Interrogating Post-Transcriptional Mechanisms of Fetal Hemoglobin Regulation

Abstract Elevated levels of fetal hemoglobin (HbF) significantly ameliorate clinical outcomes for patients with beta-hemoglobinopathies, such as sickle cell disease (SCD). The only FDA-approved drug for treating SCD through inducing HbF is hydroxyurea, however the mechanism of action is unknown with variable effectiveness among patients. Thus, there remains a strong interest to identify more robust means of upregulating HbF, such as specific inhibition of HbF repressors. BCL11A and LRF are well-characterized transcription factors that independently repress the fetal type b-globin like genes HBG1 and HBG2 but their therapeutic potential is limited by challenging druggability and critical developmental function. However, upstream regulation of these factors, such as post-transcriptional mechanisms, are not well studied and may house novel therapeutic targets. To this end, we employed a CRISPR/Cas9 based screening approach to interrogate a library of RNA binding proteins (RBP) in the context of HbF regulation. Using HUDEP2 cells, a human adult-type erythroid progenitor cell line, we screened 341 human RBPs and identified four candidate RBPs, none of which have previously been implicated in HbF regulation. Of these candidates, RNA Binding Motif 12 (RBM12) showed the greatest level of HbF induction following in vitro depletion. Depletion of RBM12 protein in HUDEP2 cells and human CD34 + hematopoietic stem and progenitor cells (HSPC) via CRISPR/Cas9 editing raised HbF production 2-4 fold as assessed by HbF flow cytometry, HBG1/2 mRNA, and protein (γ-globin). Cell viability and maturation of RBM12 perturbed cells were largely intact. Additionally, RBM12 depletion in CD34 + HSPCs derived from SCD patients resulted in reduced percentage of sickled cells under hypoxic conditions. Unexpectedly, reduction of RBM12 had minimal effect on BCL11A and LRF expression suggesting that RBM12 may regulate HbF through a pathway that is indirectly related or independent of these transcription factors. RBM12 is an RBP that is widely expressed across diverse cell types and contains multiple RNA recognition motifs (RRM). While it has been implicated in various cancers and neurological disorders, its functions are not well studied. As an RBP, RBM12 can carry out several roles of post-transcriptional regulation, such as pre-mRNA splicing, mRNA transport, stabilization, and translation. As these activities are executed in different cellular compartments, we set out to narrow down RBM12 function by assessing its subcellular localization. Immunofluorescence staining revealed strong nuclear presence of RBM12, suggesting that it functions via mRNA biogenesis and/or processing. RNASeq and LC-MS/MS analysis of RBM12 KO CD34 + HSPCs revealed modest changes in the transcriptome and proteome. In order to gain mechanistic insight into RBM12 in the context of HbF regulation, we performed cDNA rescue experiments in RBM12-deficient HUDEP2 clones. Overexpression of full length RBM12 restored HbF repression. Notably, four out of the five RRMs were dispensable for HbF silencing, but RRM1 was essential for this activity. Interestingly, an extended form of RRM1 was also sufficient for HbF silencing. Mechanistic studies of this RRM1 module are underway and will be discussed. In sum, the identification of RBM12 as a regulator of HbF production represents a previously undescribed post-transcriptional layer of hemoglobin gene regulation. In pursuing this path, we hope to gain a deeper understanding of this understudied RBP in the context of HbF regulation which might in turn lead to the identification of potential therapeutic targets for the treatment of SCD and other hemoglobinopathies. Disclosures Blobel: Pfizer: Consultancy; Fulcrum Therapeutics, Inc.: Consultancy.

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Interrogating RNA Binding Proteins as Novel Regulators of Fetal Hemoglobin Expression

Blood ◽

10.1182/blood-2019-125901 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 966-966

Author(s):

Aoi Wakabayashi ◽

Jeremy D. Grevet ◽

Xianjiang Lan ◽

Malini Sharma ◽

Junwei Shi ◽

...

Keyword(s):

Transcriptional Regulation ◽

Transcription Factors ◽

Research Funding ◽

Rna Binding ◽

Rna Binding Proteins ◽

Fetal Hemoglobin ◽

Protein Levels ◽

Hbf Induction ◽

Post Transcriptional Regulation ◽

Upstream Regulators

Elevated levels of fetal hemoglobin (HbF) can alleviate symptoms of hemoglobinopathies, such as sickle cell disease (SCD). Hydroxyurea is the only FDA approved drug that works through this mechanism of HbF induction; however, its efficacy is variable among patients and its mechanism of action is not well understood. Therefore, significant clinical benefit would arise from a more reliable treatment to upregulate HbF, such as developing inhibitors that target HbF repressors. The transcription factors, BCL11A and LRF, are two major independent repressors of HbF however, they have been challenging to control via pharmacologic means. While these transcription factors and their co-factors have been extensively studied, upstream regulation of these transcription factors, such as potential post-transcriptional regulators, are not as well studied. Exploration of these upstream regulators might yield new insights into basic mechanisms of transcriptional and post-transcriptional regulation of HbF, which has the potential to uncover novel therapeutic targets. For example, we have previously used a novel screening approach to successfully identify the protein kinase HRI as a regulator of HbF through BCL11A expression (Grevet and Lan et al., Science, 2018). Novel targets such as HRI may be more amenable to pharmacologic regulation. To uncover novel upstream regulators of HbF, we employed a CRISPR/Cas9 based screening approach to target a spectrum of RNA binding proteins (RBPs) potentially involved in post transcriptional regulation of HbF expression. Using a human erythroid progenitor cell line, termed HUDEP2, we interrogated 342 human RBPs using an sgRNA library that targets RBPs harboring RNA methyltransferase and RNA recognition motifs. This screen yielded four candidate RBPs, in which their disruption or depletion in human primary erythroid cultures and HUDEP2 cells raised HbF levels. Three of these are members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family, which have not previously been implicated in HbF regulation. Of these candidates, polypyrimidine tract binding protein 1 (PTBP1) showed the greatest level of HbF induction following in vitro depletion. Significant depletion of PTBP1 protein (>60%) in HUDEP2 cells and human CD34+ derived erythroid progenitors via CRISPR/Cas9 editing raised HbF production 2-4 fold as assessed by measuring % HbF positive cells, γ-globin (HBG, fetal β-like globin) mRNA, and HBG protein levels. Cell viability of PTBP1 perturbed samples are largely unaffected, however there is a delay in terminal differentiation as assessed by cell surface markers CD71 and CD235a (2-3 fold decrease in CD71-/CD235a+ cells at day 15 of differentiation). Unexpectedly, depletion of PTBP1 had minimal effect on BCL11A and LRF mRNA or protein levels. This suggests PTBP1 might impact the expression or activities of co-factors or upstream regulators of these transcription factors. Ongoing work is aimed at defining the mechanism of PTBP1 action by identifying its molecular targets. In sum, the identification of PTBP1 as a regulator of HbF production represents a previously undescribed layer of hemoglobin gene regulation. In pursuing this path, we hope to gain a deeper understanding of this process which might in turn lead to the identification of potential therapeutic targets for the treatment of SCD and other hemoglobinopathies. Disclosures Blobel: Bioverativ: Research Funding; Pfizer: Research Funding.

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Immunotherapeutic Potential of m6A-Modifiers and microRNAs in Controlling Acute Myeloid Leukaemia

Biomedicines ◽

10.3390/biomedicines9060690 ◽

2021 ◽

Vol 9 (6) ◽

pp. 690

Author(s):

Sunil Kumar ◽

Ravinder Nagpal ◽

Amit Kumar ◽

Muhammad Umer Ashraf ◽

Yong-Soo Bae

Keyword(s):

Acute Myeloid Leukaemia ◽

Myeloid Leukaemia ◽

Rna Binding ◽

Rna Binding Proteins ◽

Therapeutic Potential ◽

White Blood Cells ◽

Rna Modifications ◽

Hematopoietic Stem ◽

Tumour Immunity ◽

Acute Myeloid

Epigenetic alterations have contributed greatly to human carcinogenesis. Conventional epigenetic studies have been predominantly focused on DNA methylation, histone modifications, and chromatin remodelling. Epitranscriptomics is an emerging field that encompasses the study of RNA modifications that do not affect the RNA sequence but affect functionality via a series of RNA binding proteins called writer, reader and eraser. Several kinds of epi-RNA modifications are known, such as 6-methyladenosine (m6A), 5-methylcytidine (m5C), and 1-methyladenosine. M6A modification is the most studied and has large therapeutic implications. In this review, we have summarised the therapeutic potential of m6A-modifiers in controlling haematological disorders, especially acute myeloid leukaemia (AML). AML is a type of blood cancer affecting specific subsets of blood-forming hematopoietic stem/progenitor cells (HSPCs), which proliferate rapidly and acquire self-renewal capacities with impaired terminal cell-differentiation and apoptosis leading to abnormal accumulation of white blood cells, and thus, an alternative therapeutic approach is required urgently. Here, we have described how RNA m6A-modification machineries EEE (Editor/writer: Mettl3, Mettl14; Eraser/remover: FTO, ALKBH5, and Effector/reader: YTHDF-1/2) could be reformed into potential druggable candidates or as RNA-modifying drugs (RMD) to treat leukaemia. Moreover, we have shed light on the role of microRNAs and suppressors of cytokine signalling (SOCS/CISH) in increasing anti-tumour immunity towards leukaemia. We anticipate, our investigation will provide fundamental knowledge in nurturing the potential of RNA modifiers in discovering novel therapeutics or immunotherapeutic procedures.

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3'HS1 CTCF binding site in human β-globin locus regulates fetal hemoglobin expression

10.1101/2021.05.18.444713 ◽

2021 ◽

Author(s):

Pamela Himadewi ◽

Xue Qing David Wang ◽

Fan Feng ◽

Haley Gore ◽

Yushuai Liu ◽

...

Keyword(s):

Binding Site ◽

Progenitor Cells ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Ctcf Binding ◽

Ctcf Binding Site ◽

Distal Enhancer ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Ctcf Site

Mutations in the adult β-globin gene can lead to a variety of hemoglobinopathies, including sickle cell disease and β-thalassemia. An increase in fetal hemoglobin expression throughout adulthood, a condition named Hereditary Persistence of Fetal Hemoglobin (HPFH), has been found to ameliorate hemoglobinopathies. Deletional HPFH occurs through the excision of a significant portion of the 3 prime end of the β-globin locus, including a CTCF binding site termed 3'HS1. Here, we show that the deletion of this CTCF site alone induces fetal hemoglobin expression in both adult CD34+ hematopoietic stem and progenitor cells and HUDEP-2 erythroid progenitor cells. This induction is driven by the ectopic access of a previously postulated distal enhancer located in the OR52A1 gene downstream of the locus, which can also be insulated by the inversion of the 3'HS1 CTCF site. This suggests that genetic editing of this binding site can have therapeutic implications to treat hemoglobinopathies.

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Post-transcriptional regulation in hematopoiesis: RNA binding proteins take control

Biochemistry and Cell Biology ◽

10.1139/bcb-2017-0310 ◽

2019 ◽

Vol 97 (1) ◽

pp. 10-20 ◽

Cited By ~ 7

Author(s):

Laura P.M.H. de Rooij ◽

Derek C.H. Chan ◽

Ava Keyvani Chahi ◽

Kristin J. Hope

Keyword(s):

Transcriptional Regulation ◽

Stem Cell ◽

Cell Fate ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Control Of Gene Expression ◽

Hematopoietic Stem ◽

Cell Fate Decisions ◽

Post Transcriptional Regulation

Normal hematopoiesis is sustained through a carefully orchestrated balance between hematopoietic stem cell (HSC) self-renewal and differentiation. The functional importance of this axis is underscored by the severity of disease phenotypes initiated by abnormal HSC function, including myelodysplastic syndromes and hematopoietic malignancies. Major advances in the understanding of transcriptional regulation of primitive hematopoietic cells have been achieved; however, the post-transcriptional regulatory layer that may impinge on their behavior remains underexplored by comparison. Key players at this level include RNA-binding proteins (RBPs), which execute precise and highly coordinated control of gene expression through modulation of RNA properties that include its splicing, polyadenylation, localization, degradation, or translation. With the recent identification of RBPs having essential roles in regulating proliferation and cell fate decisions in other systems, there has been an increasing appreciation of the importance of post-transcriptional control at the stem cell level. Here we discuss our current understanding of RBP-driven post-transcriptional regulation in HSCs, its implications for normal, perturbed, and malignant hematopoiesis, and the most recent technological innovations aimed at RBP–RNA network characterization at the systems level. Emerging evidence highlights RBP-driven control as an underappreciated feature of primitive hematopoiesis, the greater understanding of which has important clinical implications.

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Molecular dissection of a genome defense pathway in Neurospora crassa

10.32469/10355/49006 ◽

2015 ◽

Author(s):

◽

Erin C. Boone

Keyword(s):

Neurospora Crassa ◽

Rna Binding ◽

Rna Binding Proteins ◽

Nuclear Periphery ◽

Bimolecular Fluorescence Complementation ◽

Proper Function ◽

Rnai Pathway ◽

Binding Motif ◽

Perinuclear Region ◽

A Genome

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Meiotic silencing by unpaired DNA (MSUD) is an RNA interference (RNAi) pathway in Neurospora crassa that detects genes without a homologous partner and silences them for the duration of sexual development. In this study, we have further elucidated the function of known MSUD proteins, identified novel proteins that are required for MSUD, and demonstrated the conservation of RNAi-related processes at the nuclear periphery. We began by showing SAD-2 is crucial for the localization of other MSUD proteins in the perinuclear region. These data suggest that SAD-2 works as a scaffold protein and that proper function of MSUD, like other germline RNAi-like systems, is reliant on the presence of silencing proteins in the perinuclear region. An MSUD suppression assay identified two novel MSUD proteins, SAD-Y and SAD-B'. Even though SAD-Y and its homologs contain a conserved putative RNA- binding motif, they have yet to be assigned to a biochemical pathway. Our work here has linked silencing to SAD-Y-like proteins. SAD-Y has been shown to interact with other MSUD factors in both the nucleus and at the nuclear periphery. SAD-B's homolog has been found in the nuage, an epicenter for RNA-binding proteins involved in post-transcriptional regulation for Drosophila germline cells. SAD-B interacts with core MSUD proteins and has an especially intimate association with SMS-2, which requires it for localization. Furthermore, bimolecular fluorescence complementation (BiFC) revealed that SAD-B' interacts with a Golgi retrograde transport protein and an autophagy marker protein, suggesting the importance of the endomembrane system in this RNAi process.

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Posttranscriptional regulation of human endogenous retroviruses by RNA-binding motif protein 4, RBM4

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2005237117 ◽

2020 ◽

Vol 117 (42) ◽

pp. 26520-26530

Author(s):

Amir K. Foroushani ◽

Bryan Chim ◽

Madeline Wong ◽

Andre Rastegar ◽

Patrick T. Smith ◽

...

Keyword(s):

Human Genome ◽

Posttranscriptional Regulation ◽

Rna Binding ◽

Rna Binding Proteins ◽

Ectopic Expression ◽

Transcript Level ◽

Endogenous Retroviruses ◽

Host Protein ◽

Binding Motif ◽

Sequencing Data

The human genome encodes for over 1,500 RNA-binding proteins (RBPs), which coordinate regulatory events on RNA transcripts. Most studies of RBPs have concentrated on their action on host protein-encoding mRNAs, which constitute a minority of the transcriptome. A widely neglected subset of our transcriptome derives from integrated retroviral elements, termed endogenous retroviruses (ERVs), that comprise ∼8% of the human genome. Some ERVs have been shown to be transcribed under physiological and pathological conditions, suggesting that sophisticated regulatory mechanisms to coordinate and prevent their ectopic expression exist. However, it is unknown how broadly RBPs and ERV transcripts directly interact to provide a posttranscriptional layer of regulation. Here, we implemented a computational pipeline to determine the correlation of expression between individual RBPs and ERVs from single-cell or bulk RNA-sequencing data. One of our top candidates for an RBP negatively regulating ERV expression was RNA-binding motif protein 4 (RBM4). We used photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation to demonstrate that RBM4 indeed bound ERV transcripts at CGG consensus elements. Loss of RBM4 resulted in an elevated transcript level of bound ERVs of the HERV-K and -H families, as well as increased expression of HERV-K envelope protein. We pinpointed RBM4 regulation of HERV-K to a CGG-containing element that is conserved in the LTRs of HERV-K-10, -K-11, and -K-20, and validated the functionality of this site using reporter assays. In summary, we systematically identified RBPs that may regulate ERV function and demonstrate a role for RBM4 in controlling ERV expression.

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Concurrent binding to DNA and RNA facilitates the pluripotency reprogramming activity of Sox2

Nucleic Acids Research ◽

10.1093/nar/gkaa067 ◽

2020 ◽

Vol 48 (7) ◽

pp. 3869-3887 ◽

Cited By ~ 1

Author(s):

Linlin Hou ◽

Yuanjie Wei ◽

Yingying Lin ◽

Xiwei Wang ◽

Yiwei Lai ◽

...

Keyword(s):

Dna Sequences ◽

Target Genes ◽

Rna Binding ◽

Rna Binding Proteins ◽

High Mobility ◽

Binding Motif ◽

Dna And Rna ◽

Somatic Cell Reprogramming ◽

Double Stranded Dna

Abstract Some transcription factors that specifically bind double-stranded DNA appear to also function as RNA-binding proteins. Here, we demonstrate that the transcription factor Sox2 is able to directly bind RNA in vitro as well as in mouse and human cells. Sox2 targets RNA via a 60-amino-acid RNA binding motif (RBM) positioned C-terminally of the DNA binding high mobility group (HMG) box. Sox2 can associate with RNA and DNA simultaneously to form ternary RNA/Sox2/DNA complexes. Deletion of the RBM does not affect selection of target genes but mitigates binding to pluripotency related transcripts, switches exon usage and impairs the reprogramming of somatic cells to a pluripotent state. Our findings designate Sox2 as a multi-functional factor that associates with RNA whilst binding to cognate DNA sequences, suggesting that it may co-transcriptionally regulate RNA metabolism during somatic cell reprogramming.

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Impaired Erythropoiesis Of Gfi-1 Null Hematopoietic Progenitor Cells Is Rescued By Reducing Id2 Levels

Blood ◽

10.1182/blood.v122.21.737.737 ◽

2013 ◽

Vol 122 (21) ◽

pp. 737-737

Author(s):

Wonil Kim ◽

Kimberly D Klarmann ◽

Jonathan R Keller

Keyword(s):

Transcription Factors ◽

Progenitor Cells ◽

Molecular Mechanisms ◽

Early Stage ◽

Erythroid Cell ◽

Mouse Bone Marrow ◽

Short Term ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Erythroid Development

Abstract The survival, self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPC) are tightly regulated by extrinsic signals, and intrinsically by transcription factors and their regulatory networks. The molecular and cellular mechanisms, which regulate the complex process of hematopoiesis, depend upon the correct expression of transcription factors and their regulators. One such family of regulators is the inhibitor of DNA binding/differentiation (Id), which is helix-loop-helix proteins that function by acting as dominant negative regulators of transcription factors such as E proteins, ETS, Pax, and retinoblastoma proteins. Expression of Id2, one of the Id family proteins, is regulated by growth factor independence-1 (Gfi-1) encoding a transcriptional repressor. Gfi-1 is required for the development of multiple cell lineages including HSPC and ultimately differentiated blood cells. Although genes have been identified to mediate hematopoietic defects observed in Gfi-1 knockout (Gfi-1 KO) mice including the maturational and developmental defects in granulocyte (CSF-1, RasGRP1, and PU.1) and B cell (PU.1 or Id2), and myeloid hyperplasia (Id2 or HoxA9), Gfi-1-target genes that mediate the defects in radioprotection, maintenance of HSC, and erythroid hyperplasia in Gfi-1 KO mice are unknown. Since Id2 expression is elevated in HSPC of Gfi-1 KO mice and Id2 promotes cell proliferation, we hypothesized that lowering Id2 expression could rescue the HSPC defects in the Gfi-1 KO mice. By transplanting Gfi-1 KO mouse bone marrow cells (BMC) into lethally-irradiated recipient mice, we observed that short-term reconstituting cell (STRC) activity in Gfi-1 KO BMC is rescued by transplanting Gfi-1 KO; Id2 Het (heterozygosity at the Id2 locus) BMC, while the long-term reconstitution defect of HSC was not. Interestingly, lineage- Sca-1- c-Kithi HPC, which enriched for megakaryocyte-erythroid progenitor (MEP) as one of the STRC, were fully restored in mice transplanted with Gfi-1 KO; Id2 Het BMC, in contrast to lack of the HPC in Gfi-1 KO BM-transplanted mice. The restoration of donor c-Kithi HPC was directly correlated with increased red blood cell (RBC) levels in recipient mice, which was produced after donor BM engraftment. Furthermore, we identified that reduced Id2 levels restore erythroid cell development by rescuing short-term hematopoietic stem cell, common myeloid progenitor and MEP in the Gfi-1 KO mice. In addition, burst forming unit-erythroid (BFU-E) colony assay showed that hemoglobinized BFU-E development was restored in Gfi-1 KO BM and spleen by lowering Id2 levels. Unlike Id2 reduction, reducing other Id family (Id1 or Id3) levels in Gfi-1 KO mice does not rescue the impaired development of erythroid and other hematopoietic lineages including myeloid, T and B cells. Abnormal expansion of CD71+ Ter119-/low erythroid progenitor cells was rescued in Gfi-1 KO; Id2 Het BMC compared to those in Gfi-1 KO mice. Thus, we hypothesized that erythroid development was blocked at the early stage of erythropoiesis due to the ectopic expression of Id2 in Gfi-1 KO mice. Using Id2 promoter-driven YFP reporter mice, we found that Id2 is highly expressed in the CD71+ Ter119-/low erythroid progenitors, and decreases as the cells mature to pro-erythroblasts and erythroblasts, suggesting that repression of Id2 expression is required for proper erythroid differentiation in the later stages. The dramatic changes of Id2 expression during erythroid development support our findings that the overexpression of Id2 in the absence of Gfi-1-mediated transcriptional repression causes impaired erythropoiesis at the early stage. To identify the molecular mechanisms that could account for how reduced Id2 levels rescue erythropoiesis in Gfi-1 KO mice, we compared the expression of genes and proteins in Gfi-1 KO; Id2 Het and Gfi-1 KO BMC. Using microarray, qRT-PCR and western blot, we discovered that reduction of Id2 expression in Gfi-1 KO BMC results in increased expression of Gata1, EKlf, and EpoR genes, which are required for erythropoiesis. However, the expression levels of cell cycle regulators were not altered by lowering Id2 expression in Gfi-1 KO mice. These data suggest a novel molecular mechanism in which Gfi-1 modulates erythropoiesis by repressing the expression of Id2 that reduce the levels of Id2 protein, binding to E2A and inhibiting the formation of E2A/Scl transcription enhancer complex. Disclosures: No relevant conflicts of interest to declare.

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Optimization of Bcl11a Knockdown By miRNA Scaffold Embedded Shrnas Leading to Enhanced Induction of Fetal Hemoglobin in Erythroid Cells for the Treatment of Beta-Hemoglobinopathies

Blood ◽

10.1182/blood.v124.21.2150.2150 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2150-2150

Author(s):

Christian Brendel ◽

Swaroopa Guda ◽

Raffaele Renella ◽

Peng Du ◽

Daniel E. Bauer ◽

...

Keyword(s):

Fetal Hemoglobin ◽

Erythroid Cells ◽

Guide Strand ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Pol Ii ◽

Knock Down ◽

U6 Promoter ◽

Pol Iii ◽

Murine Erythroleukemia

Abstract Repression of the BCL11A protein could represent a therapeutic target for beta-hemoglobinopathies, as its knock-down has been shown to induce the expression of the fetal HBG (y-globin) gene ultimately leading to increased levels of the fetal hemoglobin tetramer (HbF, a2y2). In mice, Bcl11a is a key repressor of the murine HBG homolog Hbb-y. RNA interference (RNAi) technology using short hairpin RNAs (shRNAs) expressed via pol III promoters has been used to modulate gene expression in a variety of mammalian cell types. However, we found a negative impact of Bcl11a knockdown on hematopoietic stem cells (HSCs), limiting the repopulation efficiency and long-term engraftment after genetic modification, which is a major impediment for its translation into human therapeutic applications. To achieve lineage-specific targeting of mRNAs in an attempt to reduce HSC toxicity, expression of shRNAs via pol II promoters is required, necessitating embedding the shRNA in mammalian microRNA (shRNAmir) sequences for expression and processing. To achieve optimal knockdown of the Bcl11a transcription factor in erythroid progenitor and precursor cells, we first compared the efficiency of mRNA modulation via pol III (U6-promoter) vs pol II (SFFV-promoter) based lentiviral vectors. We demonstrate a 100-1000 fold lower Hbb-y induction using shRNAmir vs pol III mediated shRNA vector backbones due to reduced Bcl11a knockdown efficiency. In order to understand the molecular basis for these differences, small RNA sequence analysis was performed on murine erythroleukemia cells (MEL) cells transduced by multiple shRNA–shRNAmir pairs. We show that shRNAs expressed via a U6 promoter yield guide strand sequences which differ by a 2-4 nt shift compared to pol II driven (shRNAmir) mature guide strand sequences. RNA sequencing demonstrated that the stretch of uridines making up part of the pol III termination signal is transcribed and included at the 3’ end of the shRNA. This results in the generation of mature guide strand sequences with an alternative seed sequence compared to the predicted sequence and compared to miRNA embedded shRNAs. The difference in the seed sequences between the two expression systems strongly influences the efficacy of target gene knockdown, leading to reduced knockdown in pol II based vectors. We engineered a 4bp shift into guide strands of shRNAmirs that resulted in a faithfully processed shRNA sequence (a mature guide strand sequence identical to U6-driven sh-RNAs) and improved knock-down efficiency of Bcl11a at the protein level in most cases. The improved knockdown of Bcl11a was associated with a 100-300-fold enhancement of Hbb-y induction in MEL cells. Based on these results, we propose a modified strategy for the prospective design of shRNAmirs derived from shRNA screens in pol III vector backbones to achieve lineage-specific regulation of target genes. Targeted expression of shRNAmiRs to the erythroid compartment driven by a b-globin promoter/LCR element circumvented the detrimental effect on HSC engraftment, while still mediating efficient BCL11A knockdown, leading to high y-globin induction and formation of substantial amounts of fetal hemoglobin in human CD34-derived erythroid cells in vitro. Disclosures No relevant conflicts of interest to declare.

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c-Myc is a target of RNA-binding motif protein 15 in the regulation of adult hematopoietic stem cell and megakaryocyte development

Blood ◽

10.1182/blood-2009-01-197921 ◽

2009 ◽

Vol 114 (10) ◽

pp. 2087-2096 ◽

Cited By ~ 27

Author(s):

Chao Niu ◽

Jiwang Zhang ◽

Peter Breslin ◽

Mihaela Onciu ◽

Zhigui Ma ◽

...

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Rna Binding ◽

Binding Motif ◽

B Cell Differentiation ◽

Regulation Of Expression ◽

Hematopoietic Stem ◽

Megakaryoblastic Leukemia ◽

Normal Functions ◽

Hsc Niche

Abstract RNA-binding motif protein 15 (RBM15) is involved in the RBM15-megakaryoblastic leukemia 1 fusion in acute megakaryoblastic leukemia. Although Rbm15 has been reported to be required for B-cell differentiation and to inhibit myeloid and megakaryocytic expansion, it is not clear what the normal functions of Rbm15 are in the regulation of hematopoietic stem cell (HSC) and megakaryocyte development. In this study, we report that Rbm15 may function in part through regulation of expression of the proto-oncogene c-Myc. Similar to c-Myc knockout (c-Myc-KO) mice, long-term (LT) HSCs are significantly increased in Rbm15-KO mice due to an apparent LT-HSC to short-term HSC differentiation defect associated with abnormal HSC-niche interactions caused by increased N-cadherin and β1 integrin expression on mutant HSCs. Both serial transplantation and competitive reconstitution capabilities of Rbm15-KO LT-HSCs are greatly compromised. Rbm15-KO and c-Myc-KO mice also share related abnormalities in megakaryocyte development, with mutant progenitors producing increased, abnormally small low-ploidy megakaryocytes. Consistent with a possible functional interplay between Rbm15 and c-Myc, the megakaryocyte increase in Rbm15-KO mice could be partially reversed by ectopic c-Myc. Thus, Rbm15 appears to be required for normal HSC-niche interactions, for the ability of HSCs to contribute normally to adult hematopoiesis, and for normal megakaryocyte development; these effects of Rbm15 on hematopoiesis may be mediated at least in part by c-Myc.

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