The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform

Stuart H Orkin - One of the best experts on this subject based on the ideXlab platform.

  • hemoglobin switching s surprise the versatile transcription factor BCL11A is a master repressor of fetal hemoglobin
    Current Opinion in Genetics & Development, 2015
    Co-Authors: Daniel E Bauer, Stuart H Orkin
    Abstract:

    The major disorders of β-globin, sickle cell disease and β-thalassemia, may be ameliorated by expression of the fetal gene paralog γ-globin. Uncertainty regarding the mechanisms repressing fetal hemoglobin in the adult stage has served as a puzzle of developmental gene regulation as well as a barrier to rational therapeutic design. Recent genome-wide association studies implicated the zinc-finger transcriptional repressor BCL11A in fetal hemoglobin regulation. Extensive genetic analyses have validated BCL11A as a potent repressor of fetal hemoglobin level. Studies of BCL11A exemplify how contextual gene regulation may often be the substrate for trait-associated common genetic variation. These discoveries have suggested novel rational approaches for the β-hemoglobin disorders including therapeutic genome editing.

  • optimization of BCL11A knockdown by mirna scaffold embedded shrnas leading to enhanced induction of fetal hemoglobin in erythroid cells for the treatment of beta hemoglobinopathies
    Blood, 2014
    Co-Authors: Christian Brendel, Sophia C Kamran, Daniel E Bauer, Swaroopa Guda, Raffaele Renella, Matthew C. Canver, Michael D. Milsom, James E Thornton, Helen De Boer, Stuart H Orkin
    Abstract:

    Repression of the BCL11A protein could represent a therapeutic target for beta-hemoglobinopathies, as its knock-down has been shown to induce the expression of the fetal HBG (y-globin) gene ultimately leading to increased levels of the fetal hemoglobin tetramer (HbF, a2y2). In mice, BCL11A is a key repressor of the murine HBG homolog Hbb-y. RNA interference (RNAi) technology using short hairpin RNAs (shRNAs) expressed via pol III promoters has been used to modulate gene expression in a variety of mammalian cell types. However, we found a negative impact of BCL11A knockdown on hematopoietic stem cells (HSCs), limiting the repopulation efficiency and long-term engraftment after genetic modification, which is a major impediment for its translation into human therapeutic applications. To achieve lineage-specific targeting of mRNAs in an attempt to reduce HSC toxicity, expression of shRNAs via pol II promoters is required, necessitating embedding the shRNA in mammalian microRNA (shRNAmir) sequences for expression and processing. To achieve optimal knockdown of the BCL11A transcription factor in erythroid progenitor and precursor cells, we first compared the efficiency of mRNA modulation via pol III (U6-promoter) vs pol II (SFFV-promoter) based lentiviral vectors. We demonstrate a 100-1000 fold lower Hbb-y induction using shRNAmir vs pol III mediated shRNA vector backbones due to reduced BCL11A knockdown efficiency. In order to understand the molecular basis for these differences, small RNA sequence analysis was performed on murine erythroleukemia cells (MEL) cells transduced by multiple shRNA–shRNAmir pairs. We show that shRNAs expressed via a U6 promoter yield guide strand sequences which differ by a 2-4 nt shift compared to pol II driven (shRNAmir) mature guide strand sequences. RNA sequencing demonstrated that the stretch of uridines making up part of the pol III termination signal is transcribed and included at the 3’ end of the shRNA. This results in the generation of mature guide strand sequences with an alternative seed sequence compared to the predicted sequence and compared to miRNA embedded shRNAs. The difference in the seed sequences between the two expression systems strongly influences the efficacy of target gene knockdown, leading to reduced knockdown in pol II based vectors. We engineered a 4bp shift into guide strands of shRNAmirs that resulted in a faithfully processed shRNA sequence (a mature guide strand sequence identical to U6-driven sh-RNAs) and improved knock-down efficiency of BCL11A at the protein level in most cases. The improved knockdown of BCL11A was associated with a 100-300-fold enhancement of Hbb-y induction in MEL cells. Based on these results, we propose a modified strategy for the prospective design of shRNAmirs derived from shRNA screens in pol III vector backbones to achieve lineage-specific regulation of target genes. Targeted expression of shRNAmiRs to the erythroid compartment driven by a b-globin promoter/LCR element circumvented the detrimental effect on HSC engraftment, while still mediating efficient BCL11A knockdown, leading to high y-globin induction and formation of substantial amounts of fetal hemoglobin in human CD34-derived erythroid cells in vitro. Disclosures No relevant conflicts of interest to declare.

  • Identification Of BCL11A Structure-Function Domains For Fetal Hemoglobin Silencing
    Blood, 2013
    Co-Authors: Daniel E Bauer, Cong Peng, Elenoe C. Smith, Stuart H Orkin
    Abstract:

    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.

  • genetics and epigenetics of fetal hemoglobin control
    Blood, 2013
    Co-Authors: Stuart H Orkin
    Abstract:

    Expression of fetal hemoglobin (HbF, α2γ2) greatly ameliorates the severity of the major hemoglobin disorders, sickle cell disease (SCD), and the β-thalassemias. Efforts to reactivate HbF in adults with these disorders have relied on empirical observations or therapeutic modalities that are indirect. A major goal for the field is the development of targeted reactivation of HbF through relief of γ-globin gene silencing. The regulatory factors that participate in the switch from HbF to HbA in ontogeny and in γ-gene silencing in the adult have been elusive, therefore precluding mechanism-based reactivation of HbF. Recent findings, largely derived from genome-wide association studies (GWAS), have transformed the current understanding of globin switching. This presentation will review recent evidence supporting direct involvement of the zinc-finger repressor protein BCL11A in both developmental switching of globins and HbF silencing in the adult. These studies include the impact of naturally occurring genetic variation at the BCL11A locus on HbF levels, proof-of-principle experiments in genetically engineered mice suggesting that interference with BCL11A action alone may be sufficient to provide therapeutic elevation of HbF, and the nature of protein partners of BCL11A that may mediate some aspects of BCL11A function. Recent findings on the manner in which genetic variation within the BCL11A locus influences BCL11A expression provide special insight into quantitative aspects of HbF regulation and raise the possibility of new strategies to cripple BCL11A. The opportunities and challenges for the development of mechanism-based reactivation of HbF will be discussed in the context of ongoing efforts to exploit small molecule and genetic approaches. The tools are in hand to translate an improved understanding of globin gene regulation for the benefit of patients with the major hemoglobin disorders. Disclosures: No relevant conflicts of interest to declare.

  • corepressor dependent silencing of fetal hemoglobin expression by BCL11A
    Proceedings of the National Academy of Sciences of the United States of America, 2013
    Co-Authors: Jian Xu, Daniel E Bauer, Stuart H Orkin, Marc A Kerenyi, Thuy D Vo, Jennifer J Trowbridge, Gail Mandel
    Abstract:

    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.

Vijay G Sankaran - One of the best experts on this subject based on the ideXlab platform.

  • regulation of the fetal hemoglobin silencing factor BCL11A
    Annals of the New York Academy of Sciences, 2016
    Co-Authors: Anindita Basak, Vijay G Sankaran
    Abstract:

    : The clinical severity of sickle cell disease and β-thalassemia, the major disorders of β-globin, can be ameliorated by increased production of fetal hemoglobin (HbF). Here, we provide a brief overview of the fetal-to-adult hemoglobin switch that occurs in humans shortly after birth and review our current understanding of one of the most potent known regulators of this switching process, the multiple zinc finger-containing transcription factor BCL11A. Originally identified in genome-wide association studies, multiple orthogonal lines of evidence have validated BCL11A as a key regulator of hemoglobin switching and as a promising therapeutic target for HbF induction. We discuss recent studies that have highlighted its importance in silencing the HbF-encoding genes and discuss opportunities that exist to further understand the regulation of BCL11A and its mechanism of action, which could provide new insight into opportunities to induce HbF for therapeutic purposes.

  • correction of sickle cell disease in adult mice by interference with fetal hemoglobin silencing
    Science, 2011
    Co-Authors: Cong Peng, Zhen Shao, Yuko Fujiwara, Vijay G Sankaran, Erica B. Esrick, Gregory C. Ippolito, Bryan G Chong, Benjamin L. Ebert
    Abstract:

    Persistence of human fetal hemoglobin (HbF, α2γ2) in adults lessens the severity of sickle cell disease (SCD) and the β-thalassemias. Here, we show that the repressor BCL11A is required in vivo for silencing of γ-globin expression in adult animals, yet dispensable for red cell production. BCL11A serves as a barrier to HbF reactivation by known HbF inducing agents. In a proof-of-principle test of BCL11A as a potential therapeutic target, we demonstrate that inactivation of BCL11A in SCD transgenic mice corrects the hematologic and pathologic defects associated with SCD through high-level pancellular HbF induction. Thus, interference with HbF silencing by manipulation of a single target protein is sufficient to reverse SCD.

  • Reactivation of Silenced Human HbF In Adult Mice by Inactivation of BCL11A
    Blood, 2010
    Co-Authors: Vijay G Sankaran, Erica B. Esrick, Benjamin L. Ebert, Stuart H Orkin
    Abstract:

    Abstract 643 Persistence of fetal hemoglobin (HbF) in adults ameliorates severity of sickle cell disease and β-thalassemia. The transcriptional repressor BCL11A is a newly identified critical mediator of hemoglobin switching and HbF silencing. Previously we showed that BCL11A knockout mice with a human β-globin gene cluster transgene (β-locus mice) fail to silence mouse embryonic globins and human fetal (γ-) globins in adult erythroid cells of the fetal liver. The ratio of human fetal to adult globin RNA in the fetal liver of BCL11A knockout mice is inverted compared to controls, such that γ constitutes >90% of the β-like human expression at embryonic day (E)14.5 and >75% at E18.5. These findings provide compelling evidence that BCL11A controls hemoglobin switching in vivo. These BCL11A-null mice are postnatally lethal. Thus, the extent to which developmental silencing of HbF expression is dependent on BCL11A in adult animals cannot be assessed. Here we examined by formal genetics the contribution of BCL11A to HbF silencing through conditional inactivation of BCL11A in β-locus mice. Mice harboring erythroid-specific inactivation of BCL11A develop normally. As in the conventional knockout, the hemoglobin switching fails to occur in the fetal liver, such that γ constitutes >80% of the β-like human globins. After birth, the level of γ-globin is maintained persistently and contributes 43% in newborns, 25% in 4-week-old young adults, and 12% in 30-week-old adults. Even at this late time, the level of γ-globin is >500-fold that of control mice. The viability of these mice, taken together with ostensibly normal red cell production, indicates that BCL11A has few, if any, non-critical globin targets. To determine if loss of BCL11A in the adult reactivates γ-globin genes that were previously silenced developmentally, we conditionally inactivated BCL11A through induction of Mx1-Cre. Acute loss of BCL11A in adult bone marrows leads to persistent reactivation of γ-globin (>500-fold derepression compared to controls). Thus, BCL11A is required in vivo to maintain HbF silencing in adults. Gradual silencing of γ-globin in BCL11A-null adults suggests the presence of additional silencing pathways in the mouse trans-acting environment. In support of this hypothesis, we observed that the levels of DNA methylation at the γ-globin promoters are substantially decreased in BCL11A-null erythroid precursors from E14.5 fetal livers (40%), bone marrows of young (59%) and old (66%) mice. The levels are >80% in control mice at all ages. Loss of DNA methylation at γ-promoters indicates that developmental silencing of HbF is impaired upon loss of BCL11A. The gradual increase of DNA methylation indicates that the γ-globin genes are subject to epigenetic silencing in the absence of BCL11A in the mouse trans-acting environment. Histone deacetylases (HDACs) are potential molecular targets mediating HbF induction. By high-resolution ChIP-chip analysis, we demonstrate that HDAC1 occupies the γ-globin genes in primary human adult erythroid precursors. Administration of a HDAC inhibitor (Vorinostat) to BCL11A conditional knockout mice leads to further elevation of HbF, suggesting that the combination of BCL11A downregulation and HDAC inhibition may provide a strategy for efficient HbF augmentation. Collectively, these findings provide important insight into the role of BCL11A in HbF silencing in adults and new clues for target-based therapeutics in patients with hemoglobin disorders. Disclosures: No relevant conflicts of interest to declare.

  • Transcriptional silencing of fetal hemoglobin by BCL11A
    Annals of the New York Academy of Sciences, 2010
    Co-Authors: Vijay G Sankaran, Stuart H Orkin
    Abstract:

    The beta-thalassemia syndromes are a major global health problem. Increased levels of fetal hemoglobin (HbF) ameliorate the clinical symptoms seen in this disease. By taking advantage of the natural variation in the level of HbF in various populations, we and others identified several common genetic variants in three major loci that regulate HbF levels. One of these variants resides in the gene BCL11A. We have studied the role of this gene product and established that BCL11A maintains silencing of gamma-globin expression in adult erythroid cells and functions as a direct transcriptional regulator of the fetal to adult hemoglobin switch in humans. Moreover, we found that BCL11A plays a central role in the evolutionarily divergent globin gene switches of mammals. As a factor critical for gamma-globin gene silencing, BCL11A should be considered as a therapeutic target to increase HbF in a directed manner in beta-thalassemia patients.

  • transcriptional silencing of fetal hemoglobin by BCL11A
    Annals of the New York Academy of Sciences, 2010
    Co-Authors: Jian Xu, Stuart H Orkin, Vijay G Sankaran
    Abstract:

    The β-thalassemia syndromes are a major global health problem. Increased levels of fetal hemoglobin (HbF) ameliorate the clinical symptoms seen in this disease. By taking advantage of the natural variation in the level of HbF in various populations, we and others identified several common genetic variants in three major loci that regulate HbF levels. One of these variants resides in the gene BCL11A. We have studied the role of this gene product and established that BCL11A maintains silencing of γ-globin expression in adult erythroid cells and functions as a direct transcriptional regulator of the fetal to adult hemoglobin switch in humans. Moreover, we found that BCL11A plays a central role in the evolutionarily divergent globin gene switches of mammals. As a factor critical for γ-globin gene silencing, BCL11A should be considered as a therapeutic target to increase HbF in a directed manner in β-thalassemia patients.

Jian Xu - One of the best experts on this subject based on the ideXlab platform.

  • an erythroid enhancer of BCL11A subject to genetic variation determines fetal hemoglobin level
    Science, 2013
    Co-Authors: Daniel E Bauer, Sophia C Kamran, Zhen Shao, Jian Xu, Yuko Fujiwara, Elenoe C. Smith, Matthew C. Canver, Samuel Lessard, Luca Pinello
    Abstract:

    Genome-wide association studies (GWASs) have ascertained numerous trait-associated common genetic variants, frequently localized to regulatory DNA. We found that common genetic variation at BCL11A associated with fetal hemoglobin (HbF) level lies in noncoding sequences decorated by an erythroid enhancer chromatin signature. Fine-mapping uncovers a motif-disrupting common variant associated with reduced transcription factor (TF) binding, modestly diminished BCL11A expression, and elevated HbF. The surrounding sequences function in vivo as a developmental stage–specific, lineage-restricted enhancer. Genome engineering reveals the enhancer is required in erythroid but not B-lymphoid cells for BCL11A expression. These findings illustrate how GWASs may expose functional variants of modest impact within causal elements essential for appropriate gene expression. We propose the GWAS-marked BCL11A enhancer represents an attractive target for therapeutic genome engineering for the β-hemoglobinopathies.

  • corepressor dependent silencing of fetal hemoglobin expression by BCL11A
    Proceedings of the National Academy of Sciences of the United States of America, 2013
    Co-Authors: Jian Xu, Daniel E Bauer, Stuart H Orkin, Marc A Kerenyi, Thuy D Vo, Jennifer J Trowbridge, Gail Mandel
    Abstract:

    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.

  • hbf associated genetic variation marks an erythroid regulatory element essential for BCL11A transcription and subsequent stage specific globin expression
    Blood, 2012
    Co-Authors: Daniel E Bauer, Sophia C Kamran, Zhen Shao, Richard A Voit, John A Stamatoyannopoulos, Matthew H. Porteus, Jian Xu, Yuko Fujiwara, Stuart H Orkin
    Abstract:

    Abstract 828 Genome-wide association studies (GWAS) have identified common genetic variation within a 14-kb segment of intron-2 of the BCL11A gene that is associated with the level of fetal hemoglobin (HbF) and the clinical severity of the β-globin disorders sickle cell disease and β-thalassemia. Functional studies have validated BCL11A as a major repressor of HbF. Here, we explore the consequences of genetic variation on BCL11A expression. The HbF-associated variants overlap BCL11A sequences that possess an evolutionarily conserved enhancer chromatin signature, including the presence of H3K4me1 and H3K27ac and the absence of H3K4me3 histone modifications. Moreover, these sequences show a unique pattern of DNase I hypersensitivity in erythroid precursors compared to other BCL11A-expressing lineages including fetal brain and B-lymphocytes. Occupancy of the major erythroid transcription factors GATA1 and TAL1 is observed at these DNase I hypersensitive sites (DHS). Chromosome conformation capture studies demonstrate that these DHSs form a long-range interaction with the BCL11A promoter. To address the function of the putative regulatory element, we generated transgenic mice with the 12-kb intronic region attached to a minimal promoter and reporter gene. The element enhanced reporter gene expression in an erythroid-lineage restricted, definitive erythroid stage-specific manner. The minimal enhancer activity was mapped to the +58-kb DHS. Similar enhancer activity was observed in primary human erythroid precursors. To address the possible requirement of this element for BCL11A expression, we deleted the corresponding region in adult-stage murine erythroleukemia cells with TALEN-mediated gene targeting. Expression of BCL11A was dramatically reduced and embryonic globins Hbby and Hbb-bh1 were markedly derepressed with concomitant reduction of adult globin Hbb-b1. Analysis of heterozygous primary human erythroid precursors revealed that low-HbF haplotype alleles are preferentially occupied by GATA1 and TAL1 at sequences surrounding rs1427407. A composite half-E-box–GATA motif is disrupted at this SNP in the high-HbF allele. Furthermore, the low-HbF haplotype allele of BCL11A is expressed at a 1.7-fold higher level relative to the high-HbF allele. These data demonstrate that HbF-associated genetic variation in BCL11A modulates the function of a critical erythroid enhancer of BCL11A, and consequently expression of BCL11A itself. The quantitative level of BCL11A in turn regulates the relative expression of the stage-specific globin genes. The essential erythroid enhancer of the BCL11A gene might itself serve as a target for genetic manipulation as a novel approach to the human β-globin disorders. Disclosures: Porteus: Alex9s Lemonade Stand Foundation: Research Funding.

  • functional evaluation of hbf associated region of BCL11A locus
    Blood, 2011
    Co-Authors: Daniel E Bauer, John A Stamatoyannopoulos, Jian Xu, Yuko Fujiwara, Stuart H Orkin
    Abstract:

    Abstract 2148 The severity of the major β-globin disorders, sickle cell disease and β-thalassemia, is modified by the residual level of fetal hemoglobin (HbF, α2γ2). Reactivation of HbF by derepression of γ-globin gene expression constitutes a major therapeutic goal, though an incomplete understanding of globin gene regulation has limited such efforts. The postnatal level of HbF is a highly heritable quantitative trait. Genome-wide association (GWA) studies have identified several HbF-associated loci, including the BCL11A gene. BCL11A has been demonstrated to be a bona fide repressor of γ-globin expression in mouse and man. However, the mechanisms by which common genetic variation modulate BCL11A expression remain poorly understood. Numerous GWA studies, conducted in both healthy individuals and those with hemoglobinopathies, and including populations of European, Asian, and African ancestries, have consistently found that HbF-associated variants cluster within a 14-kb region of the second intron of BCL11A . At least twenty HbF-associated SNPs have been identified within this region, with various of these genetic markers considered as “tag SNPs” most strongly associated with HbF levels depending on the particular GWA study. Based on patterns of linkage disequilibrium among SNPs, several common haplotypes have been identified. The tag SNP-dense intronic region, more than 55 kb downstream of the BCL11A transcription start site, demonstrates evolutionary sequence conservation. Here we show that this region of BCL11A is decorated by epigenetic features in human erythroid precursors indicative of distal regulatory potential. Specifically, this intronic region is marked by the presence of the histone modifications H3K4me1 and H3K27ac and the absence of H3K4me3, indicating an enhancer signature. The erythroid transcription factors GATA1 and SCL/TAL1 co-occupy discrete sequences within this region, with the binding peaks of these master erythroid regulators overlapping those of the histone marks. Furthermore, this tag SNP-dense intronic region of BCL11A displays CpG hypomethylation and exhibits DNase I hypersensitive sites restricted to the erythroid lineage but not other lineages that express BCL11A. Moreover, in in vivo transient transgenic assays, this region displays erythroid-restricted enhancer activity. A minimal 1.8-kb sequence possessing erythroid enhancer function has been mapped. This region includes GATA1 and SCL/TAL1 binding sites. We hypothesize that common polymorphisms are in linkage disequilibrium with functional variants that modulate erythroid enhancer activity. These studies further delineate pathways critical for hemoglobin switching and serve as an example of how common genetic variation may influence traits by affecting non-coding regulatory elements. Disclosures: No relevant conflicts of interest to declare.

  • transcriptional silencing of fetal hemoglobin by BCL11A
    Annals of the New York Academy of Sciences, 2010
    Co-Authors: Jian Xu, Stuart H Orkin, Vijay G Sankaran
    Abstract:

    The β-thalassemia syndromes are a major global health problem. Increased levels of fetal hemoglobin (HbF) ameliorate the clinical symptoms seen in this disease. By taking advantage of the natural variation in the level of HbF in various populations, we and others identified several common genetic variants in three major loci that regulate HbF levels. One of these variants resides in the gene BCL11A. We have studied the role of this gene product and established that BCL11A maintains silencing of γ-globin expression in adult erythroid cells and functions as a direct transcriptional regulator of the fetal to adult hemoglobin switch in humans. Moreover, we found that BCL11A plays a central role in the evolutionarily divergent globin gene switches of mammals. As a factor critical for γ-globin gene silencing, BCL11A should be considered as a therapeutic target to increase HbF in a directed manner in β-thalassemia patients.

Daniel E Bauer - One of the best experts on this subject based on the ideXlab platform.

  • Strict in vivo specificity of the BCL11A erythroid enhancer
    Blood, 2016
    Co-Authors: Elenoe C. Smith, Yuko Fujiwara, Falak Sher, Sidinh Luc, Donyell M. Croney, Mollie B. Woodworth, Luciano C. Greig, M. Nguyen, Jeffrey D. Macklis, Daniel E Bauer
    Abstract:

    BCL11A, a repressor of human fetal (γ-)globin expression, is required for immune and hematopoietic stem cell functions and brain development. Regulatory sequences within the gene, which are subject to genetic variation affecting fetal globin expression, display hallmarks of an erythroid enhancer in cell lines and transgenic mice. As such, this enhancer is a novel, attractive target for therapeutic gene editing. To explore the roles of such sequences in vivo, we generated mice in which the orthologous 10-kb intronic sequences were removed. BCL11A enhancer–deleted mice, BCL11A (Δenh), phenocopy the BCL11A-null state with respect to alterations of globin expression, yet are viable and exhibit no observable blood, brain, or other abnormalities. These preclinical findings provide strong in vivo support for genetic modification of the enhancer for therapy of hemoglobin disorders.

  • Lineage-specific BCL11A knockdown circumvents toxicities and reverses sickle phenotype
    The Journal of clinical investigation, 2016
    Co-Authors: Christian Brendel, Daniel E Bauer, Swaroopa Guda, Raffaele Renella, Matthew C. Canver, Young-jo Kim, Matthew M. Heeney, Denise Klatt, Jonathan Fogel, Michael D. Milsom
    Abstract:

    Reducing expression of the fetal hemoglobin (HbF) repressor BCL11A leads to a simultaneous increase in γ-globin expression and reduction in β-globin expression. Thus, there is interest in targeting BCL11A as a treatment for β-hemoglobinopathies, including sickle cell disease (SCD) and β-thalassemia. Here, we found that using optimized shRNAs embedded within an miRNA (shRNAmiR) architecture to achieve ubiquitous knockdown of BCL11A profoundly impaired long-term engraftment of both human and mouse hematopoietic stem cells (HSCs) despite a reduction in nonspecific cellular toxicities. BCL11A knockdown was associated with a substantial increase in S/G2-phase human HSCs after engraftment into immunodeficient (NSG) mice, a phenotype that is associated with HSC exhaustion. Lineage-specific, shRNAmiR-mediated suppression of BCL11A in erythroid cells led to stable long-term engraftment of gene-modified cells. Transduced primary normal or SCD human HSCs expressing the lineage-specific BCL11A shRNAmiR gave rise to erythroid cells with up to 90% reduction of BCL11A protein. These erythrocytes demonstrated 60%-70% γ-chain expression (vs. < 10% for negative control) and a corresponding increase in HbF. Transplantation of gene-modified murine HSCs from Berkeley sickle cell mice led to a substantial improvement of sickle-associated hemolytic anemia and reticulocytosis, key pathophysiological biomarkers of SCD. These data form the basis for a clinical trial application for treating sickle cell disease.

  • 282 circumventing hematopoietic toxicities and reversing sickle phenotype by lineage specific BCL11A knock down and γ globin induction
    Molecular Therapy, 2016
    Co-Authors: Christian Brendel, Daniel E Bauer, Swaroopa Guda, Raffaele Renella, Matthew C. Canver, Young-jo Kim, Matthew M. Heeney, Denise Klatt, Jonathan Fogel, Michael D. Milsom
    Abstract:

    The fetal hemoglobin repressor BCL11A represents a therapeutic target for β-hemoglobinopathies as reduced expression of BCL11A leads to simultaneous increased γ-globin and reduced β-globin expression. Reversing this hemoglobin switch is particularly relevant in sickle cell disease to reduce the βS concentration and increase expression of the protective fetal hemoglobin (HbF, α2γ2). Here we show that despite use of optimized shRNAs embedded into a miRNA (shRNAmiR) architecture to reduce non-specific cellular toxicities (Guda et al. MT, 2015), the knockdown of BCL11A profoundly and specifically impaired long-term engraftment of both human and mouse hematopoietic stem cells (HSCs). In competitive transplantation assays cells transduced with shRNAmiRs targeting the BCL11A mRNA were consistently underrepresented after hematopoietic reconstitution with gene modified HSCs. Although this effect was particularly pronounced in the B-cell compartment, it was also the case for all other assessed hematopoietic lineages. Mechanistically, while knock-down of BCL11A did not lead to a detectable phenotype in terms of apoptosis, growth or differentiation in human or mouse HSCs in vitro, a significant increase in S/G2-phase human HSCs after engraftment into NSG mice was found, a phenotype associated with stem cell exhaustion. To avoid this BCL11A-specific HSC toxicity, we suppressed BCL11A in erythroid cells in a lineage-specific fashion by using transcriptional regulatory elements derived from the β-globin locus. Utilizing this approach for the expression of the optimized shRNAmiRs led to stable long-term engraftment of mouse and human gene modified cells in congenic or NSG-mice, respectively. Transduced primary normal or sickle cell disease (SCD) human HSCs gave rise to erythroid cells with up to 90% reduction of BCL11A protein. These erythrocytes demonstrated 60-70% γ-chain expression and a corresponding increase in HbF at low vector copy numbers per cell (VCN<1.5). Similar results were obtained after in vitro differentiation of CD34+ cells harvested 16 weeks following engraftment in NSG mice. Transplantation of gene modified murine HSCs from BERK sickle cell mice led to a substantial improvement of sickle-associated hemolytic anemia and reticulocytosis, key phenotypes of SCD. In summary, we have shown an unexpected and severe toxicity of BCL11A knockdown in repopulating HSCs that has direct and important consequences for translation into human gene therapy trials. By utilizing erythroid lineage-specific and microRNA embedded expression of targeting shRNAs we demonstrate the capacity of lentivirus vectors to effect significant γ-globin induction leading to clinically relevant increases in HbF while obviating HSC toxicity.

  • crispr cas9 saturating mutagenesis reveals an achilles heel in the BCL11A erythroid enhancer for fetal hemoglobin induction by genome editing
    Blood, 2015
    Co-Authors: Daniel E Bauer, Elenoe C. Smith, Matthew C. Canver, Falak Sher, Luca Pinello, Neville E Sanjana, Ophir Shalem, Diane D Chen, Patrick G Schupp, Divya S Vinjamur
    Abstract:

    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.

  • crispr cas9 saturating mutagenesis reveals an achilles heel in the BCL11A erythroid enhancer for fetal hemoglobin induction by genome editing
    Blood, 2015
    Co-Authors: Daniel E Bauer, Elenoe C. Smith, Matthew C. Canver, Falak Sher, Luca Pinello, Neville E Sanjana, Ophir Shalem, Diane D Chen, Patrick G Schupp, Divya S Vinjamur
    Abstract:

    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.

Z Chen - One of the best experts on this subject based on the ideXlab platform.

  • BCL11A overexpression predicts survival and relapse in non small cell lung cancer and is modulated by microrna 30a and gene amplification
    Molecular Cancer, 2013
    Co-Authors: Ben-yuan Jiang, Xu-chao Zhang, Zhi Xie, Wei Meng, Xuening Yang, Jinji Yang, Qing Zhou, Z Chen, Shiliang Chen
    Abstract:

    Background Aberrant activation of the proto-oncogene B-cell lymphoma/leukemia 11A (BCL11A) has been implicated in the pathogenesis of leukemia and lymphoma. However, the clinical significance of BCL11A in non-small cell lung cancer (NSCLC) remains unknown.

  • BCL11A overexpression predicts survival and relapse in non small cell lung cancer and is modulated by microrna 30a and gene amplification
    Molecular Cancer, 2013
    Co-Authors: Ben-yuan Jiang, Xu-chao Zhang, Zhi Xie, Wei Meng, Xuening Yang, Jinji Yang, Qing Zhou, Z Chen, Zhihong Chen, Shiliang Chen
    Abstract:

    Aberrant activation of the proto-oncogene B-cell lymphoma/leukemia 11A (BCL11A) has been implicated in the pathogenesis of leukemia and lymphoma. However, the clinical significance of BCL11A in non-small cell lung cancer (NSCLC) remains unknown. We examined BCL11A expression at the protein and mRNA levels in a cohort (n = 114) of NSCLC patients and assessed the relationship between BCL11A expression and clinicopathological parameters. Data from array-based Comparative Genomic Hybridization (aCGH) and microRNA transfection experiments were integrated to explore the potential mechanisms of abnormal BCL11A activation in NSCLC. Compared to adjacent non-cancerous lung tissues, BCL11A expression levels were specifically upregulated in NSCLC tissues at both the mRNA (t = 9.81, P < 0.001) and protein levels. BCL11A protein levels were higher in patients with squamous histology (χ2 = 15.81, P = 0.001), smokers (χ2 = 8.92, P = 0.004), patients with no lymph node involvement (χ2 = 5.14, P = 0.029), and patients with early stage disease (χ2 = 3.91, P = 0.048). A multivariate analysis demonstrated that in early stage NSCLC (IA–IIB), BCL11A was not only an independent prognostic factor for disease-free survival (hazards ratio [HR] 0.24, 95% confidence interval [CI] 0.12-0.50, P < 0.001), but also for overall survival (HR = 0.23, 95% CI 0.09-0.61, P = 0.003). The average BCL11A expression level was much higher in SCC samples with amplifications than in those without amplifications (t = 3.30, P = 0.023). Assessing functionality via an in vitro luciferase reporter system and western blotting, we found that the BCL11A protein was a target of miR-30a. Our results demonstrated that proto-oncogene BCL11A activation induced by miR-30a and gene amplification may be a potential diagnostic and prognostic biomarker for effective management of this disease.