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Harvey F Lodish - One of the best experts on this subject based on the ideXlab platform.
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Histone deacetylase 2 is required for chromatin condensation and subsequent enucleation of cultured mouse fetal Erythroblasts
Haematologica, 2010Co-Authors: Victor Yeh, Maki Murata-hori, Tzutzuy Ramirez, Harvey F LodishAbstract:Background During the final stages of differentiation of mammalian erythroid cells, the chromatin is condensed and enucleated. We previously reported that Rac GTPases and their downstream target, mammalian homolog of Drosophila diaphanous 2 (mDia2), are required for enucleation of in vitro cultured mouse fetal liver Erythroblasts. However, it is not clear how chromatin condensation is achieved and whether it is required for enucleation. Design and Methods Mouse fetal liver Erythroblasts were purified from embryonic day 14.5 pregnant mice and cultured in erythropoietin-containing medium. Enucleation was determined by flow-cytometry based analysis after treatment with histone deacetylase inhibitors or infection with lentiviral short harirpin RNA. Results We showed that histone deacetylases play critical roles in chromatin condensation and enucleation in cultured mouse fetal liver Erythroblasts. Enzymatic inhibition of histone deacetylases by trichostatin A or valproic acid prior to the start of enucleation blocked chromatin condensation, contractile actin ring formation and enucleation. We further demonstrated that histone deacetylases 1, 2, 3 and 5 are highly expressed in mouse fetal Erythroblasts. Short hairpin RNA down-regulation of histone deacetylase 2, but not of the other histone deacetylases, phenotypically mimicked the effect of trichostatin A or valproic acid treatment, causing significant inhibition of chromatin condensation and enucleation. Importantly, knock-down of histone deacetylase 2 did not affect erythroblast proliferation, differentiation, or apoptosis. Conclusions These results identify histone deacetylase 2 as an important regulator, mediating chromatin condensation and enucleation in the final stages of mammalian erythropoiesis.
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Histone Deacetylases Are Essential for Chromatin Condensation and Enucleation in Mammalian Erythroblasts
Blood, 2008Co-Authors: Francisco J. Sánchez-rivera, Harvey F LodishAbstract:In the last steps of differentiation mammalian erythroid cells undergo chromatin condensation and enucleation; the latter process does not occur in other vertebrates. Enucleation was critical for the evolution of mammals, as it permits an enhanced hemoglobin concentration – and thus oxygen- carrying capacity – in mammalian red blood cells. We previously reported that Rac GTPases and their downstream forming target mDia2 are required for mouse fetal erythroblast enucleation. We also found that the nucleus undergoes a gradual ~10- fold decrease in volume during erythropoiesis. Since histone deacetylases (HDACs) play important roles in chromatin condensation, we hypothesized that HDACs are involved in mammalian erythroblast enucleation. To test this hypothesis, we purified E13.5 mouse fetal TER119 negative Erythroblasts and cultured them in fibronectin-coated plates. Cells were then treated with Trichostatin A (TSA), a pan-HDAC inhibitor, at different times during erythropoiesis. TSA completely blocked enucleation and kinetic studies showed this inhibitory effect occurred earlier than the step catalyzed by the Rac-mDia2 pathway. This indicates that chromatin condensation is required for the final extrusion of the nucleus. We further investigated the activity ofHDAC6 specifically, since HDAC6 is known to interact with mDia2. We found that mDia2is acetylated in vitro and in vivo and that HDAC6 interacts with and deacetylates mDia2. Treatment of purified TER119 negative mouse fetal Erythroblasts with a specific HDAC6inhibitor partially blocked enucleation. We conclude that histone deacetylase activities are essential for mammalian Erythroblasts to undergo enucleation and that HDACs act both on chromatin condensation and mDia2 deacetylation to promote enucleation.
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Enucleation of cultured mouse fetal Erythroblasts requires Rac GTPases and mDia2
Nature cell biology, 2008Co-Authors: Senthil Raja Jayapal, Harvey F LodishAbstract:Mammalian erythroid cells undergo enucleation, an asymmetric cell division involving extrusion of a pycnotic nucleus enveloped by the plasma membrane. The mechanisms that power and regulate the enucleation process have remained obscure. Here, we show that deregulation of Rac GTPase during a late stage of erythropoiesis completely blocks enucleation of cultured mouse fetal Erythroblasts without affecting their proliferation or differentiation. Formation of the contractile actin ring (CAR) on the plasma membrane of enucleating Erythroblasts was disrupted by inhibition of Rac GTPases. Furthermore, we demonstrate that mDia2, a downstream effector of Rho GTPases and a formin protein required for nucleation of unbranched actin filaments, is also required for enucleation of mouse fetal Erythroblasts. We show that Rac1 and Rac2 bind to mDia2 in a GTP-dependent manner and that downregulation of mDia2, but not mDia1, by small interfering RNA (siRNA) during the late stages of erythropoiesis blocked both CAR formation and erythroblast enucleation. Additionally, overexpression of a constitutively active mutant of mDia2 rescued the enucleation defects induced by the inhibition of Rac GTPases. These results reveal important roles for Rac GTPases and their effector mDia2 in enucleation of mammalian Erythroblasts.
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role of ras signaling in erythroid differentiation of mouse fetal liver cells functional analysis by a flow cytometry based novel culture system
Blood, 2003Co-Authors: Jing Zhang, Merav Socolovsky, Alec W Gross, Harvey F LodishAbstract:Ras signaling plays an important role in erythropoiesis. Its function has been extensively studied in erythroid and nonerythroid cell lines as well as in primary Erythroblasts, but inconclusive results using conventional erythroid colony-forming unit (CFU-E) assays have been obtained concerning the role of Ras signaling in erythroid differentiation. Here we describe a novel culture system that supports terminal fetal liver erythroblast proliferation and differentiation and that closely recapitulates erythroid development in vivo. Erythroid differentiation is monitored step by step and quantitatively by a flow cytometry analysis; this analysis distinguishes CD71 and TER119 double-stained Erythroblasts into different stages of differentiation. To study the role of Ras signaling in erythroid differentiation, different H-ras proteins were expressed in CFU-E progenitors and early Erythroblasts with the use of a bicistronic retroviral system, and their effects on CFU-E colony formation and erythroid differentiation were analyzed. Only oncogenic H-ras, not dominant-negative H-ras, reduced CFU-E colony formation. Analysis of infected Erythroblasts in our newly developed system showed that oncogenic H-ras blocks terminal erythroid differentiation, but not through promoting apoptosis of terminally differentiated erythroid cells. Rather, oncogenic H-ras promotes abnormal proliferation of CFU-E progenitors and early Erythroblasts and supports their erythropoietin (Epo)–independent growth.
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ineffective erythropoiesis in stat5a 5b mice due to decreased survival of early Erythroblasts
Blood, 2001Co-Authors: Merav Socolovsky, Carlo Brugnara, Mark D Fleming, Volker H Haase, Harvey F LodishAbstract:Erythropoietin (Epo) controls red cell production in the basal state and during stress. Epo binding to its receptor, EpoR, on erythroid progenitors leads to rapid activation of the transcription factor Stat5. Previously, fetal anemia and increased apoptosis of fetal liver erythroid progenitors were found in Stat5a−/−5b−/− mice. However, the role of Stat5 in adult erythropoiesis was not clear. The present study shows that some adult Stat5a−/−5b−/− mice have a near-normal hematocrit but are deficient in generating high erythropoietic rates in response to stress. Further, many adult Stat5a−/−5b−/− mice have persistent anemia despite a marked compensatory expansion in their erythropoietic tissue. Analysis of erythroblast maturation in Stat5a−/−5b−/− hematopoietic tissue shows a dramatic increase in early erythroblast numbers, but these fail to progress in differentiation. Decreased expression of bcl-xLand increased apoptosis in Stat5a−/−5b−/−early Erythroblasts correlate with the degree of anemia. Hence, Stat5 controls a rate-determining step regulating early erythroblast survival.
John G Conboy - One of the best experts on this subject based on the ideXlab platform.
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an important class of intron retention events in human Erythroblasts is regulated by cryptic exons proposed to function as splicing decoys
RNA, 2018Co-Authors: Marilyn Parra, Ben W Booth, Richard Weiszmann, Brian A Yee, Gene W Yeo, James B Brown, Susan E Celniker, John G ConboyAbstract:During terminal erythropoiesis, the splicing machinery in differentiating Erythroblasts executes a robust intron retention (IR) program that impacts expression of hundreds of genes. We studied IR mechanisms in the SF3B1 splicing factor gene, which expresses ∼50% of its transcripts in late Erythroblasts as a nuclear isoform that retains intron 4. RNA-seq analysis of nonsense-mediated decay (NMD)-inhibited cells revealed previously undescribed splice junctions, rare or not detected in normal cells, that connect constitutive exons 4 and 5 to highly conserved cryptic cassette exons within the intron. Minigene splicing reporter assays showed that these cassettes promote IR. Genome-wide analysis of splice junction reads demonstrated that cryptic noncoding cassettes are much more common in large (>1 kb) retained introns than they are in small retained introns or in nonretained introns. Functional assays showed that heterologous cassettes can promote retention of intron 4 in the SF3B1 splicing reporter. Although many of these cryptic exons were spliced inefficiently, they exhibited substantial binding of U2AF1 and U2AF2 adjacent to their splice acceptor sites. We propose that these exons function as decoys that engage the intron-terminal splice sites, thereby blocking cross-intron interactions required for excision. Developmental regulation of decoy function underlies a major component of the erythroblast IR program.
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an important class of intron retention events in human Erythroblasts is regulated by cryptic exons proposed to function as splicing decoys
bioRxiv, 2018Co-Authors: Marilyn Parra, Ben W Booth, Richard Weiszmann, Brian A Yee, Gene W Yeo, James B Brown, Susan E Celniker, John G ConboyAbstract:During terminal erythropoiesis, differentiating Erythroblasts execute a robust program of intron retention (IR). We studied IR mechanisms in the SF3B1 splicing factor gene, which expresses ~50% of its transcripts in late Erythroblasts as a nuclear isoform that retains intron 4. RNA-seq splice junction reads from nonsense-mediated decay (NMD)-inhibited cells revealed that highly conserved intron sequences encode cryptic cassette exons, and minigene splicing reporter assays showed that these cassettes function as decoys that promote IR. Novel decoy exons were common in large (>1kb) retained introns, and heterologous decoys promoted retention of intron 4. Although most decoys were spliced inefficiently, they exhibited substantial binding of U2AF1 and U2AF2 adjacent to their splice acceptor sites. We propose that decoy exons engage intron-terminal splice sites, blocking cross-intron interactions required for excision, and that developmental regulation of decoy function underlies a major component of the erythroblast IR program.
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sf3b1 gene expression in erythroid cells is regulated by intron retention via a posttranscriptional mechanism involving cryptic exons proposed to function as splicing decoys
Blood, 2017Co-Authors: Marilyn Parra, Ben W Booth, Richard Weiszmann, Gene W Yeo, James B Brown, Susan E Celniker, John G ConboyAbstract:Proper expression of the MDS-disease gene, SF3B1, ensures appropriate pre-mRNA splicing in erythroid progenitors and during terminal erythropoiesis. We previously showed that the SF3B1 gene is post-transcriptionally regulated in a differentiation stage-specific manner by intron retention (IR), such that ~50% of its transcripts in mature Erythroblasts retain intron 4. Based on new mechanistic studies, we propose a model in which mostly unannotated and noncoding exons within intron 4 function as splicing decoys; i.e., they promote retention of intron 4 by interacting with, and blocking splice sites of, the adjacent exons 4 and 5. A total of six putative decoy exons were revealed via RT-PCR and RNA-seq analysis of RNA from Erythroblasts treated with inhibitors of nonsense-mediated decay. That decoy exons have IR-promoting activity is suggested by several criteria. First, the frequency of interaction between constitutive exons 4 and 5 and putative decoy exons within intron 4, measured by the abundance of splice junctions in RNA-seq read data, is temporally correlated with levels of intron 4 retention during terminal erythropoiesis. Both IR and decoy splice junctions were low in early stage Erythroblasts and much higher in mature Erythroblasts. Second, selected decoy exons exhibited IR-promoting activity in the context of minigene splicing reporters expressing the exon 3-6 region of SF3B1 in transfected K562 cells. The wild type minigene reproduced the intron-specific retention phenotype, since it was fully spliced at introns 3 and 5 but exhibited substantial retention of intron 4, whereas deletion of decoy exon 4e, or mutation of its splice sites, substantially decreased IR. Third, RBP (RNA binding protein) cross-linking data from K562 cells show that 39 splice site factors including U2AF1 and U2AF2 can bind specifically to 39 splice sites of intron 49s decoy exons. Finally, several experiments showed that IR-promoting activity of decoy exons is a more general phenomenon that likely governs IR in other erythroid genes. We observed not only that SF3B1 intron 4 decoy exons could promote IR in heterologous contexts, but also that predicted decoy exons from other erythroblast transcripts could promote IR in the SF3B1 minigene. Apart from this experimental data, comparative genomics revealed that the SF3B1 decoy exons are extremely conserved among vertebrate genomes, with two of the exons being essentially identical from fish to humans. Together this data supports the hypothesis that a subset of up-regulated IR events in late Erythroblasts are controlled by decoy exons that block productive splicing at the flanking exons. We propose that regulated IR is an important post-transcriptional mechanism for adjusting cellular splicing capacity during terminal erythropoiesis by regulating expression of key splicing factors such as SF3B1. Disclosures No relevant conflicts of interest to declare.
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the erythroid intron retention program encompasses developmentally stable and dynamic networks and regulates diverse gene classes
Blood, 2015Co-Authors: Harold Pimentel, Narla Mohandas, Marilyn Parra, Lior Pachter, Sherry L Gee, John G ConboyAbstract:Computational analysis of RNA-seq data from highly purified human Erythroblasts has been instrumental in revealing changes in pre-mRNA splicing during terminal erythropoiesis. Here we report updated studies of intron retention (IR), a type of alternative splicing in which specific introns are retained in otherwise efficiently-processed transcripts, allowing post-transcriptional modulation of cellular mRNA levels. Differences in differentiation stage-specificity, degree of retention, nuclear/cytoplasmic localization, and sensitivity to nonsense-mediated decay (NMD) suggest the existence of multiple classes of erythroblast IR subject to distinct regulatory controls. Two clusters comprising ~470 "developmentally dynamic" introns in 354 genes exhibit more efficient splicing in proErythroblasts, but elevated intron retention in orthochromatic Erythroblasts prior to enucleation. Dynamic regulation of late erythroblast IR parallels previously described splicing switches involving alternative exons. Gene ontology analysis revealed that the dynamic intron group is highly enriched in genes with RNA processing functions. Among these are several spliceosomal factors including SF3B1, a commonly mutated gene in myelodysplasia patients. We also identified several clusters of "developmentally stable" introns whose IR levels are not substantially modulated during erythropoiesis. Among this latter type are two clusters containing 294 introns that are enriched in functions related to metal ion binding. Key genes include mitoferrin-1 (SC25A37; IR~50%) and mitoferrin-2 (SLC25A28; IR~20-30%), mitochondrial iron importers essential for heme biosynthesis. We observed a correlation between splice site strength and percent IR among developmentally stable but not dynamic intron clusters, indicating that splicing regulatory mechanism(s) for the latter must require additional sequence features. A search for such features revealed that IR was significantly higher adjacent to alternative 'PTC' exons containing premature termination codons than it was adjacent to other exons; moreover, by direct RT-PCR analysis we discovered novel (unannotated) PTC exons in additional retained introns. The proposed role of PTC exons in IR is being studied experimentally using an array of minigene splicing reporter constructs. Finally, we noted that while specific IR events are erythroid specific, e.g., in the alpha spectrin gene SPTA1, computational analysis of public RNA-seq data demonstrated that most erythroblast IR events were also observed in granulocytes and in 16 other tissues surveyed by the human BodyMap project. Intron retention is likely to play critical roles in gene regulation in both hematological and non-hematological tissues. Disclosures No relevant conflicts of interest to declare.
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an erythroid specific intron retention program regulates expression of selected genes during terminal erythropoiesis
Blood, 2014Co-Authors: Harold Pimentel, Narla Mohandas, Marilyn Parra, Lior Pachter, Sherry L Gee, John G ConboyAbstract:Erythroid RNAs, like their nonerythroid counterparts, are subject to post-transcriptional processing events that critically impact their coding capacity for the erythroid proteome. Previous studies have shown that differentiating human and mouse Erythroblasts execute an extensive and dynamic alternative splicing program involving regulation of numerous alternative exons. Here we report that controlled excision of selected introns is also an important component of the erythroblast alternative splicing program. Intron retention (IR) patterns in differentiating human Erythroblasts were determined via RNA-seq analysis of FACS-purified erythroblast populations. Comparison of IR among erythroblast populations and between Erythroblasts and other hematopoietic cells suggests that regulation of IR occurs in a differentiation stage- and tissue-specific manner. For example, there was little overlap of intron retention events in Erythroblasts with those reported in differentiating granulocytes. Moreover, the IR profile of proErythroblasts differed substantially from that in orthochromatic Erythroblasts, with IR generally increasing in the more mature cells that are preparing for enucleation. IR in Erythroblasts affected numerous genes functioning in RNA processing, iron homeostasis and heme biosynthesis, protein translation, and membrane properties. Mature Erythroblasts exhibited retention of introns in several human disease genes including SF3B1, a splicing factor often mutated in myelodysplasia; TFR2, encoding transferrin receptor 2 that is mutated in a form of hemochromatosis; and FUS, an RNA binding protein implicated in ALS. Inspection of intronic RNA-seq reads in >60 genes with IR revealed that single or multiple introns can be retained within a transcript; however, other introns within the same genes, and indeed the great majority of introns in erythroblast-expressed genes, are efficiently spliced with minimal or no IR. Retained introns may be flanked by either constitutively or alternatively spliced exons, suggesting different regulatory mechanisms. Ongoing studies will explore whether IR in some transcripts might function to down-regulate gene expression by introduction of premature termination codons that induce nonsense-mediated decay, or alternatively, whether IR transcripts could represent a reserve of nearly-completed mRNAs that can be processed in response to appropriate physiological stimuli. In sum, these results suggest that a highly regulated IR program plays an important role in erythroid differentiation. Disclosures No relevant conflicts of interest to declare.
Hartmut Beug - One of the best experts on this subject based on the ideXlab platform.
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igbp1 is part of a positive feedback loop in stem cell factor dependent selective mrna translation initiation inhibiting erythroid differentiation
Blood, 2008Co-Authors: Godfrey Grech, Hartmut Beug, Ernst W. Müllner, Andrea Kolbus, Montserrat Blazquezdomingo, Walter Jacob Bakker, Marieke Von LindernAbstract:Stem cell factor (SCF)-induced activation of phosphoinositide-3-kinase (PI3K) is required for transient amplification of the erythroblast compartment. PI3K stimulates the activation of mTOR (target of rapamycin) and subsequent release of the cap-binding translation initiation factor 4E (eIF4E) from the 4E-binding protein 4EBP, which controls the recruitment of structured mRNAs to polysomes. Enhanced expression of eIF4E renders proliferation of Erythroblasts independent of PI3K. To investigate which mRNAs are selectively recruited to polysomes, we compared SCF-dependent gene expression between total and polysome-bound mRNA. This identified 111 genes primarily subject to translational regulation. For 8 of 9 genes studied in more detail, the SCF-induced polysome recruitment of transcripts exceeded 5-fold regulation and was PI3K-dependent and eIF4E-sensitive, whereas total mRNA was not affected by signal transduction. One of the targets, Immunoglobulin binding protein 1 (Igbp1), is a regulatory subunit of protein phosphatase 2A (Pp2a) sustaining mTOR signaling. Constitutive expression of Igbp1 impaired erythroid differentiation, maintained 4EBP and p70S6k phosphorylation, and enhanced polysome recruitment of multiple eIF4E-sensitive mRNAs. Thus, PI3K-dependent polysome recruitment of Igbp1 acts as a positive feedback mechanism on translation initiation underscoring the important regulatory role of selective mRNA recruitment to polysomes in the balance between proliferation and maturation of Erythroblasts.
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erythroid progenitor renewal versus differentiation genetic evidence for cell autonomous essential functions of epor stat5 and the gr
Oncogene, 2006Co-Authors: Helmut Dolznig, Bianca Habermann, Eva Maria Deiner, Florian Grebien, K Stangl, Andrea Kolbus, Marc A Kerenyi, M Kieslinger, Richard Moriggl, Hartmut BeugAbstract:The balance between hematopoietic progenitor commitment and self-renewal versus differentiation is controlled by various transcriptional regulators cooperating with cytokine receptors. Disruption of this balance is increasingly recognized as important in the development of leukemia, by causing enhanced renewal and differentiation arrest. We studied regulation of renewal versus differentiation in primary murine erythroid progenitors that require cooperation of erythropoietin receptor (EpoR), the receptor tyrosine kinase c-Kit and a transcriptional regulator (glucocorticoid receptor; GR) for sustained renewal. However, mice defective for GR- (GR(dim/dim)), EpoR- (EpoR(H)) or STAT5ab function (Stat5ab(-/-)) show no severe erythropoiesis defects in vivo. Using primary erythroblast cultures from these mutants, we present genetic evidence that functional GR, EpoR, and Stat5 are essential for erythroblast renewal in vitro. Cells from GR(dim/dim), EpoR(H), and Stat5ab(-/-) mice showed enhanced differentiation instead of renewal, causing accumulation of mature cells and gradual proliferation arrest. Stat5ab was additionally required for Epo-induced terminal differentiation: differentiating Stat5ab(-/-) Erythroblasts underwent apoptosis instead of erythrocyte maturation, due to absent induction of the antiapoptotic protein Bcl-X(L). This defect could be fully rescued by exogenous Bcl-X(L). These data suggest that signaling molecules driving leukemic proliferation may also be essential for prolonged self-renewal of normal erythroid progenitors.
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Apoptosis Protection by the Epo Target Bcl-XL Allows Factor-Independent Differentiation of Primary Erythroblasts
Current Biology, 2002Co-Authors: Helmut Dolznig, Bianca Habermann, Katharina Stangl, Eva Maria Deiner, Hartmut Beug, Ernst W. MüllnerAbstract:Abstract Background: Erythropoietin (Epo) is required for correct execution of the erythroid differentiation program. Erythropoiesis requires Bcl-X L , a major late target of Epo-receptor signaling. Mice lacking Bcl-X L die around embryonic age E12.5, forming normal erythroid progenitors but lacking functional red cells. Recently, serum-free culture conditions for expansion of murine red cell progenitors were developed, yielding cells capable of in vivo-like terminal differentiation into enucleated erythrocytes, in response to Epo/insulin. Here we address whether Epo function during terminal maturation involves a cytokine-independent "default program," requiring only apoptosis inhibition through Epo-dependent upregulation of Bcl-X L . Results: Exogenous expression of Bcl-X L or Bcl-2 in primary murine Erythroblasts or clonal erythroblast lines derived from p53 −/− mice allowed these cells to undergo terminal erythroid maturation, in the complete absence of cytokines. A potential autocrine Epo loop was ruled out by respective neutralizing antibodies. Importantly, sustained proliferation of Bcl-X L -expressing immature Erythroblasts still required respective factors (Epo, stem cell factor [SCF], and the glucocorticoid receptor ligand dexamethasone [Dex]). Epo-independent differentiation in these Bcl-X L - or Bcl-2-expressing, primary Erythroblasts was thus triggered by removal of the renewal factors SCF and Dex. This initiated the maturation-specific expression cascade of erythroid transcription factors, followed by differentiation divisions (characterized by a short G1 phase and decrease in cell size), hemoglobin accumulation, and enucleation. Conclusions: During erythroid maturation, Epo regulates red cell numbers via apoptosis inhibition, caused by Epo-dependent upregulation of the antiapoptotic protein Bcl-X L . This allows "default" terminal differentiation of apoptosis-protected, committed Erythroblasts, independent of any exogenous signals.
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fli 1 inhibits differentiation and induces proliferation of primary Erythroblasts
Oncogene, 1999Co-Authors: Rui Pereira, Helmut Dolznig, Hartmut Beug, Christine Tran Quang, Isabelle Lesault, Jacques GhysdaelAbstract:Friend virus-induced erythroleukemia involves two members of the ETS family of transcriptional regulators, both activated via proviral insertion in the corresponding loci. Spi-1/PU.1 is expressed in the disease induced by the original Friend virus SFFV(F-MuLV) complex in adult mice. In contrast, FLI-1 is overexpressed in about 75% of the erythroleukemias induced by the F-MuLV helper virus in newborn mice. To analyse the consequences of the enforced expression of FLI-1 on erythroblast differentiation and proliferation and to compare its activity to that of PU.1/Spi-1, we used a heterologous system of avian primary Erythroblasts previously described to study the cooperation between Spi-1/PU.1 and the other molecular alterations observed in SFFV-induced disease. FLI-1 was found: (i) to inhibit the apoptotic cell death program normally activated in Erythroblasts following Epo deprivation; (ii) to inhibit the terminal differentiation program induced in these cells in response to Epo and; (iii) to induce their proliferation. However, in contrast to Spi-1/PU.1, the effects of FLI-1 on erythroblast, differentiation and proliferation did not require its cooperation with an abnormally activated form of the EpoR. Enhanced survival of FLI-1 expressing Erythroblasts correlated with the upregulation of bcl2 expression. FLI-1 also prevented the rapid downregulation of cyclin D2 and D3 expression normally observed during Epo-induced differentiation and delayed the downregulation of several other genes involved in cell cycle or cell proliferation control. Our results show that overexpression of FLI-1 profoundly deregulates the normal balance between differentiation and proliferation in primary Erythroblasts. Thus, the activation of FLI-1 expression observed at the onset of F-MuLV-induced erythroleukemia may provide a proliferative advantage to virus infected cells that would otherwise undergo terminal differentiation or cell death.
Marilyn Parra - One of the best experts on this subject based on the ideXlab platform.
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an important class of intron retention events in human Erythroblasts is regulated by cryptic exons proposed to function as splicing decoys
RNA, 2018Co-Authors: Marilyn Parra, Ben W Booth, Richard Weiszmann, Brian A Yee, Gene W Yeo, James B Brown, Susan E Celniker, John G ConboyAbstract:During terminal erythropoiesis, the splicing machinery in differentiating Erythroblasts executes a robust intron retention (IR) program that impacts expression of hundreds of genes. We studied IR mechanisms in the SF3B1 splicing factor gene, which expresses ∼50% of its transcripts in late Erythroblasts as a nuclear isoform that retains intron 4. RNA-seq analysis of nonsense-mediated decay (NMD)-inhibited cells revealed previously undescribed splice junctions, rare or not detected in normal cells, that connect constitutive exons 4 and 5 to highly conserved cryptic cassette exons within the intron. Minigene splicing reporter assays showed that these cassettes promote IR. Genome-wide analysis of splice junction reads demonstrated that cryptic noncoding cassettes are much more common in large (>1 kb) retained introns than they are in small retained introns or in nonretained introns. Functional assays showed that heterologous cassettes can promote retention of intron 4 in the SF3B1 splicing reporter. Although many of these cryptic exons were spliced inefficiently, they exhibited substantial binding of U2AF1 and U2AF2 adjacent to their splice acceptor sites. We propose that these exons function as decoys that engage the intron-terminal splice sites, thereby blocking cross-intron interactions required for excision. Developmental regulation of decoy function underlies a major component of the erythroblast IR program.
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an important class of intron retention events in human Erythroblasts is regulated by cryptic exons proposed to function as splicing decoys
bioRxiv, 2018Co-Authors: Marilyn Parra, Ben W Booth, Richard Weiszmann, Brian A Yee, Gene W Yeo, James B Brown, Susan E Celniker, John G ConboyAbstract:During terminal erythropoiesis, differentiating Erythroblasts execute a robust program of intron retention (IR). We studied IR mechanisms in the SF3B1 splicing factor gene, which expresses ~50% of its transcripts in late Erythroblasts as a nuclear isoform that retains intron 4. RNA-seq splice junction reads from nonsense-mediated decay (NMD)-inhibited cells revealed that highly conserved intron sequences encode cryptic cassette exons, and minigene splicing reporter assays showed that these cassettes function as decoys that promote IR. Novel decoy exons were common in large (>1kb) retained introns, and heterologous decoys promoted retention of intron 4. Although most decoys were spliced inefficiently, they exhibited substantial binding of U2AF1 and U2AF2 adjacent to their splice acceptor sites. We propose that decoy exons engage intron-terminal splice sites, blocking cross-intron interactions required for excision, and that developmental regulation of decoy function underlies a major component of the erythroblast IR program.
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sf3b1 gene expression in erythroid cells is regulated by intron retention via a posttranscriptional mechanism involving cryptic exons proposed to function as splicing decoys
Blood, 2017Co-Authors: Marilyn Parra, Ben W Booth, Richard Weiszmann, Gene W Yeo, James B Brown, Susan E Celniker, John G ConboyAbstract:Proper expression of the MDS-disease gene, SF3B1, ensures appropriate pre-mRNA splicing in erythroid progenitors and during terminal erythropoiesis. We previously showed that the SF3B1 gene is post-transcriptionally regulated in a differentiation stage-specific manner by intron retention (IR), such that ~50% of its transcripts in mature Erythroblasts retain intron 4. Based on new mechanistic studies, we propose a model in which mostly unannotated and noncoding exons within intron 4 function as splicing decoys; i.e., they promote retention of intron 4 by interacting with, and blocking splice sites of, the adjacent exons 4 and 5. A total of six putative decoy exons were revealed via RT-PCR and RNA-seq analysis of RNA from Erythroblasts treated with inhibitors of nonsense-mediated decay. That decoy exons have IR-promoting activity is suggested by several criteria. First, the frequency of interaction between constitutive exons 4 and 5 and putative decoy exons within intron 4, measured by the abundance of splice junctions in RNA-seq read data, is temporally correlated with levels of intron 4 retention during terminal erythropoiesis. Both IR and decoy splice junctions were low in early stage Erythroblasts and much higher in mature Erythroblasts. Second, selected decoy exons exhibited IR-promoting activity in the context of minigene splicing reporters expressing the exon 3-6 region of SF3B1 in transfected K562 cells. The wild type minigene reproduced the intron-specific retention phenotype, since it was fully spliced at introns 3 and 5 but exhibited substantial retention of intron 4, whereas deletion of decoy exon 4e, or mutation of its splice sites, substantially decreased IR. Third, RBP (RNA binding protein) cross-linking data from K562 cells show that 39 splice site factors including U2AF1 and U2AF2 can bind specifically to 39 splice sites of intron 49s decoy exons. Finally, several experiments showed that IR-promoting activity of decoy exons is a more general phenomenon that likely governs IR in other erythroid genes. We observed not only that SF3B1 intron 4 decoy exons could promote IR in heterologous contexts, but also that predicted decoy exons from other erythroblast transcripts could promote IR in the SF3B1 minigene. Apart from this experimental data, comparative genomics revealed that the SF3B1 decoy exons are extremely conserved among vertebrate genomes, with two of the exons being essentially identical from fish to humans. Together this data supports the hypothesis that a subset of up-regulated IR events in late Erythroblasts are controlled by decoy exons that block productive splicing at the flanking exons. We propose that regulated IR is an important post-transcriptional mechanism for adjusting cellular splicing capacity during terminal erythropoiesis by regulating expression of key splicing factors such as SF3B1. Disclosures No relevant conflicts of interest to declare.
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the erythroid intron retention program encompasses developmentally stable and dynamic networks and regulates diverse gene classes
Blood, 2015Co-Authors: Harold Pimentel, Narla Mohandas, Marilyn Parra, Lior Pachter, Sherry L Gee, John G ConboyAbstract:Computational analysis of RNA-seq data from highly purified human Erythroblasts has been instrumental in revealing changes in pre-mRNA splicing during terminal erythropoiesis. Here we report updated studies of intron retention (IR), a type of alternative splicing in which specific introns are retained in otherwise efficiently-processed transcripts, allowing post-transcriptional modulation of cellular mRNA levels. Differences in differentiation stage-specificity, degree of retention, nuclear/cytoplasmic localization, and sensitivity to nonsense-mediated decay (NMD) suggest the existence of multiple classes of erythroblast IR subject to distinct regulatory controls. Two clusters comprising ~470 "developmentally dynamic" introns in 354 genes exhibit more efficient splicing in proErythroblasts, but elevated intron retention in orthochromatic Erythroblasts prior to enucleation. Dynamic regulation of late erythroblast IR parallels previously described splicing switches involving alternative exons. Gene ontology analysis revealed that the dynamic intron group is highly enriched in genes with RNA processing functions. Among these are several spliceosomal factors including SF3B1, a commonly mutated gene in myelodysplasia patients. We also identified several clusters of "developmentally stable" introns whose IR levels are not substantially modulated during erythropoiesis. Among this latter type are two clusters containing 294 introns that are enriched in functions related to metal ion binding. Key genes include mitoferrin-1 (SC25A37; IR~50%) and mitoferrin-2 (SLC25A28; IR~20-30%), mitochondrial iron importers essential for heme biosynthesis. We observed a correlation between splice site strength and percent IR among developmentally stable but not dynamic intron clusters, indicating that splicing regulatory mechanism(s) for the latter must require additional sequence features. A search for such features revealed that IR was significantly higher adjacent to alternative 'PTC' exons containing premature termination codons than it was adjacent to other exons; moreover, by direct RT-PCR analysis we discovered novel (unannotated) PTC exons in additional retained introns. The proposed role of PTC exons in IR is being studied experimentally using an array of minigene splicing reporter constructs. Finally, we noted that while specific IR events are erythroid specific, e.g., in the alpha spectrin gene SPTA1, computational analysis of public RNA-seq data demonstrated that most erythroblast IR events were also observed in granulocytes and in 16 other tissues surveyed by the human BodyMap project. Intron retention is likely to play critical roles in gene regulation in both hematological and non-hematological tissues. Disclosures No relevant conflicts of interest to declare.
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an erythroid specific intron retention program regulates expression of selected genes during terminal erythropoiesis
Blood, 2014Co-Authors: Harold Pimentel, Narla Mohandas, Marilyn Parra, Lior Pachter, Sherry L Gee, John G ConboyAbstract:Erythroid RNAs, like their nonerythroid counterparts, are subject to post-transcriptional processing events that critically impact their coding capacity for the erythroid proteome. Previous studies have shown that differentiating human and mouse Erythroblasts execute an extensive and dynamic alternative splicing program involving regulation of numerous alternative exons. Here we report that controlled excision of selected introns is also an important component of the erythroblast alternative splicing program. Intron retention (IR) patterns in differentiating human Erythroblasts were determined via RNA-seq analysis of FACS-purified erythroblast populations. Comparison of IR among erythroblast populations and between Erythroblasts and other hematopoietic cells suggests that regulation of IR occurs in a differentiation stage- and tissue-specific manner. For example, there was little overlap of intron retention events in Erythroblasts with those reported in differentiating granulocytes. Moreover, the IR profile of proErythroblasts differed substantially from that in orthochromatic Erythroblasts, with IR generally increasing in the more mature cells that are preparing for enucleation. IR in Erythroblasts affected numerous genes functioning in RNA processing, iron homeostasis and heme biosynthesis, protein translation, and membrane properties. Mature Erythroblasts exhibited retention of introns in several human disease genes including SF3B1, a splicing factor often mutated in myelodysplasia; TFR2, encoding transferrin receptor 2 that is mutated in a form of hemochromatosis; and FUS, an RNA binding protein implicated in ALS. Inspection of intronic RNA-seq reads in >60 genes with IR revealed that single or multiple introns can be retained within a transcript; however, other introns within the same genes, and indeed the great majority of introns in erythroblast-expressed genes, are efficiently spliced with minimal or no IR. Retained introns may be flanked by either constitutively or alternatively spliced exons, suggesting different regulatory mechanisms. Ongoing studies will explore whether IR in some transcripts might function to down-regulate gene expression by introduction of premature termination codons that induce nonsense-mediated decay, or alternatively, whether IR transcripts could represent a reserve of nearly-completed mRNAs that can be processed in response to appropriate physiological stimuli. In sum, these results suggest that a highly regulated IR program plays an important role in erythroid differentiation. Disclosures No relevant conflicts of interest to declare.
Helmut Dolznig - One of the best experts on this subject based on the ideXlab platform.
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erythroid progenitor renewal versus differentiation genetic evidence for cell autonomous essential functions of epor stat5 and the gr
Oncogene, 2006Co-Authors: Helmut Dolznig, Bianca Habermann, Eva Maria Deiner, Florian Grebien, K Stangl, Andrea Kolbus, Marc A Kerenyi, M Kieslinger, Richard Moriggl, Hartmut BeugAbstract:The balance between hematopoietic progenitor commitment and self-renewal versus differentiation is controlled by various transcriptional regulators cooperating with cytokine receptors. Disruption of this balance is increasingly recognized as important in the development of leukemia, by causing enhanced renewal and differentiation arrest. We studied regulation of renewal versus differentiation in primary murine erythroid progenitors that require cooperation of erythropoietin receptor (EpoR), the receptor tyrosine kinase c-Kit and a transcriptional regulator (glucocorticoid receptor; GR) for sustained renewal. However, mice defective for GR- (GR(dim/dim)), EpoR- (EpoR(H)) or STAT5ab function (Stat5ab(-/-)) show no severe erythropoiesis defects in vivo. Using primary erythroblast cultures from these mutants, we present genetic evidence that functional GR, EpoR, and Stat5 are essential for erythroblast renewal in vitro. Cells from GR(dim/dim), EpoR(H), and Stat5ab(-/-) mice showed enhanced differentiation instead of renewal, causing accumulation of mature cells and gradual proliferation arrest. Stat5ab was additionally required for Epo-induced terminal differentiation: differentiating Stat5ab(-/-) Erythroblasts underwent apoptosis instead of erythrocyte maturation, due to absent induction of the antiapoptotic protein Bcl-X(L). This defect could be fully rescued by exogenous Bcl-X(L). These data suggest that signaling molecules driving leukemic proliferation may also be essential for prolonged self-renewal of normal erythroid progenitors.
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Apoptosis Protection by the Epo Target Bcl-XL Allows Factor-Independent Differentiation of Primary Erythroblasts
Current Biology, 2002Co-Authors: Helmut Dolznig, Bianca Habermann, Katharina Stangl, Eva Maria Deiner, Hartmut Beug, Ernst W. MüllnerAbstract:Abstract Background: Erythropoietin (Epo) is required for correct execution of the erythroid differentiation program. Erythropoiesis requires Bcl-X L , a major late target of Epo-receptor signaling. Mice lacking Bcl-X L die around embryonic age E12.5, forming normal erythroid progenitors but lacking functional red cells. Recently, serum-free culture conditions for expansion of murine red cell progenitors were developed, yielding cells capable of in vivo-like terminal differentiation into enucleated erythrocytes, in response to Epo/insulin. Here we address whether Epo function during terminal maturation involves a cytokine-independent "default program," requiring only apoptosis inhibition through Epo-dependent upregulation of Bcl-X L . Results: Exogenous expression of Bcl-X L or Bcl-2 in primary murine Erythroblasts or clonal erythroblast lines derived from p53 −/− mice allowed these cells to undergo terminal erythroid maturation, in the complete absence of cytokines. A potential autocrine Epo loop was ruled out by respective neutralizing antibodies. Importantly, sustained proliferation of Bcl-X L -expressing immature Erythroblasts still required respective factors (Epo, stem cell factor [SCF], and the glucocorticoid receptor ligand dexamethasone [Dex]). Epo-independent differentiation in these Bcl-X L - or Bcl-2-expressing, primary Erythroblasts was thus triggered by removal of the renewal factors SCF and Dex. This initiated the maturation-specific expression cascade of erythroid transcription factors, followed by differentiation divisions (characterized by a short G1 phase and decrease in cell size), hemoglobin accumulation, and enucleation. Conclusions: During erythroid maturation, Epo regulates red cell numbers via apoptosis inhibition, caused by Epo-dependent upregulation of the antiapoptotic protein Bcl-X L . This allows "default" terminal differentiation of apoptosis-protected, committed Erythroblasts, independent of any exogenous signals.
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fli 1 inhibits differentiation and induces proliferation of primary Erythroblasts
Oncogene, 1999Co-Authors: Rui Pereira, Helmut Dolznig, Hartmut Beug, Christine Tran Quang, Isabelle Lesault, Jacques GhysdaelAbstract:Friend virus-induced erythroleukemia involves two members of the ETS family of transcriptional regulators, both activated via proviral insertion in the corresponding loci. Spi-1/PU.1 is expressed in the disease induced by the original Friend virus SFFV(F-MuLV) complex in adult mice. In contrast, FLI-1 is overexpressed in about 75% of the erythroleukemias induced by the F-MuLV helper virus in newborn mice. To analyse the consequences of the enforced expression of FLI-1 on erythroblast differentiation and proliferation and to compare its activity to that of PU.1/Spi-1, we used a heterologous system of avian primary Erythroblasts previously described to study the cooperation between Spi-1/PU.1 and the other molecular alterations observed in SFFV-induced disease. FLI-1 was found: (i) to inhibit the apoptotic cell death program normally activated in Erythroblasts following Epo deprivation; (ii) to inhibit the terminal differentiation program induced in these cells in response to Epo and; (iii) to induce their proliferation. However, in contrast to Spi-1/PU.1, the effects of FLI-1 on erythroblast, differentiation and proliferation did not require its cooperation with an abnormally activated form of the EpoR. Enhanced survival of FLI-1 expressing Erythroblasts correlated with the upregulation of bcl2 expression. FLI-1 also prevented the rapid downregulation of cyclin D2 and D3 expression normally observed during Epo-induced differentiation and delayed the downregulation of several other genes involved in cell cycle or cell proliferation control. Our results show that overexpression of FLI-1 profoundly deregulates the normal balance between differentiation and proliferation in primary Erythroblasts. Thus, the activation of FLI-1 expression observed at the onset of F-MuLV-induced erythroleukemia may provide a proliferative advantage to virus infected cells that would otherwise undergo terminal differentiation or cell death.