RESUMO
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.
Assuntos
DNA/genética , Células Precursoras Eritroides/metabolismo , Hemoglobina Fetal/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Fatores de Transcrição/genética , Animais , Sítios de Ligação , Células COS , Sistemas CRISPR-Cas , Chlorocebus aethiops , DNA/metabolismo , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/transplante , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Hemoglobina Fetal/metabolismo , Feto , Edição de Genes , Células HEK293 , Xenoenxertos , Humanos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/química , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Camundongos , Modelos Moleculares , Células-Tronco Embrionárias Murinas/citologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
The switch from fetal hemoglobin (HbF) to adult hemoglobin (HbA) is a paradigm for developmental gene expression control with relevance to sickle cell disease and ß-thalassemia. Polycomb repressive complex (PRC) proteins regulate this switch, and an inhibitor of PRC2 has entered a clinical trial for HbF activation. Yet, how PRC complexes function in this process, their target genes, and relevant subunit composition are unknown. Here, we identified the PRC1 subunit BMI1 as a novel HbF repressor. We uncovered the RNA binding proteins LIN28B, IGF2BP1, and IGF2BP3 genes as direct BMI1 targets, and demonstrate that they account for the entirety of BMI1's effect on HbF regulation. BMI1 functions as part of the canonical PRC1 (cPRC1) subcomplex as revealed by the physical and functional dissection of BMI1 protein partners. Lastly, we demonstrate that BMI1/cPRC1 acts in concert with PRC2 to repress HbF through the same target genes. Our study illuminates how PRC silences HbF, highlighting an epigenetic mechanism involved in hemoglobin switching.
Assuntos
Hemoglobina Fetal , Complexo Repressor Polycomb 1 , Proteínas do Grupo Polycomb , Hemoglobina Fetal/genética , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismoRESUMO
ZNF410 is a highly-conserved transcription factor, remarkable in that it recognizes a 15-base pair DNA element but has just a single responsive target gene in mammalian erythroid cells. ZNF410 includes a tandem array of five zinc-fingers (ZFs), surrounded by uncharacterized N- and C-terminal regions. Unexpectedly, full-length ZNF410 has reduced DNA binding affinity, compared to that of the isolated DNA binding ZF array, both in vitro and in cells. AlphaFold predicts a partially-folded N-terminal subdomain that includes a 30-residue long helix, preceded by a hairpin loop rich in acidic (aspartate/glutamate) and serine/threonine residues. This hairpin loop is predicted by AlphaFold to lie against the DNA binding interface of the ZF array. In solution, ZNF410 is a monomer and binds to DNA with 1:1 stoichiometry. Surprisingly, the single best-fit model for the experimental small angle X-ray scattering profile, in the absence of DNA, is the original AlphaFold model with the N-terminal long-helix and the hairpin loop occupying the ZF DNA binding surface. For DNA binding, the hairpin loop presumably must be displaced. After combining biophysical, biochemical, bioinformatic and artificial intelligence-based AlphaFold analyses, we suggest that the hairpin loop mimics the structure and electrostatics of DNA, and provides an additional mechanism, supplementary to sequence specificity, of regulating ZNF410 DNA binding.
Assuntos
Fatores de Transcrição , Animais , Sequência de Aminoácidos , Inteligência Artificial , Mamíferos/genética , Ligação Proteica , Domínios Proteicos , Dedos de Zinco/genética , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
Histone H2B monoubiquitination (H2Bub1) is centrally involved in gene regulation. The deubiquitination module (DUBm) of the SAGA complex is a major regulator of global H2Bub1 levels, and components of this DUBm are linked to both neurodegenerative diseases and cancer. Unexpectedly, we find that ablation of USP22, the enzymatic center of the DUBm, leads to a reduction, rather than an increase, in global H2bub1 levels. In contrast, depletion of non-enzymatic components, ATXN7L3 or ENY2, results in increased H2Bub1. These observations led us to discover two H2Bub1 DUBs, USP27X and USP51, which function independently of SAGA and compete with USP22 for ATXN7L3 and ENY2 for activity. Like USP22, USP51 and USP27X are required for normal cell proliferation, and their depletion suppresses tumor growth. Our results reveal that ATXN7L3 and ENY2 orchestrate activities of multiple deubiquitinating enzymes and that imbalances in these activities likely potentiate human diseases including cancer.
Assuntos
Neoplasias da Mama/enzimologia , Proliferação de Células , Enzimas Desubiquitinantes/metabolismo , Histonas/metabolismo , Fatores de Transcrição/metabolismo , Carga Tumoral , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Enzimas Desubiquitinantes/genética , Endopeptidases/genética , Endopeptidases/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genótipo , Células HEK293 , Humanos , Células MCF-7 , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Fenótipo , Interferência de RNA , Transdução de Sinais , Tioléster Hidrolases/genética , Tioléster Hidrolases/metabolismo , Fatores de Tempo , Fatores de Transcrição/genética , Transfecção , Ubiquitina Tiolesterase , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
Reactivation of fetal hemoglobin remains a critical goal in the treatment of patients with sickle cell disease and ß-thalassemia. Previously, we discovered that silencing of the fetal γ-globin gene requires the erythroid-specific eIF2α kinase heme-regulated inhibitor (HRI), suggesting that HRI might present a pharmacologic target for raising fetal hemoglobin levels. Here, via a CRISPR-Cas9-guided loss-of-function screen in human erythroblasts, we identify transcription factor ATF4, a known HRI-regulated protein, as a novel γ-globin regulator. ATF4 directly stimulates transcription of BCL11A, a repressor of γ-globin transcription, by binding to its enhancer and fostering enhancer-promoter contacts. Notably, HRI-deficient mice display normal Bcl11a levels, suggesting species-selective regulation, which we explain here by demonstrating that the analogous ATF4 motif at the murine Bcl11a enhancer is largely dispensable. Our studies uncover a linear signaling pathway from HRI to ATF4 to BCL11A to γ-globin and illustrate potential limits of murine models of globin gene regulation.
Assuntos
Fator 4 Ativador da Transcrição/genética , Hemoglobina Fetal/genética , Proteínas Repressoras/genética , eIF-2 Quinase/genética , Anemia Falciforme/sangue , Anemia Falciforme/genética , Anemia Falciforme/terapia , Animais , Sistemas CRISPR-Cas , Células Cultivadas , Elementos Facilitadores Genéticos , Eritroblastos/metabolismo , Regulação da Expressão Gênica , Inativação Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Especificidade da Espécie , Talassemia beta/sangue , Talassemia beta/genética , Talassemia beta/terapia , gama-Globinas/biossíntese , gama-Globinas/genéticaRESUMO
The fetal-to-adult switch in hemoglobin production is a model of developmental gene control with relevance to the treatment of hemoglobinopathies. The expression of transcription factor BCL11A, which represses fetal ß-type globin (HBG) genes in adult erythroid cells, is predominantly controlled at the transcriptional level but the underlying mechanism is unclear. We identify HIC2 as a repressor of BCL11A transcription. HIC2 and BCL11A are reciprocally expressed during development. Forced expression of HIC2 in adult erythroid cells inhibits BCL11A transcription and induces HBG expression. HIC2 binds to erythroid BCL11A enhancers to reduce chromatin accessibility and binding of transcription factor GATA1, diminishing enhancer activity and enhancer-promoter contacts. DNA-binding and crystallography studies reveal direct steric hindrance as one mechanism by which HIC2 inhibits GATA1 binding at a critical BCL11A enhancer. Conversely, loss of HIC2 in fetal erythroblasts increases enhancer accessibility, GATA1 binding and BCL11A transcription. HIC2 emerges as an evolutionarily conserved regulator of hemoglobin switching via developmental control of BCL11A.
Assuntos
Hemoglobinas , Fatores de Transcrição Kruppel-Like , Proteínas Repressoras , Proteínas Supressoras de Tumor , Proteínas de Transporte/genética , Células Eritroides/metabolismo , Hemoglobinas/genética , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Globinas beta/genética , Globinas beta/metabolismo , gama-Globinas/genéticaRESUMO
The mechanisms by which the fetal-type ß-globin-like genes HBG1 and HBG2 are silenced in adult erythroid precursor cells remain a fundamental question in human biology and have therapeutic relevance to sickle cell disease and ß-thalassemia. Here, we identify via a CRISPR-Cas9 genetic screen two members of the NFI transcription factor family-NFIA and NFIX-as HBG1/2 repressors. NFIA and NFIX are expressed at elevated levels in adult erythroid cells compared with fetal cells, and function cooperatively to repress HBG1/2 in cultured cells and in human-to-mouse xenotransplants. Genomic profiling, genome editing and DNA binding assays demonstrate that the potent concerted activity of NFIA and NFIX is explained in part by their ability to stimulate the expression of BCL11A, a known silencer of the HBG1/2 genes, and in part by directly repressing the HBG1/2 genes. Thus, NFI factors emerge as versatile regulators of the fetal-to-adult switch in ß-globin production.
Assuntos
Hemoglobina Fetal , gama-Globinas , Animais , Proteínas de Transporte/genética , Células Eritroides/metabolismo , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Edição de Genes , Camundongos , Fatores de Transcrição NFI/genética , Fatores de Transcrição NFI/metabolismo , Fatores de Transcrição/genética , Globinas beta/genética , Globinas beta/metabolismo , gama-Globinas/genética , gama-Globinas/metabolismoRESUMO
Sox21 is thought to function as a counteracting partner of SoxB1 (Sox1, 2, 3) genes and is involved in cell fate determination. In this study, we comparatively analyzed the expression patterns and conserved cis-regulatory elements of the duplicated sox21 genes in zebrafish. In embryogenesis, sox21b is predominantly expressed in the telencephalon, hypothalamus, mesencephalon and lens, and sox21a is solely expressed in the midbrain-hindbrain boundary, olfactory placode and lateral line, while both genes are expressed in the hindbrain, spinal cord and ear. In adult, sox21a is expressed in the brain, skin, ovary and intestine, while sox21b is expressed in the brain and testis. Interestingly, all 16 pan-vertebrate conserved non-coding elements (CNEs) are asymmetrically preserved in the sox21b locus, whereas two fish-specific elements are kept in the sox21a locus, and this is correlated with increased evolutionary rate of the sox21a protein sequence. Transient transgenic reporter analysis revealed that six sox21b CNEs and two sox21a CNEs drove green fluorescent protein (GFP) expression in tissues correlated with the partitioning of expression in two orthologues. These results indicate that sox21a and sox21b have reciprocally lost expression domains of the ancestral gene reflected by degeneration of certain CNEs in their genomic loci and provide clear evidence for evolution of the duplicated sox21 genes by subfunctionalization. In addition, our data suggest that some CNEs-based regulatory pathways have been predominantly preserved in the sox21b locus.
Assuntos
Diferenciação Celular/fisiologia , Evolução Molecular , Duplicação Gênica/fisiologia , Loci Gênicos/fisiologia , Sistema Nervoso/metabolismo , Fatores de Transcrição SOXB2/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Estrutura Terciária de Proteína , Fatores de Transcrição SOXB2/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
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.
Assuntos
Anemia Falciforme , Hemoglobina Fetal , Anemia Falciforme/tratamento farmacológico , Eritrócitos Anormais , Células Eritroides , Hemoglobina Fetal/genética , Humanos , Hidroxiureia/farmacologiaRESUMO
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.
Assuntos
Células Eritroides/metabolismo , Hemoglobina Fetal/genética , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Adulto , Feminino , Humanos , Masculino , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Adulto JovemRESUMO
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.
Assuntos
Anemia Falciforme/genética , Proteínas de Transporte/genética , Células Eritroides/metabolismo , Hemoglobina Fetal/genética , Regulação da Expressão Gênica , Proteínas Nucleares/genética , eIF-2 Quinase/genética , Anemia Falciforme/tratamento farmacológico , Sistemas CRISPR-Cas , Proteínas de Transporte/metabolismo , Linhagem Celular , Testes Genéticos , Humanos , Terapia de Alvo Molecular , Proteínas Nucleares/metabolismo , RNA Guia de Cinetoplastídeos , Proteínas Repressoras , eIF-2 Quinase/metabolismoRESUMO
Decreased expression of the USP44 deubiquitinase has been associated with global increases in H2Bub1 levels during mouse embryonic stem cell (mESC) differentiation. However, whether USP44 directly deubiquitinates histone H2B or how its activity is targeted to chromatin is not known. We identified USP44 as an integral subunit of the nuclear receptor co-repressor (N-CoR) complex. USP44 within N-CoR deubiquitinates H2B in vitro and in vivo, and ablation of USP44 impairs the repressive activity of the N-CoR complex. Chromatin immunoprecipitation (ChIP) experiments confirmed that USP44 recruitment reduces H2Bub1 levels at N-CoR target loci. Furthermore, high expression of USP44 correlates with reduced levels of H2Bub1 in the breast cancer cell line MDA-MB-231. Depletion of either USP44 or TBL1XR1 impairs the invasiveness of MDA-MB-231 cells in vitro and causes an increase of global H2Bub1 levels. Our findings indicate that USP44 contributes to N-CoR functions in regulating gene expression and is required for efficient invasiveness of triple-negative breast cancer cells.
Assuntos
Histonas/metabolismo , Correpressor 1 de Receptor Nuclear/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Ubiquitinação , Linhagem Celular Tumoral , Células HEK293 , Humanos , Invasividade Neoplásica , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Ligação Proteica , Subunidades Proteicas/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Transducina/química , Transducina/metabolismo , Ubiquitina Tiolesterase , Repetições WD40RESUMO
The SAGA complex contains two enzymatic modules, which house histone acetyltransferase (HAT) and deubiquitinase (DUB) activities. USP22 is the catalytic subunit of the DUB module, but two adaptor proteins, ATXN7L3 and ENY2, are necessary for DUB activity toward histone H2Bub1 and other substrates. ATXN7L3B shares 74% identity with the N-terminal region of ATXN7L3, but the functions of ATXN7L3B are not known. Here we report that ATXN7L3B interacts with ENY2 but not other SAGA components. Even though ATXN7L3B localizes in the cytoplasm, ATXN7L3B overexpression increases H2Bub1 levels, while overexpression of ATXN7L3 decreases H2Bub1 levels. In vitro, ATXN7L3B competes with ATXN7L3 to bind ENY2, and in vivo, knockdown of ATXN7L3B leads to concomitant loss of ENY2. Unlike the ATXN7L3 DUB complex, a USP22-ATXN7L3B-ENY2 complex cannot deubiquitinate H2Bub1 efficiently in vitro Moreover, ATXN7L3B knockdown inhibits migration of breast cancer cells in vitro and limits expression of ER target genes. Collectively, our studies suggest that ATXN7L3B regulates H2Bub1 levels and SAGA DUB activity through competition for ENY2 binding.
Assuntos
Neoplasias da Mama/metabolismo , Citoplasma/metabolismo , Histonas/metabolismo , Tioléster Hidrolases/metabolismo , Fatores de Transcrição/metabolismo , Movimento Celular , Núcleo Celular/enzimologia , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Células MCF-7 , Fatores de Transcrição/genética , Ubiquitina Tiolesterase , Regulação para CimaRESUMO
Spinocerebellar ataxia type 7 (SCA7) is a debilitating neurodegenerative disease caused by expansion of a polyglutamine [poly(Q)] tract in ATXN7, a subunit of the deubiquitinase (DUB) module (DUBm) in the SAGA complex. The effects of ATXN7-poly(Q) on DUB activity are not known. To address this important question, we reconstituted the DUBm in vitro with either wild-type ATXN7 or a pathogenic form, ATXN7-92Q NT, with 92 Q residues at the N terminus (NT). We found that both forms of ATXN7 greatly enhance DUB activity but that ATXN7-92Q NT is largely insoluble unless it is incorporated into the DUBm. Cooverexpression of DUBm components in human astrocytes also promoted the solubility of ATXN7-92Q, inhibiting its aggregation into nuclear inclusions that sequester DUBm components, leading to global increases in ubiquitinated H2B (H2Bub) levels. Global H2Bub levels were also increased in the cerebellums of mice in a SCA7 mouse model. Our findings indicate that although ATXN7 poly(Q) expansions do not change the enzymatic activity of the DUBm, they likely contribute to SCA7 by initiating aggregates that sequester the DUBm away from its substrates.