Posttranscriptional regulation of erythropoiesis by RNA binding proteins and microRNAs

POSTTRANSCRIPTIONAL REGULATON OF ERYTHROPOIESIS BY RNA-BINDING PROTEINS AND MICRORNAS LINGBO ZHANG NATIONAL UNIVERSITY OF SINGAPORE 2013 POSTTRANSCRIPTIONAL REGULATON OF ERYTHROPOIESIS BY RNA-BINDING PROTEINS AND MICRORNAS LINGBO ZHANG (M.S., Tsinghua University; B.E., Tianjin University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN COMPUTATION AND SYSTEMS BIOLOGY (CSB) SINGAPORE-MIT ALLIANCE NATIONAL UNIVERSITY OF SINGAPORE 2013 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously ____________________ Lingbo Zhang June 2013 Acknowledgement I would like to express my sincerest gratitude to my supervisors Dr. Harvey F. Lodish and Dr. Bing Lim for their invaluable guidance and continuous support. Through these past years, Dr. Lodish has motivated me to pursue my research and as one of his former Nobel Laureate students recalled, he gave me the freedom to explore fascinating biological questions but made sure that I did not fall. He was always on hand with a variety of solutions whenever I encountered any obstacle during my research. I am also deeply grateful for Dr. Lim’s energy and enthusiasm when it comes to science. Talking to him about my scientific endeavors, I always came out of the conversation more driven and inspired to dig deeper. My two wonderful mentors have helped me to understand the beauty of life sciences and to uncover the mysteries behind them. I am truly honored to have had the opportunity to work with two such distinguished biologists. I would also like to thank my colleagues and friends at Whitehead Institute and MIT, especially Johan Flygare, Violeta Rayon Estrada, Lina Prak, Marina Bousquet, Christine Patterson, Peng Ji, Prakash Rao, Yutong Sun, Song Chou, Prathapan Thiru, Ferenc Reinhardt, Tony Chavarria, Richard Possemato, Jeong-Ah Kwon, Sumeet Gupta, Inma Barrase, Tom DiCesare, Wendy Salmon, Nicki Watson, Patti Wisniewski, Claire Mitrokostas Kitidis, Naomi Cohen, and Mary Anne Donovan for their invaluable help with discussion and assistance. Specifically, I would like to thank Johan Flygare and Violeta Rayon Estrada for sharing their experimental data on micoRNA deep sequencing (Figure 47a), the i effects of chemical compounds on BFU-E expansion and gene expression (Figure 27 and 29c), and the glucocoticoid receptor ChIP-seq data (Figure 30). I would also like to thank Ferenc Reinhardt and Tony Chavarria for their assistance with mouse bone marrow transplantation, and Prathapan Thiru for instruction in bioinformatics analysis. Part of the introduction sections ‘5.2 Hematopoiesis’ and ‘5.6 microRNAs in erythropoiesis’ is from my Leukemia paper. Finally, I would like to thank the SMA-CSB program for supporting me through my graduate studies and allowing me the opportunity to engage in scientific research. I would especially like to thank Prof. Hew Choy Leong, Prof. Gong Zhiyuan, and Prof. Paul Matsudaira, as well as my thesis committee for their continuous support and encouragement. ii Table of Contents 1. Summary 2. List of Tables 3. List of Figures 4. List of Symbols 5. Introduction 5.1 Development and stem cells 5.2 Hematopoiesis 5.3 Erythropoiesis 5.4 Terminal erythroid differentiation and erythroblast enucleation 5.5 Glucocorticoids and stress erythropoiesis 5.6 microRNAs in erythropoiesis 6. ZFP36l2 is required for self-renewal of early erythroid BFU-E progenitors 6.1 Results and discussion 6.1.1 ZFP36l2 is normally downregulated during erythroid differentiation from the BFU-E stage and this downregulation is reversed by functional GR agonists. 6.1.2 ZFP36l2 is specifically required for BFU-E self-renewal. 6.1.3 ZFP36l2 is required for erythroid lineage expansion during stress erythropoiesis in vivo. iii 6.1.4 ZFP36l2 delays erythroid differentiation and preferentially binds to several mRNAs that are induced or maintained at higher expression levels during terminal erythroid differentiation. 6.2 Materials and methods 7. miR-191 regulates mouse erythroblast enucleation by downregulating Riok3 and Mxi1 7.1 Results and discussion 7.1.1 The majority of late erythroblast CFU-E abundant microRNAs are downregulated during terminal erythroid differentiation 7.1.2 miR-191 modulates erythroblast enucleation 7.1.3 Two developmentally upregulated and erythroid enriched genes, Riok3 and Mxi1, are direct targets of miR-191 7.1.4 RIOK3 is required for erythroblast enucleation 7.1.5 MXI1 is required for erythroblast enucleation 7.1.6 Knockdown of Riok3 or Mxi1 or overexpression of miR-191 blocked Gcn5 downregulation 7.2 Materials and methods 8. Conclusions and Future Directions 9. Bibliography iv 1. Summary Humans generate 2.4 million red blood cells every second, a highly dynamic process that consists of several developmental stages regulated by multiple hormones. The earliest committed progenitor, the burst-forming unit erythroid (BFU-E), responds to multiple hormones including Erythropoietin (EPO), a principal regulator of red blood cell production. As BFU-Es divide they can generate additional BFU-Es through partial self-renewal, as well as later EPOdependent colony-forming unit erythroid (CFU-E) progenitors. EPO binds to EPO receptors on the surface of committed erythroid CFU-E progenitors, blocking apoptosis and triggering terminal erythroid differentiation1-3. While CFU-E erythroid progenitors are mainly controlled by EPO, the regulation of earlier BFU-E progenitors by a more expansive set of hormones, including glucocorticoids, which stimulate BFU-E self-renewal under stress conditions, is less understood2. Furthermore, compared to our understanding of protein-mediated mechanisms controlling the differentiation of CFU-Es to mature erythrocytes, far less is known about how microRNAs are involved in the regulation of this process4,5. To elucidate the mechanisms underlying BFU-E self-renewal, I identified the RNA binding protein Zfp36l2 as a transcriptional target of the glucocorticoid receptor (GR) in BFU-Es6. I found that Zfp36l2 is normally downregulated during erythroid differentiation from the BFU-E stage but its expression is maintained by all tested GR agonists that stimulate BFU-E self-renewal. I also showed that v Zfp36l2 is required for BFU-E self-renewal, as knockdown of Zfp36l2 disrupted glucocorticoid-induced BFU-E self-renewal in cultured BFU-E cells, and prevented expansion of erythroid lineage progenitors normally seen following induction of anemia by phenylhydrazine treatment in transplanted erythroid progenitors. Mechanistically, Zfp36l2 preferentially binds to mRNAs that are induced or maintained at high expression levels during erythroid differentiation and negatively regulates their expression levels. Thus, my research showed that Zfp36l2 functions as a molecular switch balancing BFU-E self-renewal and differentiation6. To better understand the role microRNAs play in terminal erythropoiesis, I found using RNA-seq technology, that the majority of microRNAs present in CFU-E erythroid progenitors are downregulated during terminal erythroid differentiation7. Of the developmentally downregulated microRNAs, ectopic overexpression of miR-191 blocked erythroid enucleation but had minor effects on proliferation and differentiation. I identified mRNAs encoded by two erythroid enriched and developmentally upregulated genes, Riok3 and Mxi1, as direct targets of miR-191. Knockdown of either RIOK3 or MXI1 blocked enucleation and either physiological overexpression of miR-191 or knockdown of RIOK3 or MXI1 blocked chromatin condensation. Thus my work established that downregulation of miR-191 is essential for erythroid chromatin condensation and enucleation by allowing upregulation of RIOK3 and MXI17. vi 2. List of Tables Table The summary of the experimental system used in the study of red cell formation, the normal developmental function, and the target genes of microRNAs important in red cell formation. Table Zfp36l2 targets several genes important for or related to erythropoiesis and negatively regulates their expressions. For microRNA qRT-PCR, reverse transcription was carried out by using a TaqMan® MicroRNA Reverse Transcription Kit (Appied Biosystems). The following real-time PCR analyses were performed using TaqMan® Universal PCR Master Mix (Appied Biosystems) and the 7500 Real-Time PCR System (Appied Biosystems). The real-time PCR probes were TaqMan® MicroRNA Assays from Appied Biosystems. For Western Blot, in vitro cultured erythroid cells were collected and lysed in lysis buffer (150 mM sodium chloride; 1.0% NP-40 or Triton X-100; 0.5% sodium deoxycholate; 0.1% SDS; 50 mM Tris, pH 8). SDS-PAGE was performed using the NuPAGE® Novex® Bis-Tris Gel Systems (Invitrogen). After electrophoretic transfer, the PVDF membranes with protein were incubated with the first antibody (RIOK3, Proteintech, 13593-1-AP; at dilution of 1:1000) overnight at 4°C followed by incubation with HRP conjugated secondary antibody (Jackson Immunoresearch; at dilution of 1:2000) for hour at room temperature. The intensities of bands were quantified by using Image J. 7.2.9 Luciferase reporter assay 293T cells were seeded into 96 well plates 24 hours before transfection. 10ng luciferase reporter plasmid or control vector plasmid were co-transfected with 10nM miR-191 mimic or negative control mimic (Dharmacon) into 293T cells by using Lipofectamine 2000 transfection reagent (Invitrogen). Cells were lysed 48 hours after transfection and luciferase activities were detected by using a dual luciferase kit (Promega). The 3’UTR of firefly luciferase gene with insertion of microRNA binding site was under the 110 regulation of microRNA. The Renilla luciferase was under the control of a constitutive promoter and used for normalization. 7.2.10 Student t-test One tail student's t-test was carried out to determine the statistic significances. 111 8. Conclusions and Future Directions Red blood cells account for more than 99% of total blood cells, and humans generate 2.4 million red blood cells every second to maintain the homeostasis of this large cell population. It is widely known that the hormone EPO serves as the principal regulator of red blood cell production at the CFU-E stage, through its downstream signaling pathways. However, our understanding of erythropoiesis at the earlier BFU-E stage and the regulation of erythropoiesis by means other than protein-mediated mechanisms, such as non-coding RNAs, is very limited10,16,54. In the first part of my thesis, I identified the first GR target gene, the RNA binding protein ZFP36l2, as an essential component for BFU-E self-renewal53. Under stress conditions such as acute blood loss or chronic anemia, glucocorticoids trigger self-renewal of BFU-E progenitors, leading to the production of more BFU-Es, CFU-Es, and ultimately, mature erythrocytes. ZFP36l2 is normally downregulated during erythroid differentiation from the BFU-E stage but its expression is maintained by all tested GR agonists that stimulate BFU-E self-renewal. In cultured BFU-Es, knockdown of ZFP36l2 did not affect the rate of cell division but disrupted glucocorticoid-induced BFU-E self-renewal, and in transplanted erythroid progenitors, knockdown of Zfp36l2 prevented the expansion of erythroid lineage progenitors normally seen after induction of anemia by phenylhydrazine treatment. I then demonstrated that ZFP36l2 preferentially binds to and negatively regulates expression levels of mRNAs that are induced or maintained at high expression levels during 112 erythroid differentiation. Altogether, I found that ZFP36l2 acts as an important molecular switch balancing BFU-E self-renewal and differentiation. Although ZFP36l2 is required for BFU-E self-renewal, overexpression of ZFP36l2 alone is not sufficient to trigger BFU-E self-renewal, suggesting that other genes involved in BFU-E self-renewal are yet to be identified and functionally characterized17. Moreover, self-renewal consists of both blockage of differentiation and continuation of proliferation6,7,69. However, we showed that overexpression of ZFP36l2 can partially mimic the glucocorticoid - induced differentiation delay53. We expect that other essential genes required for BFU-E self-renewal may either contribute to additional differentiation delay or continuation of proliferation. Therefore, to better understand the molecular network underlying BFU-E self-renewal, one of the most important questions for future research is to identify other novel molecular players in glucocorticoid-mediated BFU-E self-renewal. Previous research has shown that incubating BFU-Es with glucocorticoids for hours triggers the upregulation of around 80 genes that represent potential transcriptional target genes of the glucocorticoid receptor. Through an extensive literature review, I found that some of these genes are linked to the self-renewal of hematopoietic stem and progenitor cells. The leukemia-associated gene Trib2 is one of these examples. Trib2 is one of the 80 genes that are upregulated by glucocorticoids in cultured BFU-Es. Previously, Trib2 has been associated with leukemic stem and progenitor cells: Trib2 expression is elevated in a subset of human AML patient samples, and is 113 down-regulated in leukemic cells undergoing growth arrest, suggesting that Trib2 confers growth advantages to and enhances the self-renewal capacity of these cells84. These two clues suggest that Trib2 may contribute to glucocorticoid-triggered BFU-E self-renewal. To test the potential contribution of Trib2 to BFU-E self-renewal, in my own laboratory I will design shRNAs to abrogate Trib2 expression in BFU-Es and test whether Trib2 is indispensible for glucocorticoid-triggered BFU-E self-renewal, using experiments similar to the ones I conducted on Zfp36l2. If Trib2 is indeed required for BFU-E self-renewal, we will use the mice transplantation and PHZ induced stress erythropoiesis model to test the in vivo contribution of Trib2 to erythroid lineage expansion. Further analysis will focus on the underlying molecular mechanisms behind Trib2’s function in BFU-E self-renewal. In addition to Trib2, Bcl-2 is another gene that is upregulated by glucocorticoids. Bcl2 belongs to a family of anti-apoptotic genes and its upregulation may contribute to the survival of BFU-Es. In fact, one of its family members, Bcl-XL, has been widely linked with the survival of hematopoietic cells. For example, in erythroid progenitors, Epo/EpoR triggers the activation JAK-STAT5 pathway which then upregulate Bcl-XL, and Bcl-XL contributes to survival of erythroid progenitors and increased production of erythrocytes. Therefore, the upregulation of Bcl-2 may also contribute to the survival of BFU-Es. I plan to test the contribution of Bcl-2 to BFU-Es survival by shRNA medicated knockdown methods. Another important question is to further functionally characterize Zfp36l2’s role in BFU-E self-renewal. Previously, it has been shown that Zfp36l2 is a component of the 114 processing body, a subcellular structure that triggers degradation of mRNAs. Therefore, we plan to identify interaction partners of Zfp36l2 in BFU-Es through co-immunoprecipitation experiments followed by mass-spectrometry analysis to isolate proteins that physically interact with Zfp36l2. Further experiments will focus on the functional analysis of the contribution of each of these components to BFU-E self-renewal, using experiments similar to the ones I conducted on Zfp36l2. We will also monitor how the processing body behaves during BFU-E cell division, whether there is polarized localization of the processing body during BFU-E cell division, and what the localization of each of these components looks like. Finally, this research enables us to not only understand the molecular mechanisms underlying glucocorticoid- triggered BFU-E self-renewal, but also to identify novel compounds that can substitute for glucocorticoids in the treatment of Epo-resistant anemias. Therefore, we are beginning to carry out a small chemical compound screening to identify effective novel compounds. We are starting with a chemical compound library of ~2200 FDA approved compounds and other novel drug- like compounds. We will utilize a 394 well-plate primary BFU-E culture platform, and culture the cells in glucocorticoid negative medium. In each well, we will put 20µM of each single compound, and after days culture we will count the numbers of cell in each well. For the positive compounds, we will move to a more physiologically relevant in vivo pre-clinical testing and will functionally characterize how each compound positively regulates BFU-E self-renewal. 115 In the second part of my thesis, I identified microRNA-191 as a key regulator of erythroblast enucleation71. The majority of microRNAs present in CFU-E erythroid progenitors are developmentally downregulated and ectopic overexpression of miR-191 blocked erythroid enucleation but had only minor effects on proliferation and differentiation. Mechanistically, miR-191 directly targets two erythroid enriched and developmentally upregulated genes, Riok3 and Mxi1. Knockdown of either of these genes blocked enucleation and either physiological overexpression of miR-191 or knockdown of Riok3 or Mxi1 blocked chromatin condensation. Thus, my work established that miR-191 downregulation, and subsequent upregulation of Riok3 and Mxi1, is essential for erythroid chromatin condensation and enucleation. Enucleation is a complex process that requires the coordination of several cellular activities, such as terminal differentiation, cell cycle exit, chromatin condensation, and cytoskeleton rearrangement26,29,30,81,85,86. We discovered that miR-191 is required for enucleation and that it contributes to enucleation regulation by allowing the downregulation of a histone-modifying enzyme and ultimately leading to chromatin condensation. These findings will guide future studies on mechanisms behind the role of other microRNAs required for enucleation. In addition to miR-191, my functional screening identified a few other downregulated microRNAs that could also be important inhibitors of enucleation or other stages of late erythroid differentiation. For each of these microRNAs, I will first use erythroid differentiation marker genes, such as hemoglobin, heme biosynthetic enzymes, and major red cell membrane and cytoskeleton proteins, to determine whether, when 116 ectopically expressed or knocked down, each microRNA contributes solely to enucleation or to both differentiation and enucleation. After determining this, I will take advantage of microRNA target prediction databases such as Targetscan to identify potential target genes. Concurrently, we will perform microarray experiments to identify genes that are differentially expressed between control samples and microRNA knockdown/overexpression samples. By intersecting these two gene lists, we will identify functional target genes that are both predicted as a microRNA target and are differentially expressed. Further research will focus on the functional analysis of these microRNA target genes and on how they regulate erythroid enucleation and differentiation. In addition to the functions of microRNAs in normal erythroid cell development, we are also interested in their potential contribution to the pathogenesis of blood diseases related to erythroid lineage cells; one of the microRNAs we are currently testing is miR-125b. In our primary erythroid cell culture system, miR-125b is expressed at low levels in erythroid cells, and overexpression of miR-125b blocks erythroid differentiation, resulting in larger erythroid cells. Interestingly, overexpression of this microRNA has also been linked to certain types of AML, and many of these patients also have macrocytic anemia87. Therefore, we hypothesize that overexpression of miR-125b contributes not only to the pathogenesis of AML but also to macrocytic anemia. We plan to perform mouse bone marrow transplantation experiments to test whether miR-125b alone is able to cause macrocytic anemia. Mechanistically, we 117 plan to identify target genes of miR-125b and to characterize how overexpression of miR-125b leads to blockage of erythroid differentiation. In conclusion, my thesis work uncovered two novel posttranscriptional regulatory mechanisms of erythropoiesis: the RNA binding protein based mechanism facilitates BFU-E progenitor self-renewal by delaying differentiation through posttranscriptional downregulation of the expression of mRNAs critical for progression to the next differentiation stage, and the microRNA based mechanism assists erythroblast enucleation by posttranscriptional downregulation of the expression of mRNAs important for this process. 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Proc Natl Acad Sci U S A 107, 21558–21563 (2010). 125 [...]... the first cell of the organism, is pluripotent and is capable of generating the whole organism: the egg first undergoes cleavage, which leads to the formation of the blastula, followed by the gastrula and neurula, and finally, the adult body During the first few cleavages and the formation of the blastula, the division and differentiation of the pluripotent fertilized egg forms the basis of three germ... maintenance of stem cell properties and thus to ES cell self-renewal Although great progress has been achieved in understanding ES cell properties, our understanding of the self-renewal property of stem and progenitor cells in adult 11 tissues and organs is extremely limited, significantly preventing potential therapeutic uses of these cells (Figure 5)6,7 Figure 4 The regulation of ES cells by critical... factors contribute to the regulation of self-renewal of adult stem/progenitor cells Figure 5 The understanding of self-renewal of adult stem and progenitor cells is limited In different tissues and organs, a few molecules have been identified as important for self-renewal of adult stem and progenitor cells6 5.2 Hematopoiesis 13 Hematopoiesis is one of the most well studied adult stem and progenitor systems... expressing RPS19 shRNA Figure 20 microRNAs function as posttranscriptional regulators for gene expression Figure 21 microRNAs are important regulators of erythroid cell production and megakaryocyte–erythroid progenitor (MEP) lineage commitment Figure 22 miR-144/451 is a direct target gene of GATA1 Figure 23 mnr mutant Zebrafish shows defects in erythropoiesis Figure 24 The nuclear accumulation of FoxO3 in... include proteins that promote ES cells towards ectoderm differentiation, such as PAX6, MEIS1, HOXB1, LHX5, OTX1, factors that facilitate mesoderm and endoderm differentiation, such as HAND1, ONECUT1, ATBF1, and so forth Through the positive regulation of factors required for maintenance of stem cell properties and the negative regulation of factors required for differentiation, NANGO, OCT4, and SOX2... much is understood about the role of transcription factors in hematopoietic differentiation and particularly in myeloerythropoiesis Much less is known of other types of regulatory proteins and noncoding RNAs such as micro RNAs that regulate these processes 16 Figure 6 Hematopoiesis is a hierarchical differentiation process that leads to the formation of blood cells of all the blood lineages Hematopoietic... Figure 8 Erythropoiesis is regulated by a complex molecular network At different developmental stages, erythropoiesis is regulated by different hormones BFU-Es are regulated by IL-3, IL-6, SCF, Epo, glucocorticoids, and yet other hormones many of which are unknown CFU-Es are regulated by Epo Erythropoiesis is also regulated by many well- studied intracellular molecules such as transcription factors and. .. second step Molecularly, the upregulation of HDACs and the downregulation of HAT (Gcn5) is required for chromatin condensation Cytoskeleton regulators Rac and mDia2 control the second step, the formation of contractile actin ring and the extraction of nucleus26 With this novel flow cytometry method to monitor the enucleation process, a series of molecules have been identified and functionally characterized... non-coding RNAs, are involved in the regulation of this critical step31 Identifying these small regulatory molecules is a goal of this thesis 5.5 Glucocorticoids and stress erythropoiesis Compared to terminal erythropoiesis, the early stages of erythropoiesis are less understood It has been shown that IL-3, IL-6, stem cell factor (SCF) and Epo are required for the proliferation, differentiation, and survival... of in vivo terminal erythropoiesis Figure 11 Epo and EpoR regulate survival of erythroid progenitors, and STAT5 and BCL-X are important downstream mediators of EpoR signaling Figure 12 Actin cytoskeleton and nucleus structures during erythroblast enucleation Figure 13 Flow cytometry based monitoring of erythroblast enucleation Figure 14 Visualization of the flow cytometry pattern of extruded nuclei, . POSTTRANSCRIPTIONAL REGULATON OF ERYTHROPOIESIS BY RNA- BINDING PROTEINS AND MICRORNAS LINGBO ZHANG NATIONAL UNIVERSITY OF SINGAPORE 2013 POSTTRANSCRIPTIONAL. REGULATON OF ERYTHROPOIESIS BY RNA- BINDING PROTEINS AND MICRORNAS LINGBO ZHANG (M.S., Tsinghua University; B.E., Tianjin University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF. BFU-E self-renewal and differentiation 6 . To better understand the role microRNAs play in terminal erythropoiesis, I found using RNA- seq technology, that the majority of microRNAs present in