RESUMO
BCL11A, a zinc finger repressor, is a stage-specific transcription factor that controls the switch from fetal (HbF, α2γ2) to adult (HbA, α2ß2) hemoglobin in erythroid cells. While BCL11A was known as a factor critical for B-lymphoid cell development, its relationship to erythroid cells and HbF arose through genome-wide association studies (GWAS). Subsequent work validated its role as a silencer of γ-globin gene expression in cultured cells and mice. Erythroid-specific loss of BCL11A rescues the phenotype of engineered sickle cell disease (SCD) mice, thereby suggesting that downregulation of BCL11A expression might be beneficial in patients with SCD and ß-thalassemia. Common genetic variation in GWAS resides in an erythroid-specific enhancer within the BCL11A gene that is required for its own expression. CRISPR/Cas9 gene editing of the enhancer revealed a GATA-binding site that confers a large portion of its regulatory function. Disruption of the GATA site leads to robust HbF reactivation. Advancement of a guide RNA targeting the GATA-binding site in clinical trials has recently led to approval of first-in-man use of ex vivo CRISPR editing of hematopoietic stem/progenitor cells (HSPCs) as therapy of SCD and ß-thalassemia. Future challenges include expanding access and infrastructure for delivery of genetic therapy to eligible patients, reducing potential toxicity and costs, exploring prospects for in vivo targeting of hematopoietic stem cells (HSCs), and developing small molecule drugs that impair function of BCL11A protein as an alternative option.
Assuntos
Células Eritroides , Proteínas Repressoras , Animais , Humanos , Camundongos , Anemia Falciforme/genética , Anemia Falciforme/metabolismo , Talassemia beta/genética , Talassemia beta/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Sistemas CRISPR-Cas , Células Eritroides/metabolismo , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , gama-Globinas/genética , gama-Globinas/metabolismo , Edição de Genes/métodos , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
Genetic downregulation of the BCL11A transcription factor (TF) reverses the switch from fetal to adult hemoglobin and is effective in treating ß-hemoglobinopathies. Genetic ablation results in a gradual reduction in protein abundance and does not lend itself to the analysis of the immediate consequences of protein loss or the determination of the direct interactors/targets of the protein of interest. We achieved acute degradation of the largely disordered and 'undruggable' BCL11A protein by fusing it with a conditional degradation (degron) tag, FKBP12F36V, called degradable tags (dTAG). Small molecules then depleted the BCL11A-dTAG through endogenous proteolytic pathways. By integrating acute depletion with nascent transcriptomics and cell cycle separation techniques, we demonstrate the necessity of BCL11A occupancy at the target chromatin for sustained transcriptional repression in erythroid cells. We advocate for expanding the exploration of TF function to include acute depletion, which holds the potential to unveil unprecedented kinetic insights into TF mechanisms of action.
RESUMO
Although emerging evidence indicates that alterations in proteins within nuclear compartments elicit changes in chromosomal architecture and differentiation, the underlying mechanisms are not well understood. Here we investigate the direct role of the abundant nuclear complex protein Matrin3 (Matr3) in chromatin architecture and development in the context of myogenesis. Using an acute targeted protein degradation platform (dTAG-Matr3), we reveal the dynamics of development-related chromatin reorganization. High-throughput chromosome conformation capture (Hi-C) experiments revealed substantial chromatin loop rearrangements soon after Matr3 depletion. Notably, YY1 binding was detected, accompanied by the emergence of novel YY1-mediated enhancer-promoter loops, which occurred concurrently with changes in histone modifications and chromatin-level binding patterns. Changes in chromatin occupancy by Matr3 also correlated with these alterations. Overall, our results suggest that Matr3 mediates differentiation through stabilizing chromatin accessibility and chromatin loop-domain interactions, and highlight a conserved and direct role for Matr3 in maintenance of chromosomal architecture.
Assuntos
Cromatina , Elementos Facilitadores Genéticos , Proteínas Associadas à Matriz Nuclear , Proteínas de Ligação a RNA , Núcleo Celular , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Cromossomos , Regiões Promotoras Genéticas/genética , Humanos , Proteínas de Ligação a RNA/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismoRESUMO
The transcription factor BCL11A is a critical regulator of the switch from fetal hemoglobin (HbF: α 2 γ 2 ) to adult hemoglobin (HbA: α 2 ß 2 ) during development. BCL11A binds at a cognate recognition site (TGACCA) in the γ-globin gene promoter and represses its expression. DNA-binding is mediated by a triple zinc finger domain, designated ZnF456. Here, we report comprehensive investigation of ZnF456, leveraging X-ray crystallography and NMR to determine the structures in both the presence and absence of DNA. We delve into the dynamics and mode of interaction with DNA. Moreover, we discovered that the last zinc finger of BCL11A (ZnF6) plays a special role in DNA binding and γ-globin gene repression. Our findings help account for some rare γ-globin gene promoter mutations that perturb BCL11A binding and lead to increased HbF in adults (hereditary persistence of fetal hemoglobin). Comprehending the DNA binding mechanism of BCL11A opens avenues for the strategic, structure-based design of novel therapeutics targeting sickle cell disease and ß-thalassemia.