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1.
Cell Genom ; 4(9): 100630, 2024 Sep 11.
Artículo en Inglés | MEDLINE | ID: mdl-39142284

RESUMEN

Raynaud's syndrome is a dysautonomia where exposure to cold causes vasoconstriction and hypoxia, particularly in the extremities. We performed meta-analysis in four cohorts and discovered eight loci (ADRA2A, IRX1, NOS3, ACVR2A, TMEM51, PCDH10-DT, HLA, and RAB6C) where ADRA2A, ACVR2A, NOS3, TMEM51, and IRX1 co-localized with expression quantitative trait loci (eQTLs), particularly in distal arteries. CRISPR gene editing further showed that ADRA2A and NOS3 loci modified gene expression and in situ RNAscope clarified the specificity of ADRA2A in small vessels and IRX1 around small capillaries in the skin. A functional contraction assay in the cold showed lower contraction in ADRA2A-deficient and higher contraction in ADRA2A-overexpressing smooth muscle cells. Overall, our study highlights the power of genome-wide association testing with functional follow-up as a method to understand complex diseases. The results indicate temperature-dependent adrenergic signaling through ADRA2A, effects at the microvasculature by IRX1, endothelial signaling by NOS3, and immune mechanisms by the HLA locus in Raynaud's syndrome.


Asunto(s)
Estudio de Asociación del Genoma Completo , Sitios de Carácter Cuantitativo , Enfermedad de Raynaud , Enfermedad de Raynaud/genética , Enfermedad de Raynaud/inmunología , Humanos , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Femenino , Masculino
2.
bioRxiv ; 2023 Dec 21.
Artículo en Inglés | MEDLINE | ID: mdl-38187584

RESUMEN

Regulatory DNA sequences within enhancers and promoters bind transcription factors to encode cell type-specific patterns of gene expression. However, the regulatory effects and programmability of such DNA sequences remain difficult to map or predict because we have lacked scalable methods to precisely edit regulatory DNA and quantify the effects in an endogenous genomic context. Here we present an approach to measure the quantitative effects of hundreds of designed DNA sequence variants on gene expression, by combining pooled CRISPR prime editing with RNA fluorescence in situ hybridization and cell sorting (Variant-FlowFISH). We apply this method to mutagenize and rewrite regulatory DNA sequences in an enhancer and the promoter of PPIF in two immune cell lines. Of 672 variant-cell type pairs, we identify 497 that affect PPIF expression. These variants appear to act through a variety of mechanisms including disruption or optimization of existing transcription factor binding sites, as well as creation of de novo sites. Disrupting a single endogenous transcription factor binding site often led to large changes in expression (up to -40% in the enhancer, and -50% in the promoter). The same variant often had different effects across cell types and states, demonstrating a highly tunable regulatory landscape. We use these data to benchmark performance of sequence-based predictive models of gene regulation, and find that certain types of variants are not accurately predicted by existing models. Finally, we computationally design 185 small sequence variants (≤10 bp) and optimize them for specific effects on expression in silico. 84% of these rationally designed edits showed the intended direction of effect, and some had dramatic effects on expression (-100% to +202%). Variant-FlowFISH thus provides a powerful tool to map the effects of variants and transcription factor binding sites on gene expression, test and improve computational models of gene regulation, and reprogram regulatory DNA.

3.
J Proteome Res ; 21(7): 1628-1639, 2022 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-35612954

RESUMEN

Alternative splicing can lead to distinct protein isoforms. These can have different functions in specific cells and tissues or in different developmental stages. In this study, we explored whether transcripts assembled from long read, nanopore-based, direct RNA-sequencing (RNA-seq) could improve the identification of protein isoforms in human K562 cells. By comparing with Illumina-based short read RNA-seq, we showed that a large proportion of Ensembl transcripts (5949/14,326) and genes expressing alternatively spliced transcripts (486/2981) identified with long direct reads were missed by short paired-end reads. By co-analyzing proteomic and transcriptomic data, we also showed that some peptides (826/35,976), proteins (262/3215), and protein isoforms arising from distinct transcript variants (574/1212) identified with isoform-specific peptides via custom long-read-based databases were missed in Illumina-derived databases. Finally, we generated unequivocal peptide evidence for a set of protein isoforms and showed that long read, direct RNA-seq allows the discovery of novel protein isoforms not already in reference databases or custom databases built from short read RNA-seq data. Our analysis highlights the benefits of long read RNA-seq data in the generation of reference databases to increase tandem mass spectrometry (MS/MS) identification of protein isoforms.


Asunto(s)
Proteómica , Espectrometría de Masas en Tándem , Empalme Alternativo , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Péptidos/genética , Péptidos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN/metabolismo , Análisis de Secuencia de ARN , Espectrometría de Masas en Tándem/métodos , Transcriptoma
4.
Blood ; 139(14): 2107-2118, 2022 04 07.
Artículo en Inglés | MEDLINE | ID: mdl-35090172

RESUMEN

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/terapia
5.
Redox Biol ; 28: 101374, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31743887

RESUMEN

A hallmark of cancer cells is their ability to reprogram nutrient metabolism. Thus, disruption to this phenotype is a potential avenue for anti-cancer therapy. Herein we used a phenotypic chemical library screening approach to identify molecules that disrupted nutrient metabolism (by increasing cellular oxygen consumption rate) and were toxic to cancer cells. From this screen we discovered a 1,4-Naphthoquinone (referred to as BH10) that is toxic to a broad range of cancer cell types. BH10 has improved cancer-selective toxicity compared to doxorubicin, 17-AAG, vitamin K3, and other known anti-cancer quinones. BH10 increases glucose oxidation via both mitochondrial and pentose phosphate pathways, decreases glycolysis, lowers GSH:GSSG and NAPDH/NAPD+ ratios exclusively in cancer cells, and induces necrosis. BH10 targets mitochondrial redox defence as evidenced by increased mitochondrial peroxiredoxin 3 oxidation and decreased mitochondrial aconitase activity, without changes in markers of cytosolic or nuclear damage. Over-expression of mitochondria-targeted catalase protects cells from BH10-mediated toxicity, while the thioredoxin reductase inhibitor auranofin synergistically enhances BH10-induced peroxiredoxin 3 oxidation and cytotoxicity. Overall, BH10 represents a 1,4-Naphthoquinone with an improved cancer-selective cytotoxicity profile via its mitochondrial specificity.


Asunto(s)
Mitocondrias/metabolismo , Naftoquinonas/farmacología , Estrés Oxidativo/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Benzoquinonas/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/farmacología , Glucólisis/efectos de los fármacos , Humanos , Lactamas Macrocíclicas/farmacología , Mitocondrias/efectos de los fármacos , Fenotipo , Bibliotecas de Moléculas Pequeñas/farmacología , Vitamina K 3/farmacología
6.
Blood ; 133(8): 852-856, 2019 02 21.
Artículo en Inglés | MEDLINE | ID: mdl-30617196

RESUMEN

ß-hemoglobinopathies, such as sickle cell disease and ß-thalassemia, result from mutations in the adult ß-globin gene. Reactivating the developmentally silenced fetal γ-globin gene elevates fetal hemoglobin levels and ameliorates symptoms of ß-hemoglobinopathies. The continued expression of fetal γ-globin into adulthood occurs naturally in a genetic condition termed hereditary persistence of fetal hemoglobin (HPFH). Point mutations in the fetal γ-globin proximal promoter can cause HPFH. The -113A>G HPFH mutation falls within the -115 cluster of HPFH mutations, a binding site for the fetal globin repressor BCL11A. We demonstrate that the -113A>G HPFH mutation, unlike other mutations in the cluster, does not disrupt BCL11A binding but rather creates a de novo binding site for the transcriptional activator GATA1. Introduction of the -113A>G HPFH mutation into erythroid cells using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system increases GATA1 binding and elevates fetal globin levels. These results reveal the mechanism by which the -113A>G HPFH mutation elevates fetal globin and demonstrate the sensitivity of the fetal globin promoter to point mutations that often disrupt repressor binding sites but here create a de novo site for an erythroid activator.


Asunto(s)
Anemia de Células Falciformes , Hemoglobina Fetal , Factor de Transcripción GATA1/metabolismo , Regulación de la Expresión Génica , Mutación Puntual , Elementos de Respuesta , Talasemia beta , Anemia de Células Falciformes/genética , Anemia de Células Falciformes/metabolismo , Línea Celular , Hemoglobina Fetal/biosíntesis , Hemoglobina Fetal/genética , Factor de Transcripción GATA1/genética , Globinas beta/genética , Globinas beta/metabolismo , Talasemia beta/genética , Talasemia beta/metabolismo
7.
Trends Genet ; 34(12): 927-940, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30287096

RESUMEN

Disorders in hemoglobin (hemoglobinopathies) were the first monogenic diseases to be characterized and remain among the most common and best understood genetic conditions. Moreover, the study of the ß-globin locus provides a textbook example of developmental gene regulation. The fetal γ-globin genes (HBG1/HBG2) are ordinarily silenced around birth, whereupon their expression is replaced by the adult ß-globin genes (HBB primarily and HBD). Over 50 years ago it was recognized that mutations that cause lifelong persistence of fetal γ-globin expression ameliorate the debilitating effects of mutations in ß-globin. Since then, research has focused on therapeutically reactivating the fetal γ-globin genes. Here, we summarize recent discoveries, focusing on the influence of genome editing technologies, including CRISPR-Cas9, and emerging gene therapy approaches.


Asunto(s)
Terapia Genética/tendencias , Hemoglobinopatías/genética , Globinas beta/genética , gamma-Globinas/genética , Adulto , Sistemas CRISPR-Cas/genética , Edición Génica/tendencias , Hemoglobinopatías/sangre , Hemoglobinopatías/patología , Humanos , Mutación , Globinas beta/uso terapéutico , gamma-Globinas/uso terapéutico
8.
Nat Genet ; 50(4): 498-503, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29610478

RESUMEN

ß-hemoglobinopathies such as sickle cell disease (SCD) and ß-thalassemia result from mutations in the adult HBB (ß-globin) gene. Reactivating the developmentally silenced fetal HBG1 and HBG2 (γ-globin) genes is a therapeutic goal for treating SCD and ß-thalassemia 1 . Some forms of hereditary persistence of fetal hemoglobin (HPFH), a rare benign condition in which individuals express the γ-globin gene throughout adulthood, are caused by point mutations in the γ-globin gene promoter at regions residing ~115 and 200 bp upstream of the transcription start site. We found that the major fetal globin gene repressors BCL11A and ZBTB7A (also known as LRF) directly bound to the sites at -115 and -200 bp, respectively. Furthermore, introduction of naturally occurring HPFH-associated mutations into erythroid cells by CRISPR-Cas9 disrupted repressor binding and raised γ-globin gene expression. These findings clarify how these HPFH-associated mutations operate and demonstrate that BCL11A and ZBTB7A are major direct repressors of the fetal globin gene.


Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Hemoglobina Fetal/genética , Mutación , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , gamma-Globinas/genética , Anemia de Células Falciformes/genética , Anemia de Células Falciformes/terapia , Secuencia de Bases , Sitios de Unión/genética , Sistemas CRISPR-Cas , Línea Celular , Humanos , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/metabolismo , Sitio de Iniciación de la Transcripción , Talasemia beta/genética , Talasemia beta/terapia
9.
Blood ; 130(6): 803-807, 2017 08 10.
Artículo en Inglés | MEDLINE | ID: mdl-28659276

RESUMEN

ß-Hemoglobinopathies are among the most common single-locus inherited diseases. In this condition, high fetal hemoglobin (HbF) levels have been found to be beneficial, and boosting HbF expression is seen as an attractive therapy. Naturally occurring mutations in the fetal globin promoter can result in high HbF persisting into adulthood in a benign condition known as hereditary persistence of fetal hemoglobin (HPFH). Individuals with one form of HPFH, British HPFH, carry a T to C substitution at position -198 of the fetal globin gene promoter. These individuals exhibit HbF levels of up to 20%, enough to ameliorate the symptoms of ß-hemoglobinopathies. Here, we use clustered regularly interspaced short palindromic repeat-mediated genome editing to introduce the -198 substitution into human erythroid HUDEP-2 cells and show that this mutation is sufficient to substantially elevate expression of HbF. We also examined the molecular mechanism underlying the increase in fetal globin expression. Through a combination of in vitro and in vivo studies, we demonstrate that the mutation creates a de novo binding site for the important erythroid gene activator Krüppel-like factor 1 (KLF1/erythroid KLF). Our results indicate that introducing this single naturally occurring mutation leads to significantly boosted HbF levels.


Asunto(s)
Células Eritroides/metabolismo , Hemoglobina Fetal/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Mutación Puntual , Regulación hacia Arriba , gamma-Globinas/genética , Línea Celular , Humanos , Regiones Promotoras Genéticas
10.
Biochim Biophys Acta Gene Regul Mech ; 1860(5): 525-536, 2017 May.
Artículo en Inglés | MEDLINE | ID: mdl-27718361

RESUMEN

CCAAT boxes are motifs found within the proximal promoter of many genes, including the human globin genes. The highly conserved nature of CCAAT box motifs within the promoter region of both α-like and ß-like globin genes emphasises the functional importance of the CCAAT sequence in globin gene regulation. Mutations within the ß-globin CCAAT box result in ß-thalassaemia, while mutations within the distal γ-globin CCAAT box cause the Hereditary Persistence of Foetal Haemoglobin, a benign condition which results in continued γ-globin expression during adult life. Understanding the transcriptional regulation of the globin genes is of particular interest, as reactivating the foetal γ-globin gene alleviates the symptoms of ß-thalassaemia and sickle cell anaemia. NF-Y is considered to be the primary activating transcription factor which binds to globin CCAAT box motifs. Here we review recruitment of NF-Y to globin CCAAT boxes and the role NF-Y plays in regulating globin gene expression. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani.


Asunto(s)
Factor de Unión a CCAAT/metabolismo , Regulación de la Expresión Génica , Globinas/biosíntesis , Elementos de Respuesta , Talasemia beta/metabolismo , Animales , Factor de Unión a CCAAT/genética , Globinas/genética , Humanos , Talasemia beta/genética
11.
Science ; 351(6270): 285-9, 2016 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-26816381

RESUMEN

Genes encoding human ß-type globin undergo a developmental switch from embryonic to fetal to adult-type expression. Mutations in the adult form cause inherited hemoglobinopathies or globin disorders, including sickle cell disease and thalassemia. Some experimental results have suggested that these diseases could be treated by induction of fetal-type hemoglobin (HbF). However, the mechanisms that repress HbF in adults remain unclear. We found that the LRF/ZBTB7A transcription factor occupies fetal γ-globin genes and maintains the nucleosome density necessary for γ-globin gene silencing in adults, and that LRF confers its repressive activity through a NuRD repressor complex independent of the fetal globin repressor BCL11A. Our study may provide additional opportunities for therapeutic targeting in the treatment of hemoglobinopathies.


Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Hemoglobina Fetal/genética , Silenciador del Gen , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , gamma-Globinas/genética , Anemia de Células Falciformes/genética , Animales , Proteínas Portadoras/genética , Línea Celular , Cromatina/metabolismo , Proteínas de Unión al ADN/genética , Eritroblastos/citología , Eritropoyesis/genética , Humanos , Ratones , Ratones Noqueados , Proteínas Nucleares/genética , Proteínas Represoras/genética , Eliminación de Secuencia , Talasemia/genética , Factores de Transcripción/genética
12.
BMC Mol Biol ; 15: 8, 2014 May 16.
Artículo en Inglés | MEDLINE | ID: mdl-24885809

RESUMEN

BACKGROUND: Krüppel-like Factor 3 (KLF3) is a broadly expressed zinc-finger transcriptional repressor with diverse biological roles. During erythropoiesis, KLF3 acts as a feedback repressor of a set of genes that are activated by Krüppel-like Factor 1 (KLF1). Noting that KLF1 binds α-globin gene regulatory sequences during erythroid maturation, we sought to determine whether KLF3 also interacts with the α-globin locus to regulate transcription. RESULTS: We found that expression of a human transgenic α-globin reporter gene is markedly up-regulated in fetal and adult erythroid cells of Klf3-/- mice. Inspection of the mouse and human α-globin promoters revealed a number of canonical KLF-binding sites, and indeed, KLF3 was shown to bind to these regions both in vitro and in vivo. Despite these observations, we did not detect an increase in endogenous murine α-globin expression in Klf3-/- erythroid tissue. However, examination of murine embryonic fibroblasts lacking KLF3 revealed significant de-repression of α-globin gene expression. This suggests that KLF3 may contribute to the silencing of the α-globin locus in non-erythroid tissue. Moreover, ChIP-Seq analysis of murine fibroblasts demonstrated that across the locus, KLF3 does not occupy the promoter regions of the α-globin genes in these cells, but rather, binds to upstream, DNase hypersensitive regulatory regions. CONCLUSIONS: These findings reveal that the occupancy profile of KLF3 at the α-globin locus differs in erythroid and non-erythroid cells. In erythroid cells, KLF3 primarily binds to the promoters of the adult α-globin genes, but appears dispensable for normal transcriptional regulation. In non-erythroid cells, KLF3 distinctly binds to the HS-12 and HS-26 elements and plays a non-redundant, albeit modest, role in the silencing of α-globin expression.


Asunto(s)
Células Eritroides/metabolismo , Regulación de la Expresión Génica/genética , Factores de Transcripción de Tipo Kruppel/genética , Globinas alfa/genética , Animales , Sitios de Unión/genética , Células COS , Línea Celular Tumoral , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Células K562 , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Regiones Promotoras Genéticas/genética , Transcripción Genética/genética , Globinas alfa/metabolismo
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