Globin Transcripts Research Articles

Exploring Transcriptomic Landscapes in Red Blood Cells, in Their Extracellular Vesicles and on a Single-Cell Level.

Being enucleated, RBCs lack typical transcriptomes, but are known to contain small amounts of diverse long transcripts and microRNAs. However, the exact role and importance of these RNAs are lacking. Shedding of extracellular vesicles (EVs) from the plasma membrane constitutes an integral mechanism of RBC homeostasis, by which RBCs remove unnecessary cytoplasmic content and cell membrane. To study this further, we explored the transcriptomes of RBCs and extracellular vesicles (EVs) of RBCs using next-generation sequencing. Furthermore, we performed single-cell RNA sequencing on RBCs, which revealed that approximately 10% of the cells contained detectable levels of mRNA and cells formed three subpopulations based on their transcriptomes. A decrease in the mRNA quantity was observed across the populations. Qualitative changes included the differences in the globin transcripts and changes in the expression of ribosomal genes. A specific splice form of a long non-coding RNA, Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1), was the most enriched marker in one subpopulation of RBCs, co-expressing with ribosomal structural transcripts. MALAT1 expression was confirmed by qPCR in CD71-enriched reticulocytes, which were also characterized with imaging flow cytometry at the single cell level. Analysis of the RBC transcriptome shows enrichment of pathways and functional categories required for the maturation of reticulocytes and erythrocyte functions. The RBC transcriptome was detected in their EVs, making these transcripts available for intercellular communication in blood.

Down-regulation of the transcriptional repressor ZNF802 (JAZF1) reactivates fetal hemoglobin in \u03b20-thalassemia/HbE

Reactivating of fetal hemoglobin (HbF; α2γ2) can ameliorate the severity of β-thalassemia disease by compensating for adult hemoglobin deficiency in patients. Previously, microarray analysis revealed that zinc finger protein (ZNF)802 (also known as Juxta-posed with another zinc finger gene-1 (JAZF1)) was upregulated in human erythroblasts derived from adult peripheral blood compared with fetal liver-derived cells, implying a potential role as a HbF repressor. However, deficiency in ZNF802 induced by lentiviral shRNA in β0-thalassemia/hemoglobinE erythroblasts had no effect on erythroblast proliferation and differentiation. Remarkably, the induction of HBG expression was observed at the transcriptional and translational levels resulting in an increase of HbF to 35.0 ± 3.5%. Interestingly, the embryonic globin transcripts were also upregulated but the translation of embryonic globin was not detected. These results suggest ZNF802 might be a transcriptional repressor of the γ-globin gene in adult erythroid cells.

Improved nanopore direct RNA sequencing of cardiac myocyte samples by selective mt-RNA depletion

RNA sequencing is a powerful tool to analyze gene expression transcriptome wide. However, RNA sequencing in general and especially the recently developed methods of long read RNA sequencing are still low-throughput and cost-intensive. Here, one important design choice is to concentrate the sequencing capacity on specific parts of the transcriptome. Especially, abundant transcripts as ribosomal RNAs may dominate the available sequencing space, if not removed prior to sequencing. Several methods exist to reduce ribosomal RNA read numbers: either based on enrichment of the relevant fraction (polyA+ RNA) or depletion, respectively degradation of ribosomal RNAs. Furthermore, commercial kits are available to deplete globin transcripts from blood samples. However, so far, no solution exists to deal with other tissue-specific highly abundant transcripts. This is especially of interest in the heart and other muscle derived samples, where reads originating from mitochondrial RNAs make up to 30% of reads in polyA+ selected libraries and around 70% in single cell sequencing experiments. We present a simple method to diminish sequencing of mitochondrial RNAs in Oxford Nanopore direct RNA sequencing libraries by RNase H based clipping of the polyA tail. We show that mt-clipping enables enhanced detection of cytoplasmic mRNAs, among them genes involved in heart development and pathogenesis. Mt-clipping may be applied as well to other sequencing protocols that are based on oligo(dT) priming and can be easily adapted to other tissue-specific high-abundant transcripts.

Hypoxia Dependent and Independent Dysregulation of the Transcriptome in Sickle Cell Anemia

Sickle cell anemia (SCA) is associated with an increased hypoxic response from anemia and vaso-occlusion-impaired tissue perfusion. The effects of hypoxia are mediated by hypoxia transcription factors (HIFs). Chuvash erythrocytosis (CE) is an inherited condition due to homozygosity for the missense mutation in VHL gene (VHLR200W) that impairs interactions of VHL with HIF-α subunits, thereby augmenting transcription of HIF-regulated genes. CE and SCA share increased expression of erythropoietin (EPO) and other HIF target genes. As HIF-regulation of transcription is tissue and differentiation-stage specific, in this study we used reticulocytes, which are easily accessible and purified peripheral blood erythroid cells. We compared the transcriptomes of SCA and CE reticulocytes to differentiate HIF-mediated dysregulation from non-hypoxic dysregulation of SCA transcripts. Our study revealed reticulocyte gene expression changes that are common to both diseases as well as SCA-specific changes. The reticulocytes were purified from 5 HbSS and 5 age- and gender-matched HbAA African-American individuals, and from 17 VHLR200W homozygotes from Chuvashia and 13 wild type Chuvash individuals. Total RNA was depleted of ribosomal RNA and globin transcripts, and reverse transcribed. Strand-specific libraries were constructed for 100 or 125 bp paired-end sequencing to 30-45 million reads using Illumina HiSeq 2500 or 4000 platform. The sequencing data were mapped to human reference genome version GRCh37 using the splice-aware aligner STAR and analyzed using DESeq2. In separate analyses of the two diseases, we identified 1435 genes differentially expressed in SCA among 6965 analyzed genes, 848 increased and 587 decreased in SCA relative to control individuals. We also identified 1498 genes differentially expressed in CE among 8989 analyzed genes, 862 increased and 636 decreased in CE relative to control individuals. Across all analyzed genes, there was a moderate correlation (r=0.30) of expression changes between the two diseases. Among genes differentially expressed, 258 up-regulated and 155 down-regulated genes overlapped between the two diseases, representing a 1.4-fold enrichment. In a combined analysis of the two diseases, we identified 1228 genes among 6924 analyzed genes that shared altered regulation in both diseases. The 693 genes increased in both diseases were enriched (adjusted P < 0.05) in multiple metabolic, inflammatory, and oxidative pathways. The 535 genes decreased in both diseases were enriched in a cell cycle pathway. Among the commonly increased genes, the expression level of ERFE encoding erythroferrone was increased by 9.4-fold in CE and by 4.3-fold in SCA, suggesting markedly altered iron regulation in CE and SCA. This would be expected as both SCA and CE share upregulated erythropoiesis that is associated with augmented erythroferrone. Among the commonly decreased genes, RPL3L encoding Ribosomal Protein L3 Like was decreased by 68% in CE and by 93% in SCA. These common expression changes reflect hypoxic regulation related to chronic anemia in SCA. To assess disease-specific gene expression change, we tested the disease (SCA versus CE) by genotype (mutation versus wildtype) interaction effect. We identified 822 genes that showed disease-specific expression changes among 6924 analyzed genes. Of these disease-specific genes, 304 were increased and 153 decreased in SCA (adjusted P <0.05) but not in CE (nominal P >0.05). For example, SLC16A1, encoding Solute Carrier Family 16 Member 1 and associated with an erythrocyte lactate transporter defect, was increased in SCA by 11-fold whereas PPBP encoding Pro-Platelet Basic Protein was decreased in SCA by 92%; both genes showing no change in CE. The 304 genes with SCA-specific increased expression were enriched in "Thyroid hormone signaling" (5.1 fold) and "Glioma" (7.0-fold) pathways. The 153 genes with SCA-specific decreased expression were enriched in "Ribosome" (12-fold) pathway. Among the disease-specific genes, only 34 increased and 28 decreased in CE but not in SCA. Our study demonstrates high HIF transcriptional activity in both CE and SCA reticulocytes but also reveals hypoxia-independent gene expression changes in SCA reticulocytes. These results suggest that HIF might be a therapeutic target of SCA. These data also shed light on the different molecular mechanisms underlying SCA complications. Disclosures Gordeuk: Pfizer: Research Funding; Modus Therapeutics: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Emmaus: Consultancy, Honoraria; Global Blood Therapeutics: Consultancy, Honoraria, Research Funding; Inctye: Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Ironwood: Research Funding; Imara: Research Funding.

RNA Sequencing (RNA-Seq) Reveals Extremely Low Levels of Reticulocyte-Derived Globin Gene Transcripts in Peripheral Blood From Horses (Equus caballus) and Cattle (Bos taurus).

RNA-seq has emerged as an important technology for measuring gene expression in peripheral blood samples collected from humans and other vertebrate species. In particular, transcriptomics analyses of whole blood can be used to study immunobiology and develop novel biomarkers of infectious disease. However, an obstacle to these methods in many mammalian species is the presence of reticulocyte-derived globin mRNAs in large quantities, which can complicate RNA-seq library sequencing and impede detection of other mRNA transcripts. A range of supplementary procedures for targeted depletion of globin transcripts have, therefore, been developed to alleviate this problem. Here, we use comparative analyses of RNA-seq data sets generated from human, porcine, equine, and bovine peripheral blood to systematically assess the impact of globin mRNA on routine transcriptome profiling of whole blood in cattle and horses. The results of these analyses demonstrate that total RNA isolated from equine and bovine peripheral blood contains very low levels of globin mRNA transcripts, thereby negating the need for globin depletion and greatly simplifying blood-based transcriptomic studies in these two domestic species.

Induced Pluripotent Stem Cell (iPSC)-Based Mapping of Globin Expression throughout Human Erythropoietic Development

It has recently become clear that in vitro hematopoietic differentiation protocols using pluripotent stem cells mainly capture the primitive wave of hematopoietic development with limited induction of the definitive wave that gives rise to cells with adult-type characteristics such as the ability to express adult β-globin. Recent efforts from our group in optimizing our original differentiation protocol to derive more definitively patterned erythroid cells from iPSCs have resulted in a significant increase in β-globin transcripts as well as robust β-globin protein expression at the most mature stage of differentiation. To better quantify our progress in augmenting β-globin expression and to track globin ontogeny in real-time, we created a β-globin reporter iPSC line that allows for the mapping of β-globin expression throughout erythroid development.To create this tool, TALEN were used to target the β-globin locus in iPSCs where a promoterless GFP cassette was fused in frame to the first codon of the β-globin gene allowing for visualization of β-globin expression at single cell resolution via GFP expression.Interestingly, using our optimized protocol, only about 1% of cells exceeded the GFP detection threshold at the most mature stage of differentiation, suggesting that the several log-fold increase in β-globin transcripts seen at the population level as the cells progress through erythroid differentiation could be the result of high levels of β-globin transcription in just a small fraction of cells. Single cell RNA sequencing of GFP- and GFP+ sorted fractions showed significantly greater levels of β-globin transcripts in the GFP+ fraction in line with this hypothesis, thereby validating our reporter line as a tool to visualize and enrich for β-globin expressing cells.Despite lacking β-globin protein expression, the GFP- cells were not completely devoid of β-globin transcripts. As recent studies suggest that translation is dynamically controlled in maturing red blood cells, our results might indicate that posttranscriptional mechanisms could impact the translation of these globin transcripts. In fact, mining of the genes differentially expressed between both populations revealed several transcripts enriched in the GFP+ fraction that code for proteins involved in the dynamic translational control of transcripts essential for maturing red blood cells. These findings suggest advanced maturation of the GFP+ fraction, as well as a role for posttranscriptional mechanisms in the regulation of β-globin protein expression during erythroid development from iPSCs.Establishment of the developmental time frame of iPSC-derived erythroid cells is challenging as in vitro differentiation cultures lack the spatiotemporal separation of the different hematopoietic waves present in the embryo. Due to the possibility of multiple hematopoietic programs co-existing in one well, bulk expression analyses should be interpreted with caution when used to ascribe primitive or definitive characteristics to cell populations. Considering that the complexity of globin regulation in development might currently be underappreciated due to these factors, we also used single cell RNA sequencing to dissect the globin expression profiles of individual cells. Looking at the co-expression of different globins in individual cells, we found that the majority of the cells express a combination of embryonic (ε), fetal (γ) and adult (β) globins, indicative of a definitive yolk sac identity. Moreover, as β-globin transcripts increased, we observed a decrease in ε-globin transcripts, indicating that primitive/embryonic characteristics are gradually lost as cells gain more definitive/adult features.Further interrogation of the ‘β-globin expression signature’ that we distilled from the single cell RNA sequencing data will be instructive for future strategies aimed at increasing β-globin protein levels in iPSC-derived erythroid cells. Progress in this area will improve the resolution with which we can study hemoglobinopathies such as β-thalassemia and sickle cell disease and such strategies can then immediately be tested using the β-globin reporter iPSC line as a screening platform. DisclosuresNo relevant conflicts of interest to declare.

Gene Expression Profiling of Reticulocytes in Patients with Hereditary Spherocytosis after Depleting Globin Gene Transcripts

IntroductionGlobal transcriptome analysis of circulating reticulocytes using microarrays is challenging due to the high abundance of globin transcripts. In order to accurately measure the transcriptome in reticulocytes, we planned to study the reticulocytes transcriptome profile before and after globin depletion. Patients with Hereditary Spherocytosis (HS) were recruited because of high reticulocytosis and also there was no global trancriptome profile available for the disease to the best of our knowledge.MethodsReticulocytes were purified by passing EDTA anticoagulated red cell suspensions through a column of microcrystalline cellulose and α-cellulose mixture. The extraction of total RNA was done fresh prior to microarray analysis using TRIzol reagent (Invitrogen) and RNeasy columns as per manufacturer’s instruction. The globin transcripts were depleted with starting quantity of 3 µg of total RNA.using GLOBINclearTMHuman kit (Ambion, Austin, TX). RNA quality were assessed before and after depletion using Agilent Technologies 2100 Bioanalyzer in each sample used for the study.The Agilent Low Input Quick Amp Labelling kit was used to generate cRNA with a sample input of 200 ng total RNA in single-color microarray analysis (SurePrint G3 Human Gene Expression v3 8x60K Microarray, G4858A- 072363). cRNA was hybridized for 17 hours at 65˚C. After thorough washing, the results were extracted with Feature Extraction Project software and analysed using GeneSpring v13.0. For this study, differentially expressed genes were identified according to the criteria: t-test unpaired p (Corr) cut-off = 0.05 and fold-change cut-off of 2.0. These differentially expressed genes were imported in PANTHER (Protein Analysis Through Evolutionary Relationships) classification system to classify the genes.ResultsTranscriptome profiling of reticulocyte RNA from HS patients revealed 5318 annotated transcripts and 2744 lnc-RNA/uncharacterized transcripts to be differentially regulated before globin depletion. After globin depletion increase (8196 annotated transcripts and 4292 lnc-RNA) in the number of differentially regulated genes were observed. An increase of 54% and 56% was seen in annotated transcripts and noncoding transcripts respectively after globin depletion. This increased coverage may be attributed to the removal of the globin transcript. Gene Ontology analysis for molecular function, biological process, cellular component and protein class did not reveal any difference in the percentage of the category, though the number of transcripts was more after depletion. There were approximately 7% more pathways in the globin transcript depleted samples. Namely, 2-arachidonoylglycerol biosynthesis, biotin biosynthesis, carnitine metabolism, cobalamin biosynthesis, coenzyme a linked carnitine metabolism, cysteine biosynthesis, flavin biosynthesis, phenylalanine biosynthesis, sulfate assimilation, tyrosine biosynthesis pathways, etc. were elucidated only after globin depletion.ConclusionsThe globin transcript reduction followed by microarray analysis represents a robust methodology for studying pathophysiology of hematologic diseases which are not related to globin chain disorders. Furthermore we describe the global transcriptome profile of HS for the first time. Globin transcript depletion elucidates the better number of the transcripts and eventually the pathways which may have been earlier masked under the abundance of globin mRNAs. Pathway analysis and validation experiments are required to explain the role of these differentially expressed genes in the pathophysiology of HS. DisclosuresNo relevant conflicts of interest to declare.

Crispr/Cas9- Mediated Genome Editing of Human CD34+ Cells Upregulate Fetal Hemoglobin to Clinically Relevant Levels in Single Cell-Derived Erythroid Colonies

Fetal hemoglobin (HbF) is abundant in the late stage fetus and newborns, but is progressively lost over the first 6 months of life as the genes encoding the g-globin subunit of HbF are repressed. HbF expression can provide clinical benefit to patients with deficient or defective b-globin, for example in β-thalassemia and Sickle Cell Disease (SCD), respectively. CRISPR-Cas9 technology offers a unique treatment modality that can be used to ex vivo edit regulatory DNA sequences in patient CD34+ hematopoietic stem and progenitor cells (HSPC) containing hematopoietic stem and progenitor cells in order to upregulate HbF. Reinfusion of edited CD34+ HSPC would be expected to lead to the production of erythrocytes expressing high levels of HbF, and amelioration of disease.There are a number of reports of naturally occurring genetic variants that are associated with Hereditary Persistence of Fetal Hemoglobin (HPFH), presumably through the loss of regulatory sequences that would otherwise bind regulatory proteins that developmentally down-regulate the expression of HbF. Thus, by using the CRISPR-Cas9 system to re-create the DNA sequence variations associated with HPFH in CD34+ HSPC, we aim to relieve transcriptional inhibition of γ-globin, resulting in upregulation of HbF.Here, we transfected healthy primary human CD34+ HSPC with Cas9 and guide RNAs in order to re-create naturally occurring HPFH genetic variants. Transfected CD34+ HSPC were sorted as single cells post-editing and expanded as erythroid colonies. These colonies were then genotyped to confirm editing and further assessed for globin transcript and protein. Clonal analysis demonstrated Non-Homologous End Joining (NHEJ) mediated editing in greater than 90% of the colonies. These editing events re-created the intended HPFH genetic variants in up to 75% of the colonies. For some, but not all of the HPFH genetic variants, both mono-and bi-allelic genetic modifications led to significant upregulation of HbF. These results provide us with an editing strategy that support a viable therapeutic approach for the treatment of β-thalassemia and SCD. DisclosuresMishra:CRISPR Therapeutics: Employment. Chou:CRISPR Therapeutics: Employment. Lin:CRISPR Therapeutics: Employment. Paik:CRISPR Therapeutics: Employment. Zhang:CRISPR Therapeutics: Employment, Equity Ownership. Liang:CRISPR Therapeutics: Employment. Tomkinson:CRISPR Therapeutics: Employment. Pettiglio:CRISPR Therapeutics: Employment. Sanginario:CRISPR Therapeutics: Employment. Allen:CRISPR Therapeutics: Employment. Cradick:CRISPR Therapeutics: Employment. John:CRISPR Therapeutics: Employment. Chakraborty:CRISPR Therapeutics: Employment. Cowan:CRISPR Therapeutics: Employment, Equity Ownership. Novak:CRISPR Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Lundberg:CRISPR Therapeutics: Employment, Equity Ownership.

Sickle Cell Anemia in a Child with Three β-Globin Clusters (β-S/β-S, β-A): Loss of LCR and 3' HS1 in the Duplicated Wild-Type β-Globin Cluster Does Not Fully Abrogate Its Transcript

Transcriptional regulation of β-globin cluster genes follows a complex, highly conserved system of gene expression with developmental and tissue-specific control. The DNase I hypersensitivity (HS) sites in the upstream locus control region (LCR) and 3' HS1 element are thought to interact with β-globin cluster genes involving long range DNA interactions mediated by various transcription factors to drive the regulation of β-like globin gene expression. The majority of studies have focused on the role of the LCR on active transcription and globin gene switching. Various in vitro and in vivo studies have shown that the LCR interacts with one gene at a time and that absence of the LCR results in a dramatic decrease (10 -100 fold) in globin transcripts (Kiefer et. al., Mutat. Res. 2008, 647:68, Noordermeer et. al., IUBMB Life 2008, 60: 824). However, the role of the 3'HS1 downstream and other distal cis -regulatory elements are not entirely understood, with recent studies in mouse and cell-culture models suggesting they play a role in the insulation of globin genes from silencing chromatin (Bender et. al., Blood 2008, 106: 1395). Our knowledge of the β-globin LCR and 3'HS1 function is still incomplete and much can still be learned from human mutations affecting these regulatory elements.We report a unique head-to-tail duplication of the β-globin cluster in a patient phenotypically expressing homozygous HbS (sickle-cell anemia, SCA) that provides insight into the regulatory role of the β-LCR and 3'HS1 on wild-type β-globin (β-A) expression in a background of SCA. The studies were driven by an apparent discrepancy between hemoglobin analysis of an infant with a SCA phenotype and no detectable HBA, and Sanger sequencing of the β-globin genes which showed a heterozygous genotype.Analyses of parents' blood samples and DNA revealed that each were carriers for sickle cell allele. Hemoglobin analysis showed the father expressed HbS fraction at 41.3% and the mother at 33.3%, while the proband had a majority of HbS, some HbF and no detectable HbA. The reduced HbS fraction in the mother could be explained by co-inheritance of α-thalassemia (αα/α-3.7). The proband did not inherit α-thalassemia from the mother.Multiplex ligation-dependent probe amplification analysis of the proband's DNA suggested duplication of the β-globin cluster, resulting in three copies of the HBB gene in the genome. Subsequent next-generation sequencing confirmed that the duplication occurred immediately adjacent to the first iteration of sequence, in head-to-tail orientation and resulted in an intact β-S cluster having both LCR and HS1 elements, followed by the duplicated β-A cluster (β-S, β-A) that excluded a part of HBE (epsilon globin) and the upstream β-LCR regions, extending through to LINE L1LBP1 ([hg19] chr11:4640332-5290168). Further analyses revealed that the duplicated β-A cluster, which encompassed approximately 650 kb sequence, lacked a DNA segment containing the 3'HS1 element (figure 1). The proband's β genotype is thus (β-S/β-S, β-A). DNA analysis showed that the father carried the duplicated β-globin cluster with genotype (β-A/β-S, β-A), and the mother, a heterozygous HbS genotype (β-A/β-S).Reverse transcription, quantitative polymerase chain reaction (RT-qPCR) was used to assess transcription levels of β-A and β-S mRNA for each family member. Analysis of the parents' reticulocyte RNA showed that the β-globin (β-A and β-S) transcript levels were nearly balanced. RT-qPCR of proband's reticulocyte RNA showed no convincing detection of β-A transcript, but the β-A transcript was clearly detected by digitalPCR, albeit at a very low level (0.4% of total HBB transcript) (figure 2).The β-globin cluster duplication on one chromosome in a background of a phenotypically homozygous (HbSS) SCA patient has provided a unique opportunity to assess the effect of the LCR and 3'HS1 regions on the transcription of a β-A gene in an unbiased environment. Prior studies in transformed cell-lines or mouse models have shown the down regulatory effect of LCR loss and potential protective effect of the 3'HS1 element. Within the human model, the observed transcription from the duplicated, distally displaced (~650 kb) β-A cluster demonstrates that the loss of LCR and flanking HS sites does not lead to complete silencing of β-globin transcription.VD was supported by project IGA MzCR NT13587. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Increasing gene discovery and coverage using RNA-seq of globin RNA reduced porcine blood samples.

BackgroundTranscriptome analysis of porcine whole blood has several applications, which include deciphering genetic mechanisms for host responses to viral infection and vaccination. The abundance of alpha- and beta-globin transcripts in blood, however, impedes the ability to cost-effectively detect transcripts of low abundance. Although protocols exist for reduction of globin transcripts from human and mouse/rat blood, preliminary work demonstrated these are not useful for porcine blood Globin Reduction (GR). Our objectives were to develop a porcine specific GR protocol and to evaluate the GR effects on gene discovery and sequence read coverage in RNA-sequencing (RNA-seq) experiments.ResultsA GR protocol for porcine blood samples was developed using RNase H with antisense oligonucleotides specifically targeting porcine hemoglobin alpha (HBA) and beta (HBB) mRNAs. Whole blood samples (n = 12) collected in Tempus tubes were used for evaluating the efficacy and effects of GR on RNA-seq. The HBA and HBB mRNA transcripts comprised an average of 46.1% of the mapped reads in pre-GR samples, but those reads reduced to an average of 8.9% in post-GR samples. Differential gene expression analysis showed that the expression level of 11,046 genes were increased, whereas 34 genes, excluding HBA and HBB, showed decreased expression after GR (FDR <0.05). An additional 815 genes were detected only in post-GR samples.ConclusionsOur porcine specific GR primers and protocol minimize the number of reads of globin transcripts in whole blood samples and provides increased coverage as well as accuracy and reproducibility of transcriptome analysis. Increased detection of low abundance mRNAs will ensure that studies relying on transcriptome analyses do not miss information that may be vital to the success of the study.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-954) contains supplementary material, which is available to authorized users.

Defects of protein production in erythroid cells revealed in a zebrafish Diamond-Blackfan anemia model for mutation in RPS19.

Diamond–Blackfan anemia (DBA) is a rare congenital red cell aplasia that classically presents during early infancy in DBA patients. Approximately, 25% of patients carry a mutation in the ribosomal protein (RP) S19 gene; mutations in RPS24, RPS17, RPL35A, RPL11, and RPL5 have been reported. How ribosome protein deficiency causes defects specifically to red blood cells in DBA has not been well elucidated. To genetically model the predominant ribosome defect in DBA, we generated an rps19 null mutant through the use of TALEN-mediated gene targeting in zebrafish. Molecular characterization of this mutant line demonstrated that rps19 deficiency reproduced the erythroid defects of DBA, including a lack of mature red blood cells and p53 activation. Notably, we found that rps19 mutants' production of globin proteins was significantly inhibited; however, globin transcript level was either increased or unaffected in rps19 mutant embryos. This dissociation of RNA/protein levels of globin genes was confirmed in another zebrafish DBA model with defects in rpl11. Using transgenic zebrafish with specific expression of mCherry in erythroid cells, we showed that protein production in erythroid cells was decreased when either rps19 or rpl11 was mutated. L-Leucine treatment alleviated the defects of protein production in erythroid cells and partially rescued the anemic phenotype in both rps19 and rpl11 mutants. Analysis of this model suggests that the decreased protein production in erythroid cells likely contributes to the blood-specific phenotype of DBA. Furthermore, the newly generated rps19 zebrafish mutant should serve as a useful animal model to study DBA. Our in vivo findings may provide clues for the future therapy strategy for DBA.

Structural determinants of human ζ-globin mRNA stability.

BackgroundThe normal accumulation of adult α and β globins in definitive erythrocytes is critically dependent upon processes that ensure that the cognate mRNAs are maintained at high levels in transcriptionally silent, but translationally active progenitor cells. The impact of these post-transcriptional regulatory events on the expression of embryonic ζ globin is not known, as its encoding mRNA is not normally transcribed during adult erythropoiesis. Recently, though, ζ globin has been recognized as a potential therapeutic for α thalassemia and sickle-cell disease, raising practical questions about constitutive post-transcriptional processes that may enhance, or possibly prohibit, the expression of exogenous or derepresssed endogenous ζ-globin genes in definitive erythroid progenitors.MethodsThe present study assesses mRNA half-life in intact cells using a pulse-chase approach; identifies cis-acting determinants of ζ-globin mRNA stability using a saturation mutagenesis strategy; establishes critical 3′UTR secondary structures using an in vitro enzymatic mapping method; and identifies trans-acting effector factors using an affinity chromatographical procedure.ResultsWe specify a tetranucleotide 3′UTR motif that is required for the high-level accumulation of ζ-globin transcripts in cultured cells, and formally demonstrate that it prolongs their cytoplasmic half-lives. Surprisingly, the ζ-globin mRNA stability motif does not function autonomously, predicting an activity that is subject to structural constraints that we subsequently specify. Additional studies demonstrate that the ζ-globin mRNA stability motif is targeted by AUF1, a ubiquitous RNA-binding protein that enhances the half-life of adult β-globin mRNA, suggesting commonalities in post-transcriptional processes that regulate globin transcripts at all stages of mammalian development.ConclusionsThese data demonstrate a mechanism for ζ-globin mRNA stability that exists in definitive erythropoiesis and is available for therapeutic manipulation in α thalassemia and sickle-cell disease.

Variation in RNA-Seq Transcriptome Profiles of Peripheral Whole Blood from Healthy Individuals with and without Globin Depletion

BackgroundThe molecular profile of circulating blood can reflect physiological and pathological events occurring in other tissues and organs of the body and delivers a comprehensive view of the status of the immune system. Blood has been useful in studying the pathobiology of many diseases. It is accessible and easily collected making it ideally suited to the development of diagnostic biomarker tests. The blood transcriptome has a high complement of globin RNA that could potentially saturate next-generation sequencing platforms, masking lower abundance transcripts. Methods to deplete globin mRNA are available, but their effect has not been comprehensively studied in peripheral whole blood RNA-Seq data. In this study we aimed to assess technical variability associated with globin depletion in addition to assessing general technical variability in RNA-Seq from whole blood derived samples.ResultsWe compared technical and biological replicates having undergone globin depletion or not and found that the experimental globin depletion protocol employed removed approximately 80% of globin transcripts, improved the correlation of technical replicates, allowed for reliable detection of thousands of additional transcripts and generally increased transcript abundance measures. Differential expression analysis revealed thousands of genes significantly up-regulated as a result of globin depletion. In addition, globin depletion resulted in the down-regulation of genes involved in both iron and zinc metal ion bonding.ConclusionsGlobin depletion appears to meaningfully improve the quality of peripheral whole blood RNA-Seq data, and may improve our ability to detect true biological variation. Some concerns remain, however. Key amongst them the significant reduction in RNA yields following globin depletion. More generally, our investigation of technical and biological variation with and without globin depletion finds that high-throughput sequencing by RNA-Seq is highly reproducible within a large dynamic range of detection and provides an accurate estimation of RNA concentration in peripheral whole blood. High-throughput sequencing is thus a promising technology for whole blood transcriptomics and biomarker discovery.

Pharmacological Inhibition Of Histone Deacetylase (HDAC) 1, 2 Or 3 Have Distinct Effects On Cellular Viability, Erythroid Differentiation, and Fetal Globin (HbG) Induction

Induction of HbG is an established therapeutic strategy for the treatment of sickle cell disease (SCD), and could also be effective in treating beta-thalassemia (bT). Genetic ablation of HDAC1 or HDAC2, but not HDAC3, results in the induction of HbG expression (Bradner JE, Proc Natl Acad Sci, 2010). Furthermore, we have previously shown that selective chemical inhibitors of HDAC1 and 2 elicit a dose and time dependent induction of HbG mRNA and fetal hemoglobin (HbF) protein in cultured human CD34+ bone marrow cells undergoing erythroid differentiation (Shearstone JS, ASH Annual Meeting Abstracts, 2012). While a variety of selective HDAC inhibitors have been used successfully to induce HbF, further clinical development has been limited by variable efficacy and concerns over off-target side-effects observed in clinical trials, potentially due to inhibition of HDAC3. Additionally, it remains to be determined if HDAC1 or HDAC2 is the preferred therapeutic target. In this work we present data that investigates the effects of selective inhibitors of HDAC1, 2, or 3 on cytotoxicity, erythroid differentiation, and HbG induction in cultured human CD34+ bone marrow cells.Acetylon Pharmaceuticals has generated a library of structurally distinct compounds with a range of selectivity for each of HDAC1, 2, or 3 (Class I HDAC) as determined in a biochemical assay platform. From our initial chemical series, we identified ACY-822 as a Class I HDAC inhibitor with IC50 values of 5, 5, and 8 nM against HDAC1, 2, and 3, respectively. In contrast, ACY-1112 is 30-fold selective for HDAC1 and 2, with IC50 values of 38, 34, and 1010 nM against HDAC1, 2, and 3, respectively. Treatment of cells for 4 days with ACY-822 (1 μM) resulted in a 20-fold decrease in cell viability, while ACY-1112 (1 μM) treatment resulted in a minimal reduction in viability (1.2-fold) and a 2-fold increase in the percentage of HbG relative to other beta-like globin transcripts. This result suggests that pharmacological inhibition of HDAC3 is cytotoxic and is consistent with the therapeutic rationale for the design selective inhibitors of HDAC1 and 2.To investigate if HDAC1 or HDAC2 is the preferred therapeutic target, we utilized a second series of structurally distinct compounds. We identified ACY-957 as an HDAC1/2 selective compound biased towards HDAC1 with IC50 values of 4, 15, and 114 nM for HDAC1, 2, and 3, respectively. In contrast ACY-1071 showed balanced HDAC1 and 2 selectivity with IC50 values of 27, 24, and 247 nM for HDAC1, 2, and 3, respectively. Treatment of cells for 6 days with 1 μM of ACY-957 or ACY-1071 resulted in a 3-fold increase in the percentage of HbG relative to other beta-like globin transcripts. However, we found that ACY-957 treatment resulted in an approximately 3-fold decrease in cell viability after 6 days of treatment, while ACY-1071 treatment resulted in a minimal reduction (1.2-fold) in cell viability. Decreased cell viability observed with ACY-957 was associated with a reduction of cells positive for the erythroid differentiation markers CD71 and glycophorinA. This result is consistent with the Mx-Cre mouse model where HDAC1KO; HDAC2het had reduced numbers of erythrocytes, thrombocytes, and total bone marrow cells, while the HDAC1het; HDAC2KO was unaffected (Wilting RH, EMBO Journal, 2010).Our results suggest that compounds with a pharmacological profile of increased selectivity towards HDAC2 inhibition versus HDAC1 may be less cytotoxic and minimize effects on differentiation, while still inducing HbG in human CD34+ bone marrow cells. Disclosures:Shearstone:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. van Duzer:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership.

αCP Poly(C) Binding Proteins Act as Global Regulators of Alternative Polyadenylation

We have previously demonstrated that the KH-domain protein αCP binds to a 3' untranslated region (3'UTR) C-rich motif of the nascent human alpha-globin (hα-globin) transcript and enhances the efficiency of 3' processing. Here we assess the genome-wide impact of αCP RNA-protein (RNP) complexes on 3' processing with a specific focus on its role in alternative polyadenylation (APA) site utilization. The major isoforms of αCP were acutely depleted from a human hematopoietic cell line, and the impact on mRNA representation and poly(A) site utilization was determined by direct RNA sequencing (DRS). Bioinformatic analysis revealed 357 significant alterations in poly(A) site utilization that could be specifically linked to the αCP depletion. These APA events correlated strongly with the presence of C-rich sequences in close proximity to the impacted poly(A) addition sites. The most significant linkage was the presence of a C-rich motif within a window 30 to 40 bases 5' to poly(A) signals (AAUAAA) that were repressed upon αCP depletion. This linkage is consistent with a general role for αCPs as enhancers of 3' processing. These findings predict a role for αCPs in posttranscriptional control pathways that can alter the coding potential and/or levels of expression of subsets of mRNAs in the mammalian transcriptome.

Genome-wide comparison of two RNA-stabilizing reagents for transcriptional profiling of peripheral blood

Peripheral whole blood is relatively easily obtained for monitoring gene expression for biomarker discovery using transcriptomic platforms such as genome-wide microarrays. However, whole blood provides challenges caused by sensitivity for exvivo incubation and overrepresentation of globin mRNAs. We compared the performance of 2 commercial whole blood preservation methods, TEMPUS (Applied Biosystems, Foster City, CA) and PAXgene (PreAnalytiX, Qiagen BD, Valencia, CA), using 2 RNA amplification protocols and high-density microarrays. Performance of commercial globin mRNA reduction protocol also was studied. Human peripheral blood samples collected with TEMPUS and PAXgene Blood RNA tubes were amplified with the RiboAmp OA 1 Round RNA Amplification Kit (Arcturus; Applied Biosystems) and the Affymetrix (Santa Clara, CA) small sample protocol. Affymetrix globin reduction protocol was applied for total RNA samples. Samples amplified with RiboAmp were hybridized on Illumina Sentrix HumanRef-8 Expression BeadChips (Illumina Inc, San Diego, CA) and subjected to statistical analyses. RiboAmp mRNA amplification did not notably amplify globin mRNA that is overrepresented in RNA isolated by both TEMPUS and PAXgene preservation. Enzymatic depletion of globin transcript reduced the quality of total RNA and is thus not recommendable. Microarray analysis showed acceptable correlation within and between the RNA preservation methods, but altogether 443 transcripts were differentially expressed between RNA samples preserved in TEMPUS and PAXgene tubes. We demonstrated that the 2 tested blood RNA-preservation methods combined with RiboAmp mRNA amplification may be used for microarray experiments without the need for a prior globin RNA reduction. However, because genes involved in immune cell functions and gene regulatory pathways were differentially expressed as a result of the technical bias between the preservation methods, they should not be used in the same analytic setting.

Increased sensitivity of next generation sequencing-based expression profiling after globin reduction in human blood RNA

BackgroundTranscriptome analysis is of great interest in clinical research, where significant differences between individuals can be translated into biomarkers of disease. Although next generation sequencing provides robust, comparable and highly informative expression profiling data, with several million of tags per blood sample, reticulocyte globin transcripts can constitute up to 76% of total mRNA compromising the detection of low abundant transcripts. We have removed globin transcripts from 6 human whole blood RNA samples with a human globin reduction kit and compared them with the same non-reduced samples using deep Serial Analysis of Gene Expression.ResultsGlobin tags comprised 52-76% of total tags in our samples. Out of 21,633 genes only 87 genes were detected at significantly lower levels in the globin reduced samples. In contrast, 11,338 genes were detected at significantly higher levels in the globin reduced samples. Removing globin transcripts allowed us to also identify 2112 genes that could not be detected in the non-globin reduced samples, with roles in cell surface receptor signal transduction, G-protein coupled receptor protein signalling pathways and neurological processes.ConclusionsThe reduction of globin transcripts in whole blood samples constitutes a reproducible and reliable method that can enrich data obtained from next generation sequencing-based expression profiling.

Evolution and Function of the Globin Intergenic Regulatory Regions of the Antarctic Dragonfishes (Notothenioidei: Bathydraconidae)

As the Southern Ocean cooled to -1.8 °C over the past 40 My, the teleostean clade Notothenioidei diversified and, under reduced selection pressure for an oxygen-transporting apparatus, became less reliant on hemoglobin and red blood cells. At the extreme of this trend, the crown group of Antarctic icefishes (Channichthyidae) lost both components of oxygen transport. Under the decreased selection scenario, we hypothesized that the Antarctic dragonfishes (Bathydraconidae, the red-blooded sister clade to the icefishes) evolved lower blood hemoglobin concentrations because their globin gene complexes (α- and β-globin gene pairs linked by a regulatory intergene) transcribe globin mRNAs less effectively than those of basal notothenioids (e.g., the Nototheniidae [notothens]). To test our hypothesis, we 1) sequenced the α/β-intergenes of the adult globin complexes of three notothen and eight dragonfish species and 2) measured globin transcript levels in representative species from each group. The typical nototheniid intergene was ∼3-4 kb in length. The bathydraconid intergenes resolved into three subclasses (long [3.8 kb], intermediate [3.0 kb], and short [1.5-2.3 kb]) that corresponded to the three subclades proposed for the taxon. Although they varied in length due to indels, the three notothen and eight dragonfish intergenes contained a conserved ∼90-nt element that we have previously shown to be required for globin gene transcription. Using the quantitative polymerase chain reaction, we found that globin mRNA levels in red cells from one notothen species and from one species of each dragonfish subclade were equivalent statistically. Thus, our results indicate that the bathydraconids have evolved adult globin loci whose regulatory intergenes tend to be shorter than those of the more basal nototheniids yet are equivalent in transcriptional efficacy. Their low blood hemoglobin concentrations are most likely due to reduction in hematocrit.

A transient definitive erythroid lineage with unique regulation of the β-globin locus in the mammalian embryo

AbstractA transient erythromyeloid wave of definitive hematopoietic progenitors (erythroid/myeloid progenitors [EMPs]) emerges in the yolk sac beginning at embryonic day 8.25 (E8.25) and colonizes the liver by E10.5, before adult-repopulating hematopoietic stem cells. At E11.5, we observe all maturational stages of erythroid precursors in the liver and the first definitive erythrocytes in the circulation. These early fetal liver erythroblasts express predominantly adult β-globins and the definitive erythroid-specific transcriptional modifiers c-myb, Sox6, and Bcl11A. Surprisingly, they also express low levels of “embryonic” βH1-, but not εy-, globin transcripts. Consistent with these results, RNA polymerase and highly modified histones are found associated with βH1- and adult globin, but not εy-globin, genes. E11.5 definitive proerythroblasts from mice transgenic for the human β-globin locus, like human fetal erythroblasts, express predominately human γ-, low β-, and no ε-globin transcripts. Significantly, E9.5 yolk sac–derived EMPs cultured in vitro have similar murine and human transgenic globin expression patterns. Later liver proerythroblasts express low levels of γ-globin, while adult marrow proerythroblasts express only β-globin transcripts. We conclude that yolk sac–derived EMPs, the first of 2 origins of definitive erythropoiesis, express a unique pattern of globin genes as they generate the first definitive erythrocytes in the liver of the mammalian embryo.

NSR-seq transcriptional profiling enables identification of a gene signature of Plasmodium falciparum parasites infecting children

Malaria caused by Plasmodium falciparum results in approximately 1 million annual deaths worldwide, with young children and pregnant mothers at highest risk. Disease severity might be related to parasite virulence factors, but expression profiling studies of parasites to test this hypothesis have been hindered by extensive sequence variation in putative virulence genes and a preponderance of host RNA in clinical samples. We report here the application of RNA sequencing to clinical isolates of P. falciparum, using not-so-random (NSR) primers to successfully exclude human ribosomal RNA and globin transcripts and enrich for parasite transcripts. Using NSR-seq, we confirmed earlier microarray studies showing upregulation of a distinct subset of genes in parasites infecting pregnant women, including that encoding the well-established pregnancy malaria vaccine candidate var2csa. We also describe a subset of parasite transcripts that distinguished parasites infecting children from those infecting pregnant women and confirmed this observation using quantitative real-time PCR and mass spectrometry proteomic analyses. Based on their putative functional properties, we propose that these proteins could have a role in childhood malaria pathogenesis. Our study provides proof of principle that NSR-seq represents an approach that can be used to study clinical isolates of parasites causing severe malaria syndromes as well other blood-borne pathogens and blood-related diseases.