RESUMO
Key mechanisms of fetal hemoglobin (HbF) regulation and switching have been elucidated through studies of human genetic variation, including mutations in the HBG1/2 promoters, deletions in the ß-globin locus, and variation impacting BCL11A. While this has led to substantial insights, there has not been a unified understanding of how these distinct genetically-nominated elements, as well as other key transcription factors such as ZBTB7A, collectively interact to regulate HbF. A key limitation has been the inability to model specific genetic changes in primary isogenic human hematopoietic cells to uncover how each of these act individually and in aggregate. Here, we describe a single-cell genome editing functional assay that enables specific mutations to be recapitulated individually and in combination, providing insights into how multiple mutation-harboring functional elements collectively contribute to HbF expression. In conjunction with quantitative modeling and chromatin capture analyses, we illustrate how these genetic findings enable a comprehensive understanding of how distinct regulatory mechanisms can synergistically modulate HbF expression.
Assuntos
Edição de Genes , Hemoglobinas/genética , Hemoglobinas/metabolismo , Sistemas CRISPR-Cas , Cromatina , Cromossomos , Proteínas de Ligação a DNA/metabolismo , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Expressão Gênica , Globinas , Humanos , Mutação , Proteínas Repressoras , Fatores de Transcrição/metabolismo , Globinas beta/genéticaRESUMO
Increased production of fetal hemoglobin (HbF) can ameliorate the severity of sickle cell disease and ß-thalassemia1. BCL11A represses the genes encoding HbF and regulates human hemoglobin switching through variation in its expression during development2-7. However, the mechanisms underlying the developmental expression of BCL11A remain mysterious. Here we show that BCL11A is regulated at the level of messenger RNA (mRNA) translation during human hematopoietic development. Despite decreased BCL11A protein synthesis earlier in development, BCL11A mRNA continues to be associated with ribosomes. Through unbiased genomic and proteomic analyses, we demonstrate that the RNA-binding protein LIN28B, which is developmentally expressed in a pattern reciprocal to that of BCL11A, directly interacts with ribosomes and BCL11A mRNA. Furthermore, we show that BCL11A mRNA translation is suppressed by LIN28B through direct interactions, independently of its role in regulating let-7 microRNAs, and that BCL11A is the major target of LIN28B-mediated HbF induction. Our results reveal a previously unappreciated mechanism underlying human hemoglobin switching that illuminates new therapeutic opportunities.
Assuntos
Hemoglobinas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/genética , Adulto , Animais , Sítios de Ligação , Células Cultivadas , Células Eritroides/metabolismo , Eritropoese/genética , Regulação da Expressão Gênica , Hemoglobinas/genética , Humanos , Recém-Nascido , MicroRNAs/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA Ribossômico 18S/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/metabolismo , Ribossomos/genética , Ribossomos/metabolismoRESUMO
Diamond-Blackfan anemia (DBA) is a rare hematopoietic disease characterized by a block in red cell differentiation. Most DBA cases are caused by mutations in ribosomal proteins and characterized by higher than normal activity of the tumor suppressor p53. Higher p53 activity is thought to contribute to DBA phenotypes by inducing apoptosis during red blood cell differentiation. Currently, there are few therapies available for patients with DBA. We performed a chemical screen using zebrafish ribosomal small subunit protein 29 (rps29) mutant embryos that have a p53-dependent anemia and identified calmodulin inhibitors that rescued the phenotype. Our studies demonstrated that calmodulin inhibitors attenuated p53 protein amount and activity. Treatment with calmodulin inhibitors led to decreased p53 translation and accumulation but does not affect p53 stability. A U.S. Food and Drug Administration-approved calmodulin inhibitor, trifluoperazine, rescued hematopoietic phenotypes of DBA models in vivo in zebrafish and mouse models. In addition, trifluoperazine rescued these phenotypes in human CD34+ hematopoietic stem and progenitor cells. Erythroid differentiation was also improved in CD34+ cells isolated from a patient with DBA. This work uncovers a potential avenue of therapeutic development for patients with DBA.
Assuntos
Anemia de Diamond-Blackfan , Anemia de Diamond-Blackfan/tratamento farmacológico , Animais , Apoptose , Calmodulina , Eritropoese , Humanos , Proteína Supressora de Tumor p53 , Peixe-ZebraRESUMO
The clinical severity of sickle cell disease and ß-thalassemia, the major disorders of ß-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.
Assuntos
Proteínas de Transporte/fisiologia , Hemoglobina Fetal/fisiologia , Inativação Gênica/fisiologia , Proteínas Nucleares/fisiologia , Anemia Falciforme/genética , Anemia Falciforme/terapia , Animais , Técnicas de Silenciamento de Genes/métodos , Técnicas de Silenciamento de Genes/tendências , Estudo de Associação Genômica Ampla/métodos , Estudo de Associação Genômica Ampla/tendências , Humanos , Proteínas Repressoras , Talassemia beta/genética , Talassemia beta/terapiaRESUMO
A transition from fetal hemoglobin (HbF) to adult hemoglobin (HbA) normally occurs within a few months after birth. Increased production of HbF after this period of infancy ameliorates clinical symptoms of the major disorders of adult ß-hemoglobin: ß-thalassemia and sickle cell disease. The transcription factor BCL11A silences HbF and has been an attractive therapeutic target for increasing HbF levels; however, it is not clear to what extent BCL11A inhibits HbF production or mediates other developmental functions in humans. Here, we identified and characterized 3 patients with rare microdeletions of 2p15-p16.1 who presented with an autism spectrum disorder and developmental delay. Moreover, these patients all exhibited substantial persistence of HbF but otherwise retained apparently normal hematologic and immunologic function. Of the genes within 2p15-p16.1, only BCL11A was commonly deleted in all of the patients. Evaluation of gene expression data sets from developing and adult human brains revealed that BCL11A expression patterns are similar to other genes associated with neurodevelopmental disorders. Additionally, common SNPs within the second intron of BCL11A are strongly associated with schizophrenia. Together, the study of these rare patients and orthogonal genetic data demonstrates that BCL11A plays a central role in silencing HbF in humans and implicates BCL11A as an important factor for neurodevelopment.
Assuntos
Sequência de Bases , Proteínas de Transporte/metabolismo , Transtornos Globais do Desenvolvimento Infantil/metabolismo , Hemoglobina Fetal/biossíntese , Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleotídeo Único , Deleção de Sequência , Adulto , Proteínas de Transporte/genética , Criança , Transtornos Globais do Desenvolvimento Infantil/genética , Transtornos Globais do Desenvolvimento Infantil/patologia , Pré-Escolar , Hemoglobina Fetal/genética , Humanos , Lactente , Íntrons , Masculino , Proteínas Nucleares/genética , Proteínas Repressoras , Esquizofrenia/genética , Esquizofrenia/metabolismo , Esquizofrenia/patologiaRESUMO
Although pseudouridine nucleobases are abundant in tRNAs, rRNAs, and small nuclear RNAs (snRNAs), they are not known to have physiologic roles in cell differentiation. We have identified a pseudouridine residue (Ψ28) on spliceosomal U6 snRNA that is induced during filamentous growth of Saccharomyces cerevisiae. Pus1p catalyzes this modification and is upregulated during filamentation. Several U6 snRNA mutants are strongly pseudouridylated at Ψ28. Remarkably, these U6 mutants activate pseudohyphal growth, dependent upon Pus1p, arguing that U6-Ψ28 per se can initiate at least part of the filamentous growth program. We confirmed this by using a designer small nucleolar RNA (snoRNA) targeting U6-U28 pseudouridylation. Conversely, mutants that block U6-U28 pseudouridylation inhibit pseudohyphal growth. U6-U28 pseudouridylation changes the splicing efficiency of suboptimal introns; thus, Pus1p-dependent pseudouridylation of U6 snRNA contributes to the filamentation growth program.