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1.
Mol Cell ; 81(2): 239-254.e8, 2021 01 21.
Artículo en Inglés | MEDLINE | ID: mdl-33301730

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 Transcripcional
2.
Genes Dev ; 31(16): 1704-1713, 2017 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-28916711

RESUMEN

Chromatin structure is tightly intertwined with transcription regulation. Here we compared the chromosomal architectures of fetal and adult human erythroblasts and found that, globally, chromatin structures and compartments A/B are highly similar at both developmental stages. At a finer scale, we detected distinct folding patterns at the developmentally controlled ß-globin locus. Specifically, new fetal stage-specific contacts were uncovered between a region separating the fetal (γ) and adult (δ and ß) globin genes (encompassing the HBBP1 and BGLT3 noncoding genes) and two distal chromosomal sites (HS5 and 3'HS1) that flank the locus. In contrast, in adult cells, the HBBP1-BGLT3 region contacts the embryonic ε-globin gene, physically separating the fetal globin genes from the enhancer (locus control region [LCR]). Deletion of the HBBP1 region in adult cells alters contact landscapes in ways more closely resembling those of fetal cells, including increased LCR-γ-globin contacts. These changes are accompanied by strong increases in γ-globin transcription. Notably, the effects of HBBP1 removal on chromatin architecture and gene expression closely mimic those of deleting the fetal globin repressor BCL11A, implicating BCL11A in the function of the HBBP1 region. Our results uncover a new critical regulatory region as a potential target for therapeutic genome editing for hemoglobinopathies and highlight the power of chromosome conformation analysis in discovering new cis control elements.


Asunto(s)
Cromatina/química , Eritroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Elementos Reguladores de la Transcripción , Globinas beta/genética , Adulto , Proteínas Portadoras/genética , Feto , Silenciador del Gen , Humanos , Región de Control de Posición , Proteínas Nucleares/genética , Seudogenes , Proteínas Represoras , Transcriptoma , gamma-Globinas/genética
3.
J Biol Chem ; 288(9): 6140-53, 2013 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-23300080

RESUMEN

CALHM1 (calcium homeostasis modulator 1) forms a plasma membrane ion channel that mediates neuronal excitability in response to changes in extracellular Ca(2+) concentration. Six human CALHM homologs exist with no homology to other proteins, although CALHM1 is conserved across >20 species. Here we demonstrate that CALHM1 shares functional and quaternary and secondary structural similarities with connexins and evolutionarily distinct innexins and their vertebrate pannexin homologs. A CALHM1 channel is a hexamer, comprised of six monomers, each of which possesses four transmembrane domains, cytoplasmic amino and carboxyl termini, an amino-terminal helix, and conserved extracellular cysteines. The estimated pore diameter of the CALHM1 channel is ∼14 Å, enabling permeation of large charged molecules. Thus, CALHMs, connexins, and pannexins and innexins are structurally related protein families with shared and distinct functional properties.


Asunto(s)
Canales de Calcio/química , Canales de Calcio/metabolismo , Conexinas/química , Conexinas/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Animales , Canales de Calcio/genética , Línea Celular Tumoral , Conexinas/genética , Humanos , Glicoproteínas de Membrana/genética , Ratones , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología Estructural de Proteína
4.
Blood Adv ; 4(18): 4560-4572, 2020 09 22.
Artículo en Inglés | MEDLINE | ID: mdl-32956454

RESUMEN

Increasing fetal hemoglobin (HbF) provides clinical benefit in patients with sickle cell disease (SCD). We recently identified heme-regulated inhibitor (HRI, EIF2AK1), as a novel HbF regulator. Because HRI is an erythroid-specific protein kinase, it presents a potential target for pharmacologic intervention. We found that maximal HbF induction required >80% to 85% HRI depletion. Because it remains unclear whether this degree of HRI inhibition can be achieved pharmacologically, we explored whether HRI knockdown can be combined with pharmacologic HbF inducers to achieve greater HbF production and minimize potential adverse effects associated with treatments. Strongly cooperative HbF induction was observed when HRI depletion was combined with exposure to pomalidomide or the EHMT1/2 inhibitor UNC0638, but not to hydroxyurea. Mechanistically, reduction in the levels of the HbF repressor BCL11A reflected the cooperativity of HRI loss and pomalidomide treatment, whereas UNC0638 did not modulate BCL11A levels. In conjunction with HRI loss, pomalidomide maintained its HbF-inducing activity at 10-fold lower concentrations, in which condition there were minimal observed detrimental effects on erythroid cell maturation and viability, as well as fewer alterations in the erythroid transcriptome. When tested in cells from patients with SCD, combining HRI depletion with pomalidomide or UNC0638 achieved up to 50% to 60% HbF and 45% to 50% HbF, respectively, as measured by high-performance liquid chromatography, and markedly counteracted cell sickling. In summary, this study provides a foundation for the exploration of combining future small-molecule HRI inhibitors with additional pharmacologic HbF inducers to maximize HbF production and preserve erythroid cell functionality for the treatment of SCD and other hemoglobinopathies.


Asunto(s)
Anemia de Células Falciformes , Hemoglobina Fetal , Anemia de Células Falciformes/tratamiento farmacológico , Eritrocitos Anormales , Células Eritroides , Hemoglobina Fetal/genética , Humanos , Hidroxiurea/farmacología
5.
Blood Adv ; 3(10): 1586-1597, 2019 05 28.
Artículo en Inglés | MEDLINE | ID: mdl-31126914

RESUMEN

Reactivation of fetal hemoglobin (HbF) production benefits patients with sickle cell disease and ß-thalassemia. To identify new HbF regulators that might be amenable to pharmacologic control, we screened a protein domain-focused CRISPR-Cas9 library targeting chromatin regulators, including BTB domain-containing proteins. Speckle-type POZ protein (SPOP), a substrate adaptor of the CUL3 ubiquitin ligase complex, emerged as a novel HbF repressor. Depletion of SPOP or overexpression of a dominant negative version significantly raised fetal globin messenger RNA and protein levels with minimal detrimental effects on normal erythroid maturation, as determined by transcriptome and proteome analyses. SPOP controls HbF expression independently of the major transcriptional HbF repressors BCL11A and LRF. Finally, pharmacologic HbF inducers cooperate with SPOP depletion during HbF upregulation. Our study implicates SPOP and the CUL3 ubiquitin ligase system in controlling HbF production in human erythroid cells and may offer new therapeutic strategies for the treatment of ß-hemoglobinopathies.


Asunto(s)
Células Eritroides/metabolismo , Hemoglobina Fetal/genética , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Adulto , Femenino , Humanos , Masculino , Proteínas Nucleares/genética , Proteínas Represoras/genética , Adulto Joven
6.
Science ; 361(6399): 285-290, 2018 07 20.
Artículo en Inglés | MEDLINE | ID: mdl-30026227

RESUMEN

Increasing fetal hemoglobin (HbF) levels in adult red blood cells provides clinical benefit to patients with sickle cell disease and some forms of ß-thalassemia. To identify potentially druggable HbF regulators in adult human erythroid cells, we employed a protein kinase domain-focused CRISPR-Cas9-based genetic screen with a newly optimized single-guide RNA scaffold. The screen uncovered the heme-regulated inhibitor HRI (also known as EIF2AK1), an erythroid-specific kinase that controls protein translation, as an HbF repressor. HRI depletion markedly increased HbF production in a specific manner and reduced sickling in cultured erythroid cells. Diminished expression of the HbF repressor BCL11A accounted in large part for the effects of HRI depletion. Taken together, these results suggest HRI as a potential therapeutic target for hemoglobinopathies.


Asunto(s)
Anemia de Células Falciformes/genética , Proteínas Portadoras/genética , Células Eritroides/metabolismo , Hemoglobina Fetal/genética , Regulación de la Expresión Génica , Proteínas Nucleares/genética , eIF-2 Quinasa/genética , Anemia de Células Falciformes/tratamiento farmacológico , Sistemas CRISPR-Cas , Proteínas Portadoras/metabolismo , Línea Celular , Pruebas Genéticas , Humanos , Terapia Molecular Dirigida , Proteínas Nucleares/metabolismo , ARN Guía de Kinetoplastida , Proteínas Represoras , eIF-2 Quinasa/metabolismo
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