HbF cells during the later period of culture as the cells underwent terminal differentiation. During this later culture period, hemoglobinization occurred, and cells grown in the high-HbF condition revealed a pancellular distribution of HbF compared with a heterocellular distribution in the low-HbF culture condition. Chromatin immunoprecipitation further demonstrated that the addition of HbF-inducing cytokines caused a nearly complete loss of BCL11A chromatin occupancy within the beta-globin locus under the high-HbF culture condition. Specifically, the loss of chromatin occupancy was detected in a region approximately 3 kb downstream of the (A)gamma-globin gene. Further examination of this genomic region demonstrated several BCL11A binding domains located on a cluster of non-coding, intronless RNAs previously named “BGL3” that possess an expression pattern in vivo that is largely restricted to the fetal-liver. In addition to increased and pancellular expression of fetal hemoglobin in the high-HbF erythroblasts, the loss of BCL11A chromatin occupancy in that region of the beta-globin locus was associated with increased expression of BGL3 mRNA (GenBank: AY034471) measured by RT-PCR. These findings demonstrate that defined combinations of cytokines regulate the expression level and chromatin occupancy of BCL11A in adult human erythroblasts as they undergo terminal differentiation. In addition to inheritance and ontogeny, the data also support a role for BCL11A in the regulation of HbF by cytokine signal transduction. Disclosures:No relevant conflicts of interest to declare.