RÉSUMÉ
BACKGROUND: Blood typing is essential for safe transfusions and is performed serologically or genetically. Genotyping predominantly focuses on coding regions, but non-coding variants may affect gene regulation, as demonstrated in the ABO, FY and XG systems. To uncover regulatory loci, we expanded a recently developed bioinformatics pipeline for discovery of non-coding variants by including additional epigenetic datasets. METHODS: Multiple datasets including ChIP-seq with erythroid transcription factors (TFs), histone modifications (H3K27ac, H3K4me1), and chromatin accessibility (ATAC-seq) were analyzed. Candidate regulatory regions were investigated for activity (luciferase assays) and TF binding (electrophoretic mobility shift assay, EMSA, and mass spectrometry, MS). RESULTS: In total, 814 potential regulatory sites in 47 blood-group-related genes were identified where one or more erythroid TFs bound. Enhancer candidates in CR1, EMP3, ABCB6, and ABCC4 indicated by ATAC-seq, histone markers, and co-occupancy of 4 TFs (GATA1/KLF1/RUNX1/NFE2) were investigated but only CR1 and ABCC4 showed increased transcription. Co-occupancy of GATA1 and KLF1 was observed in the KEL promoter, previously reported to contain GATA1 and Sp1 sites. TF binding energy scores decreased when three naturally occurring variants were introduced into GATA1 and KLF1 motifs. Two of three GATA1 sites and the KLF1 site were confirmed functionally. EMSA and MS demonstrated increased GATA1 and KLF1 binding to the wild-type compared to variant motifs. DISCUSSION: This combined bioinformatics and experimental approach revealed multiple candidate regulatory regions and predicted TF co-occupancy sites. The KEL promoter was characterized in detail, indicating that two adjacent GATA1 and KLF1 motifs are most crucial for transcription.
Sujet(s)
Antigènes de groupe sanguin , Épigenèse génétique , Humains , Antigènes de groupe sanguin/génétique , Facteur de transcription GATA-1/génétique , Facteurs de transcription Krüppel-like/génétique , Séquences d'acides nucléiques régulatrices/génétique , Facteurs de transcription/génétique , Facteurs de transcription/métabolismeRÉSUMÉ
Genetic determinants underlying most human blood groups are now clarified but variation in expression levels remains largely unexplored. By developing a bioinformatics pipeline analyzing GATA1/Chromatin immunoprecipitation followed by sequencing (ChIP-seq) datasets, we identify 193 potential regulatory sites in 33 blood-group genes. As proof-of-concept, we aimed to delineate the low-expressing complement receptor 1 (CR1) Helgeson phenotype on erythrocytes, which is correlated with several diseases and protects against severe malaria. We demonstrate that two candidate CR1 enhancer motifs in intron 4 bind GATA1 and drive transcription. Both are functionally abolished by naturally-occurring SNVs. Erythrocyte CR1-mRNA and CR1 levels correlate dose-dependently with genotype of one SNV (rs11117991) in two healthy donor cohorts. Haplotype analysis of rs11117991 with previously proposed markers for Helgeson shows high linkage disequilibrium in Europeans but explains the poor prediction reported for Africans. These data resolve the longstanding debate on the genetic basis of inherited low CR1 and form a systematic starting point to investigate the blood group regulome.
Sujet(s)
Cellules érythroïdes , Facteur de transcription GATA-1 , Récepteurs au C3b du complément , Humains , Africains , Biologie informatique , Facteur de transcription GATA-1/génétique , Facteur de transcription GATA-1/métabolisme , Génotype , Introns , Phénotype , Récepteurs au C3b du complément/génétique , Récepteurs au C3b du complément/métabolisme , Séquençage après immunoprécipitation de la chromatine , Cellules érythroïdes/métabolisme , EuropéensRÉSUMÉ
BACKGROUND: The PBDX/XG gene encoding the Xga blood group antigen was described in 1994, but the genetic determinant of XG expression on RBCs was reported only in 2018. However, the frequencies of Xg(a-) individuals could not explain the rarity of anti-Xga makers. We therefore sought to elucidate the molecular basis of the Xg(a-) phenotype in people producing anti-Xga . STUDY DESIGN AND METHODS: Two genomic DNA (gDNA) and 13 plasma-derived cell-free DNA (cfDNA) samples from anti-Xga makers were investigated (14 males and one female). PBDX/XG exon sequencing was attempted on one gDNA sample. Polymerase chain reaction assays were developed and bioinformatics used to define a suspected deletion in all samples. RESULTS: Investigation of one gDNA sample revealed a 114-kb deletion (esv2662319) on the X chromosome that spans XG exons 4 through 10 and the downstream GYG2 gene. A 3555-bp fragment bridging this deletion was amplified to confirm its presence. Another deletion-specific polymerase chain reaction of 714 bp enabled identification of esv2662319 in both gDNA samples and eight cfDNA samples while ruling it out in one cfDNA. Males were hemizygous for esv2662319 and the female likely homozygous. Four cfDNA sample results were inconclusive, probably due to poor sample quality. Sanger sequencing recognized the recombination junctions as a heterogeneous LTR6B sequence. CONCLUSION: We identified a large deletion on the X chromosome, resulting in a true, tissue-wide Xgnull phenotype. This deletion was found in 10 of 11 anti-Xga makers from which DNA could be amplified. One sample remained unexplained, indicating further heterogeneity to be explored.
Sujet(s)
Antigènes de groupe sanguin/génétique , Chromosomes X humains/génétique , Délétion de gène , Chromosomes Y humains/génétique , Exons/génétique , Femelle , Humains , Mâle , Phénotype , Réaction de polymérisation en chaîneRÉSUMÉ
The Xga blood group is differentially expressed on erythrocytes from men and women. The underlying gene, PBDX, was identified in 1994, but the molecular background for Xga expression remains undefined. This gene, now designated XG, partly resides in pseudoautosomal region 1 and encodes a protein of unknown function from the X chromosome. By comparing calculated Xga allele frequencies in different populations with 2612 genetic variants in the XG region, rs311103 showed the strongest correlation to the expected distribution. The same single-nucleotide polymorphism (SNP) had the most significant impact on XG transcript levels in whole blood (P = 2.0 × 10-22). The minor allele, rs311103C, disrupts a GATA-binding motif 3.7 kb upstream of the transcription start point. This silences erythroid XG messenger RNA expression and causes the Xg(a-) phenotype, a finding corroborated by SNP genotyping in 158 blood donors. Binding of GATA1 to biotinylated oligonucleotide probes with rs311103G but not rs311103C was observed by electrophoretic mobility shift assay and proven by mass spectrometry. Finally, a luciferase reporter assay indicated this GATA motif to be active for rs311103G but not rs311103C in HEL cells. By using an integrated bioinformatic and molecular biological approach, we elucidated the underlying genetic basis for the last unresolved blood group system and made Xga genotyping possible.
Sujet(s)
Sites de fixation , Antigènes de groupe sanguin/génétique , Molécules d'adhérence cellulaire/génétique , Facteur de transcription GATA-1/métabolisme , Régulation de l'expression des gènes , Motifs nucléotidiques , Allèles , Antigènes de groupe sanguin/métabolisme , Molécules d'adhérence cellulaire/métabolisme , Érythrocytes/métabolisme , Femelle , Fréquence d'allèle , Gènes rapporteurs , Études d'associations génétiques , Génotype , Humains , Mâle , Phénotype , Polymorphisme de nucléotide simple , Site d'initiation de la transcriptionRÉSUMÉ
Malaria, despite being one of the world's oldest infectious diseases, remains difficult to eradicate because the parasite is rapidly developing resistance to frontline chemotherapies. Previous studies have shown that the parasite exhibits features resembling programmed cell death upon treatment with drugs that disrupt its digestive vacuole (DV), providing a phenotypic readout that can be detected using the imaging flow cytometer. Large compound collections can thus be screened to identify drugs that are able to disrupt the DV of the malaria parasite using this high-content high-throughput screening platform. As a proof-of-concept, 4440 compounds were screened using this platform in 4months and 254 hits (5.7% hit rate) were obtained. Additionally, 25 compounds (0.6% top hit rate) were observed to retain potent DV disruption activity that was comparable to the canonical DV disruptive drug chloroquine when tested at a ten-fold lower concentration from the original screen. This pilot study demonstrates the robustness and high-throughput capability of the imaging flow cytometer and we report herein the methodology of this screening assay.
Sujet(s)
Érythrocytes/parasitologie , Cytométrie en flux/méthodes , Cytométrie en images/méthodes , Étapes du cycle de vie/effets des médicaments et des substances chimiques , Plasmodium falciparum/effets des médicaments et des substances chimiques , Vacuoles/effets des médicaments et des substances chimiques , Dérivés de l'aniline/composition chimique , Antipaludiques/composition chimique , Antipaludiques/pharmacologie , Benzimidazoles/composition chimique , Carbocyanines/composition chimique , Cellules cultivées , Érythrocytes/effets des médicaments et des substances chimiques , Érythrocytes/ultrastructure , Colorants fluorescents/composition chimique , Tests de criblage à haut débit , Humains , Étapes du cycle de vie/physiologie , Plasmodium falciparum/croissance et développement , Plasmodium falciparum/ultrastructure , Bibliothèques de petites molécules/composition chimique , Bibliothèques de petites molécules/pharmacologie , Coloration et marquage/méthodes , Vacuoles/ultrastructure , Xanthènes/composition chimiqueRÉSUMÉ
Phenotypic assays are increasingly employed to provide clues about drug mechanisms. In antimalarial drug screening, however, the majority of assays are designed to only measure parasite-killing activity. We describe here a high-content assay to detect drug-mediated perturbation of the digestive vacuole integrity in the trophozoite stage of Plasmodium falciparum, using the ImageStream imaging flow cytometer.
Sujet(s)
Cytométrie en flux/méthodes , Cytométrie en images/méthodes , Plasmodium falciparum/cytologie , Plasmodium falciparum/effets des médicaments et des substances chimiques , Vacuoles/effets des médicaments et des substances chimiques , Humains , Paludisme à Plasmodium falciparum/traitement médicamenteux , Paludisme à Plasmodium falciparum/parasitologie , Paludisme à Plasmodium falciparum/anatomopathologie , Plasmodium falciparum/croissance et développementRÉSUMÉ
The mechanism of action of artemisinin and its derivatives, the most potent of the anti-malarial drugs, is not completely understood. Here we present an unbiased chemical proteomics analysis to directly explore this mechanism in Plasmodium falciparum. We use an alkyne-tagged artemisinin analogue coupled with biotin to identify 124 artemisinin covalent binding protein targets, many of which are involved in the essential biological processes of the parasite. Such a broad targeting spectrum disrupts the biochemical landscape of the parasite and causes its death. Furthermore, using alkyne-tagged artemisinin coupled with a fluorescent dye to monitor protein binding, we show that haem, rather than free ferrous iron, is predominantly responsible for artemisinin activation. The haem derives primarily from the parasite's haem biosynthesis pathway at the early ring stage and from haemoglobin digestion at the latter stages. Our results support a unifying model to explain the action and specificity of artemisinin in parasite killing.
Sujet(s)
Antipaludiques/pharmacologie , Artémisinines/pharmacologie , Hème/métabolisme , Plasmodium falciparum/effets des médicaments et des substances chimiques , Artémisinines/composition chimique , Génie chimique , Hème/composition chimique , Modèles moléculaires , Structure moléculaire , Conformation des protéines , Protéines de protozoaire/composition chimique , Protéines de protozoaire/génétique , Protéines de protozoaire/métabolismeRÉSUMÉ
Chloroquine was a cheap, extremely effective drug against Plasmodium falciparum until resistance arose. One approach to reversing resistance is the inhibition of chloroquine efflux from its site of action, the parasite digestive vacuole. Chloroquine accumulation studies have traditionally relied on radiolabelled chloroquine, which poses several challenges. There is a need for development of a safe and biologically relevant substitute. We report here a commercially-available green fluorescent chloroquine-BODIPY conjugate, LynxTag-CQGREEN, as a proxy for chloroquine accumulation. This compound localized to the digestive vacuole of the parasite as observed under confocal microscopy, and inhibited growth of chloroquine-sensitive strain 3D7 more extensively than in the resistant strains 7G8 and K1. Microplate reader measurements indicated suppression of LynxTag-CQGREEN efflux after pretreatment of parasites with known reversal agents. Microsomes carrying either sensitive- or resistant-type PfCRT were assayed for uptake; resistant-type PfCRT exhibited increased accumulation of LynxTag-CQGREEN, which was suppressed by pretreatment with known chemosensitizers. Eight laboratory strains and twelve clinical isolates were sequenced for PfCRT and Pgh1 haplotypes previously reported to contribute to drug resistance, and pfmdr1 copy number and chloroquine IC50s were determined. These data were compared with LynxTag-CQGREEN uptake/fluorescence by multiple linear regression to identify genetic correlates of uptake. Uptake of the compound correlated with the logIC50 of chloroquine and, more weakly, a mutation in Pgh1, F1226Y.
Sujet(s)
Composés du bore/métabolisme , Chloroquine/pharmacologie , Résistance aux substances/effets des médicaments et des substances chimiques , Colorants fluorescents/métabolisme , Techniques de sonde moléculaire/instrumentation , Adénosine triphosphate/pharmacologie , Animaux , Marqueurs biologiques/métabolisme , Variations de nombre de copies de segment d'ADN/génétique , Résistance aux substances/génétique , Gènes de protozoaire , Concentration inhibitrice 50 , Mibéfradil/pharmacologie , Microsomes/effets des médicaments et des substances chimiques , Microsomes/métabolisme , Parasites/effets des médicaments et des substances chimiques , Parasites/métabolisme , Plasmodium falciparum/effets des médicaments et des substances chimiques , Plasmodium falciparum/génétique , Polymorphisme génétique , Reproductibilité des résultats , Vérapamil/pharmacologieRÉSUMÉ
Plasmodium falciparum is the etiological agent of malignant malaria and has been shown to exhibit features resembling programmed cell death. This is triggered upon treatment with low micromolar doses of chloroquine or other lysosomotrophic compounds and is associated with leakage of the digestive vacuole contents. In order to exploit this cell death pathway, we developed a high-content screening method to select compounds that can disrupt the parasite vacuole, as measured by the leakage of intravacuolar Ca(2+). This assay uses the ImageStream 100, an imaging-capable flow cytometer, to assess the distribution of the fluorescent calcium probe Fluo-4. We obtained two hits from a small library of 25 test compounds, quinacrine and 3',4'-dichlorobenzamil. The ability of these compounds to permeabilize the digestive vacuole in laboratory strains and clinical isolates was validated by confocal microscopy. The hits could induce programmed cell death features in both chloroquine-sensitive and -resistant laboratory strains. Quinacrine was effective at inhibiting field isolates in a 48-h reinvasion assay regardless of artemisinin clearance status. We therefore present as proof of concept a phenotypic screening method with the potential to provide mechanistic insights to the activity of antimalarial drugs.