RESUMEN
Metazoan transcription factors typically regulate large numbers of genes. Here we identify via a CRISPR-Cas9 genetic screen ZNF410, a pentadactyl DNA-binding protein that in human erythroid cells directly activates only a single gene, the NuRD component CHD4. Specificity is conveyed by two highly evolutionarily conserved clusters of ZNF410 binding sites near the CHD4 gene with no counterparts elsewhere in the genome. Loss of ZNF410 in adult-type human erythroid cell culture systems and xenotransplantation settings diminishes CHD4 levels and derepresses the fetal hemoglobin genes. While previously known to be silenced by CHD4, the fetal globin genes are exposed here as among the most sensitive to reduced CHD4 levels.. In vitro DNA binding assays and crystallographic studies reveal the ZNF410-DNA binding mode. ZNF410 is a remarkably selective transcriptional activator in erythroid cells, and its perturbation might offer new opportunities for treatment of hemoglobinopathies.
Asunto(s)
ADN/genética , Células Precursoras Eritroides/metabolismo , Hemoglobina Fetal/genética , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Factores de Transcripción/genética , Animales , Sitios de Unión , Células COS , Sistemas CRISPR-Cas , Chlorocebus aethiops , ADN/metabolismo , Células Precursoras Eritroides/citología , Células Precursoras Eritroides/trasplante , Sangre Fetal/citología , Sangre Fetal/metabolismo , Hemoglobina Fetal/metabolismo , Feto , Edición Génica , Células HEK293 , Xenoinjertos , Humanos , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/química , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Ratones , Modelos Moleculares , Células Madre Embrionarias de Ratones/citología , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
The benign condition hereditary persistence of fetal hemoglobin (HPFH) is known to ameliorate symptoms of co-inherited ß-hemoglobinopathies, such as sickle cell disease and ß-thalassemia. The condition is sometimes associated with point mutations in the fetal globin promoters that disrupt the binding of the repressors BCL11A or ZBTB7A/LRF, which have been extensively studied. HPFH is also associated with a range of deletions within the ß-globin locus that all reside downstream of the fetal HBG2 gene. These deletional forms of HPFH are poorly understood and are the focus of this study. Numerous different mechanisms have been proposed to explain how downstream deletions can boost the expression of the fetal globin genes, including the deletion of silencer elements, of genes encoding noncoding RNA, and bringing downstream enhancer elements into proximity with the fetal globin gene promoters. Here we systematically analyze the deletions associated with both HPFH and a related condition known as δß-thalassemia and propose a unifying mechanism. In all cases where fetal globin is upregulated, the proximal adult ß-globin (HBB) promoter is deleted. We use clustered regularly interspaced short palindromic repeats-mediated gene editing to delete or disrupt elements within the promoter and find that virtually all mutations that reduce ΗΒΒ promoter activity result in elevated fetal globin expression. These results fit with previous models where the fetal and adult globin genes compete for the distal locus control region and suggest that targeting the ΗΒΒ promoter might be explored to elevate fetal globin and reduce sickle globin expression as a treatment of ß-hemoglobinopathies.
Asunto(s)
Globinas , Talasemia beta , Proteínas Portadoras/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Expresión Génica , Globinas/metabolismo , Humanos , Factores de Transcripción/genética , Globinas beta/genética , Globinas beta/metabolismo , Talasemia beta/genética , Talasemia beta/terapiaRESUMEN
Here, we describe protocols to interrogate the binding of the zinc fingers of the transcription factor ZBTB7A to the fetal γ-globin (HBG) promoter. We detail the steps for performing electrophoretic mobility shift assays (EMSAs), X-ray crystallography, and isothermal titration calorimetry (ITC) to explore this interaction. These techniques could readily be applied to the structural studies of other zinc finger transcription factors and cognate DNA sequences. For complete details on the use and execution of this protocol, please refer to Yang et al. (2021).
Asunto(s)
Proteínas de Unión al ADN , Factores de Transcripción , Línea Celular Tumoral , ADN/genética , Proteínas de Unión al ADN/genética , Factores de Transcripción/genética , Dedos de ZincRESUMEN
Elevated levels of fetal globin protect against ß-hemoglobinopathies, such as sickle cell disease and ß-thalassemia. Two zinc-finger (ZF) repressors, BCL11A and ZBTB7A/LRF, bind directly to the fetal globin promoter elements positioned at -115 and -200, respectively. Here, we describe X-ray structures of the ZBTB7A DNA-binding domain, consisting of four adjacent ZFs, in complex with the -200 sequence element, which contains two copies of four consecutive C:G base pairs. ZF1 and ZF2 recognize the 5' C:G quadruple, and ZF4 contacts the 3' C:G quadruple. Natural non-coding DNA mutations associated with hereditary persistence of fetal hemoglobin (HPFH) impair ZBTB7A DNA binding, with the most severe disruptions resulting from mutations in the base pairs recognized by ZF1 and ZF2. Our results firmly establish ZBTB7A/LRF as a key molecular regulator of fetal globin expression and inform genome-editing strategies that inhibit repressor binding and boost fetal globin expression to treat hemoglobinopathies.