A. Red blood cell development:
Introduction Erythropoietin (Epo) is the principal regulator of red blood cell production; Epo is produced by the kidney in response to low oxygen pressure in the blood. Epo binds to Epo receptors on the surface of committed erythroid CFU-E progenitors, blocking apoptosis (programmed cell death), their usual fate, and triggering them to undergo a program of 4 – 5 terminal erythroid cell divisions and differentiation. We showed that the first two cell divisions, concomitant with differentiation from CFU-Es to late basophilic erythroblasts, are highly Epo-dependent; differentiation beyond this stage, involving chromatin condensation, ~1-2 terminal cell divisions, and enucleation, is no longer dependent on Epo but is enhanced by adhesion of the cells to a fibronectin matrix. Following condensation of chromatin and subsequent enucleation reticulocytes (immature red cells) are released into the blood.
The earliest committed progenitor, termed the burst- forming unit erythroid (BFU-E), can divide and generate additional BFU-Es (that is, undergo self- renewal) as well as generate later Epo- dependent CFU-E progenitors. Several cytokines and hormones are known to support BFU-E proliferation and formation of CFU-Es, including stem cell factor (SCF, the ligand for the c-kit protein tyrosine receptor) as well as IL-3, IL-6, and IGF-1. However, regulation of BFU-E proliferation and differentiation during basal and stress conditions is not well understood. We decided to focus on this important area based on the clinical observation that many patients with bone marrow failure disorders such as Diamond-Blackfan anemia are helped by glucocorticoids (GCs) rather than Epo treatment. These patients already have very high Epo levels in the blood, but do not have sufficient Epo-responsive CFU-E cells in the bone marrow.
Mechanisms of stress erythropoiesis
In situations of severe loss of red blood cells mammals respond by a process known as stress erythropoiesis (SE), in which there is increased formation of erythroid progenitors. Glucocorticoids (GCs) are known to be very potent enhancers of SE. This stimulatory effect of GCs on SE is utilized in the therapeutic regimen of Diamond-Blackfan Anemia (DBA), an erythropoietin-resistant congenital red cell aplasia. While an Epo-dependent balance of late red cell precursor survival normally maintains red cell homeostasis, findings by a former postdoc, Johan Flygare, indicate that the physiology of SE involves a stimulation of earlier erythroid progenitors, which when activated are able to rescue red cell production in conditions such as DBA, where erythropoietin has little effect. Johan showed that glucocorticoids stimulate self-renewal of early Epo-independent progenitor cells (burst-forming units erythroid or BFU-Es), over time increasing production of colony-forming units erythroid (CFU-E) erythroid progenitors from the BFU-E cells, and enhance the numbers of terminally differentiated red cells. GCs do not affect CFU-E cells or erythroblasts. In mRNA-seq experiments, he found that glucocorticoids induced expression of ~86 genes more than 2- fold in BFU-E cells. Computational analyses indicated that, of all transcription factors, binding sites for hypoxia-induced factor 1 alpha (HIF1α) were most enriched in the promoter regions of these genes, suggesting that activation of HIF1α may enhance or replace the effect of glucocorticoids on BFU-E self-renewal. Indeed, HIF1α activation by the pan-prolyl hydroxylase inhibitor (PHI) DMOG synergized with glucocorticoids and enhanced production of CFU-Es and later erythroblasts over 170-fold. PHI-induced stimulation of BFU-E progenitors thus represents a conceptually new therapeutic window for treating Epo-resistant anemia. Johan proposes a physiological model of stress erythropoiesis where increased levels of GCs –systemic stress hormones - and reduced oxygen – local stress - help maintain the earliest erythroid progenitors, increase CFU-E output, and at the same time stimulate terminal differentiation, thus promoting both a rapid and long-lasting increase in red blood cell production.
Since the main action of the activated GCR is to interact with chromatin and regulate transcription Johan and his technical assistant Violeta Rayon Estrada together with a graduate student, Lingbo Zhang, used ChIP-Seq on BFU-E cells to map the locations of the activated GCR along the genome, determine which binding partners it has, and how transcription is repressed and/or activated at these sites in BFU-E cells. Johan recently established his own group at the Lund Stem Cell Center in Sweden, where he will search for genes, molecular pathways and compounds that modify the red cell progenitor defect in Diamond Blackfan anemia. The aim of this work is to develop novel treatments for this disorder.
Zfp36l2 is required for self-renewal of early erythroid BFU-E progenitors
Lingbo Zhang, together with his technical assistant Lina Prak, identified the RNA- binding protein Zfp36l2 as essential for corticosteroid- induced self-renewal of early burst forming unit-erythroid (BFU-E) progenitors. Lingbo and Violeta showed that Zfp36l2 is a transcriptional target of the glucocorticoid receptor (GR) in BFU-Es: their Chip-seq experiments showed that the activated glucocorticoid receptor binds to several genomic regions near the transcription start site of Zfp36l2, and luciferase reporter assays demonstrated that these regions are indeed glucocorticoid inducible. These data suggest this gene as a direct transcriptional target of GR. Zfp36l2 is normally downregulated during erythroid differentiation from the BFU-E stage but its expression was maintained by all tested GR agonists that stimulate BFU-E self-renewal. Knockdown of Zfp36l2 in cultured BFU-E cells completely disrupted glucocorticoid mediated BFU-E self-renewal, but had no effects on cell division rates or cell survival. Lingbo further found that knockdown of Zfp36l2 in transplanted erythroid progenitors prevented expansion of erythroid lineage progenitors normally seen following induction of anaemia by phenylhydrazine treatment. Mechanistically, Zfp36l2 preferentially binds to messenger RNAs that are induced or maintained at high expression levels during terminal erythroid differentiation and negatively regulates their expression levels, including the mRNA for a key transcription factor required for erythroid differentiation. Zfp36l2 therefore functions as part of a molecular switch promoting BFU-E self-renewal and a subsequent increase in the total numbers of CFU-E progenitors and erythroid cells that are generated.
Chemicals that stimulate BFU-E self-renewal and increase production of red blood cells.
Lingbo and Lina recently completed a high throughput screening of a library of >2000 tested and approved therapeutic compounds for those that can stimulate murine red cell production in culture, either at the BFU-E or CFU-E level, and identified 45 potential “hits.” We chose 23 for detailed characterization, mainly on the basis that they were FDA- approved drugs used therapeutically for other indications. Thus far they have shown that four non-steroid drugs and 6 steroid drugs stimulate expansion of murine BFU-E progenitors in culture and stimulate red cell production to the same extent as does the corticosteroid dexamethasone. We are characterizing the actions of each of these drugs in detail, initially on murine cells in culture and presently on cultures of differentiating human erythroid progenitors. We are optimistic that this work will identify compounds less toxic than glucocorticoids for treatment of Diamond-Blackfan Anemia and potentially other EPO unresponsive anemias.
Hypoxia and stress erythropoiesis
Hypoxia - inadequate oxygen supply to cells and tissues - is a strong regulator of gene expression in both eukaryotic and prokaryotic cells; that hypoxia results in an increased number of red blood cells in humans and animals was observed over 100 years ago. Synthesis of Epo in the kidney is induced by hypoxia and mediated by stabilization of the transcription factor HIF1α in low oxygen; additionally HIF1α synergizes with glucocorticoids (GCs) to stimulate red blood cell formation by promoting self-renewal of erythroid burst-forming unit progenitors (BFU-Es). As noted above, the promoter/enhancers of many genes induced in BFU-Es by GCs are enriched for HIF1α binding sites, supporting the notion that HIF1α is likely to play an essential role in promoting self-renewal of BFU-Es. However, the mechanism by which HIF1α stimulates self-renewal of BFU-Es is unknown. Xiaofei Gao has shown that two clinically-tested HIF1α activators – inhibitors of specific prolyl hydroxylases (PHIs) – also synergize with corticosteroids to stimulate both human and mouse BFU-E self renewal and at lower concentrations that DMOG. Additionally he has identified several transcriptional factors and chromatin modifiers up-regulated significantly only by GCs together with PHI treatment, and they re now conducting further experiments to study their functions in BFU-E self-renewal.
Mechanistic study of activation of normal and pathogenic Janus kinase 2 and their associations with the erythropoietin receptor
A point mutation in the Janus kinase 2 (JAK2) pseudo-kinase domain, V617F, is found in most patients with Polycythemia Vera and those with other myeloproliferative disorders. This mutation enables cytokine-independent activation of JAK2 in cells that express a homodimeric cytokine receptor such as the erythropoietin receptor (EpoR) or related receptors including those for thrombopoietin and G-CSF. The activation mechanisms of both normal and pathogenic JAK2 are poorly understood. Jiahai Shi is studying the interaction between JAK2 and the EpoR cytoplasmic domain by X-ray crystallography. In particular he will determine the binding interface between the EpoR BOX 1 motif and JAK2 FERM domain. This interface would be a novel and specific drug target against JAK2-V617F positive myeloproliferative disorders. This work is being done in collaboration with Prof. Thomas Schwartz of the MIT Biology Department.
JAK2-V617F enhances red cell production by triggering CFU-E self-renewal
As noted above, JAK2-V617F is a mutant activated JAK2 kinase found in most polycythemia vera (PV) patients; it skews lineage determination of hematopoietic stem and progenitor cells towards the erythroid lineage and increases the number of erythroid progenitors. This leads to overproduction of red cells, consistent with the high percentage of erythroid progenitors from most PV patients that express JAK2-V617F. However, in some PV patients JAK2-V617F is found in only 10-30% of erythroid progenitors, implying that JAK2-V617F might also stimulate terminal erythropoiesis after the erythropoietin (Epo) dependent CFU-E stage. To confirm this hypothesis, Jiahai Shi showed that expression of JAK2-V617F in murine CFU-Es allows then to divide ~6 rather than the normal ~4 times in the presence of Epo, initially increasing the numbers of CFU-Es and delaying cell cycle exit. Over time the number of red cells formed from each CFU-E is increased ~4 fold; similar to human PV pathology, JAK2-V617F erythroid progenitors eventually differentiate to normal reticulocytes. Microarray analyses comparing JAK2 and JAK2-V617F erythroblasts indicate that JAK2-V617F not only activates EpoR-JAK2 signaling pathways, but also transiently induces non-erythroid-signaling pathways that delay terminal erythroid differentiation and permit extended cell divisions. These results provide a more complete understanding of PV pathogenesis, in particular in patients in with low numbers of JAK2-V617F expressing erythroid progenitors.
Production of large numbers of enucleated human erythroblasts in culture.
Jiahai Shi set out to optimize our system of in vitro culture of murine fetal liver Epo- responsive CFU-E progenitors to generate the maximum numbers of enucleated erythroid cells, i.e. reticulocytes. Careful examination of media constituents led to the discovery that the ferro- transferrin concentration indeed limits terminal erythropoiesis, and supplementation with as much as 500 µg/ml ferrotransferrin greatly enhance production of reticulocytes. Fetal bovine serum was essential, and ferro transferrin could be partially but not completely replaced by a water- soluble iron chelating agent.
Sherry Lee then turned her attention to erythroid culture of mobilized human blood CD34+ erythroid progenitor cells; these cells could easily be cultured under conditions such that nucleated hemoglobin- containing erythroid cells were formed but little terminal maturation and enucleation occurred. Sherry developed a four- stage culture system yielding an approximately 30,000-fold expansion after a 21- day culture. At the end of the culture over 50% of the cells had undergone enucleation and all of the cells were hemoglobinized; each enucleated reticulocyte contained ~30 pg hemoglobin, similar to the amount in normal human red blood cells. Some serum is essential for enucleation. Her experiments indicate that timely supply and withdrawal of cytokines required for each developmental stage is important for erythroid differentiation and synchronizing the cell population in culture. She has been conducting further analyses to characterize the proteome and transcriptome of the cells. This culture system enables many studies on terminal differentiation of human erythrocytes.
Engineered erythrocytes generated by sortase-mediated modification (“sortagging”) of erythroid membrane proteins.
For several reasons red blood cells are an attractive vehicle for delivering proteins and small molecules and for targeting to specific tissues: They lack a nucleus, and therefore, there will no concerns about remnants of genes that could alter normal cell physiology, and they have a lifespan of around 120 days in the blood stream. In collaboration with Dr. Lenka Kundrat in Prof. Hidde Ploegh’s laboratory at the Whitehead Institute, Jiahai Shi, Sherry Lee, and Nova Pishesha are generating specific types of genetically engineered erythrocytes (eRBCs) in culture, and are using the sortase technique to covalently link proteins and small molecules to these eRBCs which then can be used for multiple types of applications. The sortase technique for cell and protein engineering was developed in the Ploegh laboratory. Sortases are transpeptidases derived from bacteria – they are involved in cell wall biogenesis – that normally link together two proteins that contain at their ends particular short amino acid sequences. While the sortases from different bacteria recognize different sequences, the most commonly used sortase cleaves between the T and G residues in the flexible motif: LPXTG, generating a thioacyl enzyme intermediate at the C-terminus of the threonine residue. This is resolved by nucleophilic attack in a reaction that involves an N- terminal glycine-initiated peptide or probe, (G)n-Y, where n =1 - 5. Therefore, the sortase-based approach (sortagging) covalently links the peptide-based motifs, X-LPXTG and (G)n-Y, resulting in a new structure: X-LPXT(G)n-Y, where X or Y could be any protein, peptide, high molecular weight polymer, or small molecule. For proof of concept, they expressed the sortaggable versions of two erythroid membrane proteins - Glycophorin A, a Type I transmembrane protein. and the Type II transmembrane protein Kell, on the surface of human CD34+ cells via lentiviral transduction. The expression of sortaggable Glycophorin A was sustained as erythroid differentiation proceeded. The cells undergo terminal erythroid differentiation and can readily be sortagged with a biotin label at very high efficiency.
There are many ways to use these sortase- and genetically modified erythrocytes produced in culture; initially we will focus on linking fibrinolytic proteins to red cells that could be used therapeutically. The engineered red cells with fibrinolytic protein, like plasminogen activators (PA), should have a long half-life in the circulation. These cells will aggregate around a nascent blood clot, as would normal erythrocytes, and increase the local PA concentration to dissolve the blood clot. This therapeutic should be very useful in preventing thrombosis in patients having a high risk for life-threatening thrombosis like coronary thrombosis. Importantly, there are many other potential uses that we will explore as time permits, including novel therapeutics, stabilizing otherwise unstable red cells, novel immune modulators and vaccines, and novel imaging modalities.
Transcriptional control of gene expression during terminal erythroid differentiation
In the vertebrate, late erythroblasts must undergo terminal differentiation, which involves terminal cell cycle exit and chromatin condensation, to become reticulocytes. In mammals, there is an additional step requiring extrusion of the pycnotic nucleus via an asymmetric cell division. Many aspects of transcriptional regulation of this process remain unknown. Previous RNA sequencing studies on late erythroblasts identified several genes encoding DNA- binding proteins and whose expression is up-regulated during terminal differentiation. Recent results by Xiaofei Gao and Sherry Lee showed that knocking down nuclear coactivator 4 (NCOA4) interrupts the terminal differentiation in both our human CD34 ex vivo differentiation system and cultured mouse fetal liver cells.
Their chromatin immunoprecipitation linked to deep sequencing (ChIP-Seq) results showed that the chromatin occupancy of NCOA4 is enriched at intronic and distal intergenic regions (defined as > 3 kb from transcription start site) in human erythroblasts. Of note, RNA polymerase II proximally occupies more than half of NCOA4-assoiated chromatin sites. De Novo motif discovery of NCOA4-binding chromatin regions also uncovered binding motifs of several transcription factors (TFs) essential for human development. We are currently investigating the function of these TFs and their interaction with NCOA4 during red blood cell terminal differentiation.
Chromatin condensation and enucleation
Mammalian erythroid cells undergo enucleation during a late stage of differentiation, a process that does not occur in other vertebrates. This process has critical physiological and evolutional significance for the morphogenesis and hemoglobin enrichment of mature mammalian red blood cells. Although enucleation has been known for decades, the mechanisms that regulate this process remain obscure. Peng Ji began studying enucleation in our lab, and identified key roles for Rac GTPase and fir the formin (actin nucleating protein) mDia2 in the final step of erythroblast enucleation – the formation of the contractile actin ring on the plasma membrane of late-stage erythroblasts at the boundary between the cytoplasm and nucleus of enucleating cells.
In collaboration with Tzutzuy Ramirez and Junxia Wang, fellows with Maki Murata Hori of the Temasek Life Sciences Laboratory, Singapore, Peng investigated the roles of many cytoskeletal and other proteins in nuclear migration and enucleation of these cells, in part using video microscopy of cells expressing fluorescent- tagged proteins. Initial results show that, unlike conventional cytokinesis, the nucleus is squeezed out by formation of a bleb-like protrusion from a limited area of the erythroblast cell cortex; the bleb increases in size by dynamic contractions of asymmetrically distributed actomyosin. Importantly, they showed that enucleation requires establishment of cell polarization that is regulated by microtubule-dependent local activation of phosphoinositide 3-kinase (PI(3)K), displacing the nucleus to one side of the cell, and restricting actin to the other side. Dynamic actin- mediated cytoplasmic contractions generate pressure that pushes the viscoelastic nucleus through a narrow constriction in the cell surface, forming a bud. The PI3K products PtdIns(3,4)P2 and PtdIns(3,4,5)P3 are highly localized at the cytoplasmic side of the plasma membrane. PI3K inhibition caused impaired cell polarization, leading to a severe delay in enucleation. Also depolymerization of microtubules reduced PI3K activity, resulting in impaired cell polarization and enucleation.
Peng also focused on the role of histone deacetylases (HDACs) in chromatin and nuclear condensation and enucleation of late erythroid cells. He showed that inhibition of HDACs by Trichostatin A (TSA) or Valproic acid (VPA) prior to the start of enucleation blocked chromatin condensation, contractile actin ring formation, and enucleation. He further demonstrated that HDAC1, HDAC2, HDAC3 and HDAC5 are highly expressed in mouse fetal erythroblasts. shRNA down-regulation of HDAC2, but not the other HDACs, phenotypically mimicked TSA and VPA treated cells with significant inhibition of chromatin condensation and enucleation. Importantly, knockdown of HDAC2 does not affect erythroblast proliferation, differentiation, or apoptosis. These results identify HDAC2 as an important regulator in mediating chromatin condensation and enucleation in the final stages of mammalian erythropoiesis. Peng is continuing to work on these and related projects in his new position as Assistant Professor of Pathology at the Northwestern University Medical School.
Histones to the cytosol: Exportin 7 is essential for erythroid nuclear condensation and enucleation.
Recently, along with her undergraduate student, Austin Gromatzky, Shilpa Hattangadi has begun to uncover the function of an unusual regulator of erythroid chromatin condensation and enucleation, the nuclear export protein, Xpo7. Xpo7 is highly erythroid specific and induced markedly during terminal differentiation; its expression is regulated by some of the master erythroid transcriptional regulators. It is unusual for a nuclear export protein in that it does not require a specific nuclear export signal, as do all other exportins. Interestingly, except for Xpo7 transcripts of all other nuclear exportins are repressed during terminal erythropoiesis. Shilpa studied the function of Xpo7 by shRNA knockdown and discovered that erythroblast nuclei from Xpo7-kd cells were less condensed and larger than control nuclei, as judged by confocal immunofluorescence microscopy. Enucleation was blocked, and Xpo7-KD nuclei retained almost all nuclear proteins while normal extruded nuclei had very little protein, as judged both by silver stained gels and mass spectrometry. This suggested that Xpo7 is a nonspecific nuclear export protein that removes all nuclear proteins from the erythroid nucleus in order to allow chromatin to condense. Strikingly, DNA binding proteins such as histones H2A and H3 accumulated in the cytoplasm of normal late erythroblasts prior to and during enucleation, but not in Xpo7-knockdown cells. Thus chromatin condensation during erythroid development involves removal of histones from the nucleus facilitated by Xpo7. Along with Austin, she is using immunoprecipitation and yeast-2-hybrid methods to uncover the erythroid-specific cargos of Xpo7. She is continuing this work as Assistant Professor in the Departments of Pediatrics and Pathology at Yale University School of Medicine.
The pre-mRNA splicing factor Muscleblind-like 1(Mbnl1) is required for terminal erythroid differentiation
The scope and role of regulated exon use in pre-mRNAs during erythroid development is poorly understood. Using their mRNA- seq data sets from erythroid progenitors and mature Ter-119+ erythroblasts, Bill Wong, working with Albert W. Cheng in Prof. Chris Burge’s lab and UROPs Katherine Luo and Paula Trepman, identified hundreds of differentiation-associated isoform changes during terminal erythropoiesis. During differentiation there were major changes in eight classes of alternative isoform expression events involving alternative splicing, alternative 3’ end cleavage and polyadenylation, and/or alternative promoter usage; these included skipped exons, mutually exclusive exons, alternative 5′ and 3′ splice sites, alternative first exons, alternative last exons, tandem 3′ untranslated regions, and retained introns. They focused on alternative exon use during differentiation; many of these changes in usage coincided with induction of ~400 erythroid-important genes as well as repression of about 6000 early- stage genes, suggesting that both large-scale transcriptional and post-transcriptional programs are critical to ensure proper erythroid differentiation.Segments in pre mRNAs surrounding regulated exons were enriched in motifs corresponding to the splicing factor, muscleblind-like1 (Mbnl1). Knockdown of Mbnl1 in cultured murine fetal liver erythroid progenitors resulted in a strong block in erythroid differentiation and disrupted the developmentally regulated exon skipping of several pre mRNAs, including Ndel1 mRNA, which they showed is a direct target of Mbnl1 and required for enucleation. These findings reveal an unanticipated scope of the alternative splicing program and the importance of Mbnl1 during erythroid differentiation.
The Genetics of Human Erythropoiesis
Vijay Sankaran along with laboratory colleagues Leif Ludwig and Jenn Eng¬ have been dissecting the genetic architecture of human erythropoiesis, along with colleagues at the Broad Institute. This work is being performed using a combination of complex trait genetics, Mendelian genetics, and analysis of rare human syndromes. Additionally, this group is using the insight from such approaches to refine current culture conditions of human erythroid cells.
Elevated levels of fetal hemoglobin can ameliorate the major disorders of beta-hemoglobin, sickle cell disease and beta-thalassemia. They followed up on a several decades old observation that patients with trisomy 13 have elevated levels of fetal hemoglobin and used mapping of partial trisomy cases to show that elevated levels of microRNAs 15a and 16-1 appear to mediate this phenotype. A direct target of these microRNAs, MYB, plays an important role in silencing the fetal and embryonic hemoglobin genes. Thus they have demonstrated how the developmental regulation of a clinically important human trait can be better understood through the genetic and functional study of aneuploidy syndromes, and suggest that miR-15a, 16-1, and MYB may be important therapeutic targets to increase HbF levels in patients with sickle cell disease and β-thalassemia. Following up on this work, this group is defining the physiological function of these microRNAs and their targets using a variety of approaches in primary mouse and human erythroid progenitor cells. Ongoing work is aimed at understanding the mechanistic basis for alterations in hemoglobin expression in the context of other rare human syndromes and clinical conditions.
Using complex trait genetics, this group has been defining new regulators of human erythropoiesis. By using readily measured erythrocyte traits and following up on the results of genome-wide association studies (GWAS), new mechanisms underlying the regulation of erythropoiesis are being defined. Using such approaches, they have recently defined a role for the pleiotropic cell cycle regulator, cyclin D3, in regulating the number of divisions that occur during terminal erythropoiesis, thereby controlling erythrocyte size and number. Specifically, this GWAS variant affects an erythroid-specific enhancer of CCND3. A Ccnd3 knockout mouse phenocopies these erythroid phenotypes, with a dramatic increase in erythrocyte size and a concomitant decrease in erythrocyte number. By examining cultures of differentiating human and mouse primary erythroid progenitor cells, they demonstrated that the CCND3 gene product, cyclin D3, regulates the number of cell divisions that erythroid precursors undergo during terminal differentiation before enucleation, thereby controlling erythrocyte size and number. Ongoing studies are aimed at broadening these approaches to other loci across the genome.
Finally, to gain further insight into important regulators of erythropoiesis, this group has been using Mendelian genetic approaches to identify genes involved in erythropoiesis that are mutated in rare human diseases. With collaborators at a number of institutions, new candidate genes mediating these diseases have been defined and functional work is being performed to understand the nature and mechanism of action of these genes. For example, with close collaborator Hanna Gazda, this group has recently defined the first non-ribosomal protein gene involved in Diamond-Blackfan anemia, GATA1. Ongoing studies are defining the mechanisms by which this and other mutations affect human erythropoiesis.
Carrying on a 45-year-old tradition of close interactions and scientific exchanges between the Division of Hematology/Oncology at Boston Children’s Hospital and the Lodish laboratory (that began when David Nathan was a sabbatical visitor with Harvey in 1970), Vijay is continuing this work in his laboratory at Boston Children’s Hospital as an Assistant Professor of Pediatrics at Harvard Medical School.
Modeling disorders of erythropoiesis in primary human erythroid cells
In work currently being started by new post-doc Hojun Li, in collaboration with Feng Zhang at the Broad Institute and Dan Bauer, Jian Xu, and Elenoe Smith from Stuart Orkin’s laboratory at Boston Children’s Hospital, they are attempting to model human diseases of erythropoiesis in our human erythroid culture system. Hojun is utilizing the CRISPR/Cas9 nuclease system to target various genes known to be mutated in human anemias, and is working with Sherry Lee’s CD45 cell culture system to determine the stages of erythropoiesis are affected by these mutations.
B. Long non-coding RNAs (lincRNAs) that regulate the differentiation and function of erythroid and myeloid cells and of white and brown adipose cells.
Introduction Long non-coding RNAs (lncRNAs) are transcripts longer than 200nt that do not encode proteins. Many are capped, polyadenylated and often spliced, and presumably transcribed by RNA Polymerase 2 (Pol2). LncRNAs constitute a significant fraction of the mammalian transcriptome. Compared to mRNAs, lncRNAs tend to be shorter and less well conserved at the primary sequence level. Expression of lncRNAs is often restricted to specific tissues and developmental stages, suggesting that many may regulate cell fate specification .A few dozen intergenic lncRNAs (lincRNAs) have been functionally characterized in mammals, and they have been associated with important developmental processes such as apoptosis, proliferation and lineage commitment However, the biological functions of most of these genes and their potential roles in disease still remain uncharacterized.
An erythroid-specific long non-coding RNA prevents apoptosis of erythroid progenitors and promotes terminal proliferation.
Erythropoiesis is regulated at multiple levels by different factors to ensure the proper generation of red blood cells in response to various physiological and pathological stimuli. Although the regulation of erythropoiesis by transcription factors and microRNAs is becoming well understood, the modulation of red blood cell development by lncRNAs is still unknown. LncRNAs can regulate gene expression via multiple mechanisms and many lncRNAs are differentially expressed in many developmental and pathological processes, suggesting that they play important biological roles.
Wenqian Hu identified one erythroid-specific lncRNA, LincRNA-EPS, with potent anti-apoptotic activity. Expression of LincRNA-EPS is largely confined to terminally differentiating fetal erythroid cells and its expression is induced in CFU-E progenitors by Epo. Inhibition of this lncRNA blocks erythroid differentiation and promotes apoptosis. Ectopic expression of this lncRNA in CFU-E progenitors prevents erythroid progenitor cells from the apoptosis that is normally induced by erythropoietin deprivation. This lncRNA represses expression of several proapoptotic genes including the one encoding Pycard, an activator of caspases, explaining in part the inhibition of programmed cell death. These findings reveal a novel layer of regulation of cell differentiation and apoptosis by a lncRNA. Currently Wenqian with Juan R. Alvarez-Dominguez are identifying and cloning the human LincRNA-EPS ortholog and characterizing its putative antiapoptotic functions.
Multiple Types of Long Non-coding RNAs Regulate Red Blood Cell Development
To obtain a comprehensive view of how lncRNAs contribute to erythropoiesis, Wenqian Hu and Juan R. Alvarez-Dominguez, together with two undergraduates, Staphany Park and Austin Gromatzky, performed and analyzed data from high depth RNA-sequencing on both Poly(A)+ RNAs and Poly(A)- RNAs from erythroid progenitor cells and terminal differentiating erythroblasts. They combined genome-wide surveys of expression levels, chromatin states, and transcription factor occupancy in differentiating primary mouse fetal liver erythroid cells with computational analyses to systematically characterize all lncRNA subclasses by a spectrum of >30 features covering structural, conservation, regulation and expression traits. They uncovered global features of the biogenesis and in the coordination of chromatin and transcription dynamics of lncRNAs during erythropoiesis, as well as subclass-specific patterns in conservation and tissue and developmental stage specificity. Importantly, they discovered that binding of the key erythroid transcription factors GATA1 and TAL1 at both lncRNA and mRNA promoters is typically accompanied by gain of H3K4me2 along with transcriptional activation.
They then focused on differentiation-induced lncRNAs, including novel erythroid-specific lncRNAs conserved in humans that are nuclear-localized. They identified 13 erythroid-specific lncRNAs that are greatly induced during erythroid terminal differentiation; importantly, shRNA-mediated loss-of-function assays reveal that all 13 are important for this developmental process. One of them, alncRNA-EC7, is specifically needed for activation of the neighboring gene encoding a major erythrocyte membrane protein. Thus diverse types of lncRNAs participate in the regulatory circuitry underlying lineage-specific development. Currently, they are using biochemical and informatic approaches to determine how these lncRNAs control erythroid terminal differentiation.
LincRNAs in fat cell development and function.
Many protein coding genes, mRNAs, and microRNAs have been implicated in regulating adipocyte development; however, the global expression patterns and functional contributions of long intergenic noncoding RNA (lincRNA) during adipogenesis have not been explored. Lei Sun and Ryan Alexander, collaborating with John Rinn’s group at the Broad Institute, examined the roles of large intergenic non-coding RNAs (lincRNAs) in adipogenesis. To begin, they profiled the transcriptome of primary brown and white adipocytes, pre- brown and white adipocytes and cultured adipocytes and identified 175 lincRNAs that are specifically regulated during both brown and white adipogenesis. Many lincRNAs are adipose-enriched, strongly induced during adipogenesis, and bound at their promoters by key adipogenic transcription factors such as PPARγ and CEBPα. RNAi-mediated loss of function screens identified 9 functional lincRNAs required for adipogenesis; mRNA analyses showed that each of these lincRNAs is essential for normal induction of a discrete set of adipocyte- induced mRNAs and for down regulation of a discrete set of mRNAs expressed in adipocyte progenitor cells.
They further focused on an X-linked lincRNA required for proper adipogenesis; both the human and mouse orthologs of this lincRNA contain numerous copies of a conserved RNA sequence motif. Collectively, they have identified numerous lincRNAs that comprise a critical transcriptional regulatory layer that is functionally required for proper differentiation of both brown and white adipocytes.
Marko Knoll, a new postdoctoral fellow, is determining the mechanism by which these 9 functional lincRNAs influence adipogenesis. He determined the ends of each of these lincRNAs by 5´ and 3´ RACE, respectively, and is cloning them into a doxycycline inducible overexpression vector. Then these lincRNAs will be overexpressed in C2C12 myoblast cells, which do not express these lincRNAs and changes in gene expression will be monitored by microarrays. Further, using the CRISPR/Cas system, Marko aims to knock out or mutate the lincRNAs in the preadipocytic 3T3-L1 cell line. Lei Sun is continuing work on other lncRNAs in his own laboratory in the Duke- NUS Medical School in Singapore.
MicroRNAs in fat cell development and obesity
Mammals have two principal types of fat: white adipose tissue (WAT) primarily serves to store extra energy as triglycerides, while brown adipose tissue (BAT) is specialized to burn lipids for heat generation and energy expenditure as a defense against cold and obesity. Recent studies demonstrate that brown adipocytes arise in vivo from a Myf5-positive, bipotential myoblastic progenitor by the action of the Prdm16 (PR domain containing 16) transcription factor. Former postdocs Lei Sun and Huangming Xie, and UROP student Ryan Alexander investigated the role of miRNAs in brown fat adipogenesis and identified a brown fat-enriched miRNA cluster, miR-193b-365, as a key regulator of brown fat development. Blocking miR-193b and/or miR-365 in primary brown preadipocytes dramatically impaired brown adipocyte adipogenesis by enhancing expression of Runx1t1 (runt-related transcription factor 1; translocated to 1) whereas myogenic markers were significantly induced. In contrast, forced expression of miR-193b and/or miR-365 in C2C12 myoblasts blocked the entire program of myogenesis, and, in adipogenic conditions, miR-193b induced myoblasts to differentiate into brown adipocytes. MiR-193b-365 was upregulated by Prdm16 partially through the action of the transcription factor PPARγ. Taken together, these results underlie the importance of tissue enriched miRNAs 193b-365 in regulating lineage specification between brown fat and muscle, and also suggest that these or other miRNAs may have therapeutic potential in inducing expression of brown fat-specific genes.
Another miRNA, miR-203 was also enriched in brown fat. Marko Knoll is following up work by Ryan Alexander showing that a knock down of miR-203 results in a block of adipogenesis in primary brown pre-adipocytes. Further, forced expression of miR-203 in C2C12 myoblasts blocked the myogenic program and induced differentiation into adipocytes. Marko aims to search for the target of miR-203. With the development of the CRISPR/Cas system it is possible to insert multiple mutations of up to 25 base pairs. Marko wants to use the CRISPR/Cas approach to generate mice that have a mutated seed sequence in the miR-203 and analyze the effect of miR-203 seed region mutation on the development of white and brown adipose tissue.
C. Adipocyte biology and insulin resistance
A novel kinase in the development of adipocytes
Marko Knoll, a new post-doc is investigating how this specific kinase regulates the development of adipocytes using a floxed mice for the kinase and breeding these mice to a mouse expressing a fat cell specific Cre. Marko found that aged adipocyte-specific kinase knock out mice have reduced subcutaneous and epididymal adipose tissue compared to age matched wild type littermates. Protein isolated from subcutaneous and epididymal adipose tissue from knock out or wild type mice express the same amount of the kinase, indicating a counter selection in vivo therefore resulting in a reduced fat mass. Further experiments aim to dissect the signaling pathways by this kinase and the possible role in the development of white adipose tissue.
A second novel kinase that mediates signaling by oxidative stress and in vivo insulin resistance
Heide Christine Patterson, a post-doc in the laboratory and a pathologist at Brigham and Women’s Hospital, together with her UROPs Cher Huang and Michael Harden, is investigating whether a kinase important for signal transduction in immune cells also mediates activation of oxidative stress induced pathways, in what subcellular compartment the signaling occurs and what components regulate activation of this kinase. Her model systems are primary murine embryonic fibroblasts and mouse splenic B cells stimulated in vitro by hydrogen peroxide.
Ji, P. and H. Lodish Ankyrin and Band 3 Differentially Affect Expression of Membrane Glycoproteins But Are Not required for Erythroblast Enucleation Biochem Biophys Res Comm 417: 1188 – 1192 (2012).
Wang, J., T. Ramirez, P. Ji, S. Jayapal, H. F. Lodish and M. Murata-Hori. Mammalian erythroblast enucleation requires PI(3)K-dependent cell polarization . J. Cell. Science. 125: 340 - 349 (2012).
Zhang, L., Sankaran, V. and H. Lodish MicroRNAs in erythroid and megakaryocytic differentiation and megakaryocyte-erythroid progenitor lineage commitment. Leukemia 26: 2310-2316 (2012).
Lodish, H. F., and N. Fedoroff. Retrospective: Norton Zinder (1928-2012) Science 335:6074 (2012).
Sankaran, V., L. S. Ludwig, E. Sicinska, J. Xu, D. E. Bauer, J. C. Eng, H. C. Patterson, R. A. Metcalf, Y. Natkunam, S. H. Orkin, P. Sicinski, E. S. Lander, and H. F. Lodish. Cyclin D3 coordinates the cell cycle during differentiation to regulate erythrocyte size and number. Genes and Development 26: 2075- 2087 (2012).
Bousquet, M., D. Nguyen, C. Chen, L. Shields, and H. Lodish miR-125b transforms myeloid progenitors by repressing multiple mRNA targets Haematologica 97: 1713-1721 (2012).
Liu, Q, B. Yuan, K. Lo, H. Patterson, Y. Sun, and H. Lodish Adiponectin regulates expression of hepatic genes critical for glucose and lipid metabolism PNAS 109: 14568 - 14573 (2012).
Hu, W., J. Alvarez-Dominguez, and H. Lodish Regulation of Mammalian Cell Differentiation by Long Noncoding RNAs. EMBO Reports 13: 971 - 983 (2012).
Lodish, H. F., A. Berk, C. Kaiser, M. Krieger, A. Bretscher, H. Ploegh, A. Amon, and M. Scott, Molecular Cell Biology, 7th ed. W. H.. Freeman and Company, N.Y. (2012).
Duraisingh, M. and H. Lodish Sickle cell microRNAs inhibit the malaria parasite. Cell Host and Microbe 12: 127-128 (2012).
Lodish, H. Reflections, Translational Control of Protein Synthesis: The Early Years. J. Biol. Chem. 287: 36528 - 36535 (2012).
Alvarez-Dominguez, J., W. Hu, and H. Lodish, Regulation of Eukaryotic Cell Differentiation by Long Noncoding RNAs. In The Molecular Biology of Long Non-coding RNAs, J. Coller and A. Khalil, eds. Springer Science, pp 15- 67 (2013) ISBN 978-1-4614-8621-3
Chen, C., D. Garcia-Santos, Y. Ishikawa, A. Seguin, L. Li, K. Fegan, G. Hildick-Smith, D. Shah, J. D. Cooney, W. Chen, M. King, Y. Yien, I. Schultz, H. Anderson, A. Dalton, M. Freedman, P. Kingsley, J. Palis, S. Hattangadi, H. Lodish, D. Ward, J. Kaplan, T. Maeda, P. Ponka, and B. Paw. Snx3 regulates recycling of the transferrin receptor and iron assimilation. Cell Metabolism. 17:343 - 352 (2013).
Lo, K. A., A. Labadorf, N. J. Kennedy, M. S. Han, Y. S. Yap, B. Matthews, X. Xin, L. Sun, R. J. Davis, H. F. Lodish, and E. Fraenkel. Analysis of In Vitro Insulin-Resistance Models and Their Physiological Relevance to In Vivo Diet-Induced Adipose Insulin Resistance. Cell Reports 5:259-270 (2013) S2211-1247(13)00480-4 [pii] 10.1016/j.celrep.2013.08.039
Okuyama, K., T. Ikawa, B. Gentner, K. Hozumi, R. Harnprasopwat, J. Lu, R. Yamashita, D. Ha, T. Toyoshima, B. Chanda, T. Kawamata, K. Yokoyama, S. Wang, K. Ando, H. F. Lodish, A. Tojo, H. Kawamoto, and A. Kotani. MicroRNA-126-mediated control of cell fate in B-cell myeloid progenitors as a potential alternative to transcriptional factors. Proc Natl Acad Sci U S A 110:13410-5. (2013).
Sun, L., L. Goff, C. Trapnell, R. Alexander, A. Lo, E. Hacisuleyman, M. Savageau, B. Tazon-Vega, D. Kelley, D. Hendrickson, B. Yuan, M. Kellis, H. Lodish, and J. Rinn, Long noncoding RNAs regulate adipogenesis. PNAS 110:3387 - 3392 (2013).
Chou, S., J. Flygare, and H. Lodish Fetal Hepatic Progenitors Support Long-term Expansion of Hematopoietic Stem Cells. Experimental Hematology 41: 479-490 (2013).
Zhang, L., L. Prak, J. Flygare, V. R. Estrada, P. Thiru, B. Lim, and H. F. Lodish. Zfp36l2 is required for
Dang, M., N. Armbruster, M. Miller, E. Cermeno-Blondet, M. Hartmann, G. Bell, D. Root, D. Lauffenburger, H. Lodish, and A. Herrlich. Regulated ADAM17 dependent EGF family ligand release through separate and distinct signaling pathways without increase of protease activity PNAS 110: 9776 – 9781 (2013).
Trajkovski, M., and H. Lodish. MicroRNA networks regulate development of brown adipocytes. Trends Endocrinol Metab 24:442-450 (2013).
Lodish, H. Cloning Expeditions: Risky but Rewarding. Mol Cell Biol. 33:4620 – 4627 (2013).
Ludwig, L., H. Gazda, J. Eng, S. Eichhorn, P. Thiru, R. Ghazvinian, T. George, J. Gotlib, A. Beggs, C. Sieff, H. Lodish, E. Lander, and V. Sankaran, Altered GATA1 Translation Underlies Diamond-Blackfan Anemia. Nature Medicine submitted (2014).
Hattangadi, S., H. C. Patterson, J. Shi, K. Burke, C. Huang, J. Wang, M. Murata-Hori, and H. Lodish. Histones to the cytosol: Exportin 7 is essential for erythroid nuclear condensation and enucleation. Blood submitted (2014).
Hacisuleyman, E., L. Goff, C. Trapnell, L. Sun, A. Williams6, J.Henao-Mejia, P. McClanahan, D. Hendrickson, M. Sauvageau, D. Kelley, M. Morse, J. Engreitz, M. Guttman, E. Lander, R. Flavell, H. Lodish, A. Raj, and J. Rinn Topological Organization of Multi-chromosomal Domains by linc-Firre Nature Structure Molecular Biol. In the press (2014).
Alvarez-Dominguez, J., Hu, W., Yuan, B., Shi, J., Park, S., Gromatsky, A., van Oudenaarden, A., and Lodish, H. Global discovery of erythroid long non-coding RNAs reveals novel regulators of red cell maturation Blood 123: 570 - 581 (2014).
Chen, C., and H. Lodish Global analysis of induced transcription factors and cofactors identifies Tfdp2 as an essential coregulator during terminal erythropoiesis. Exp. Hematol in the press (2014).
Pishesha, N., P. Thiru, J. Shi, J. Eng, V. Sankaran, and H. Lodish Transcriptional divergence and conservation of human and mouse erythropoiesis PNAS in the press (2014).
Cho, H. L. Ludwig, J. Eng, M. Fleming, H. Lodish, and V. Sankaran Genome-wide association study follow-up identifies cyclin A2 as a regulator of the transition through cytokinesis during terminal erythropoiesis Blood submitted (2014).
Alvarez, J. R., W. Hu. A. Gromatzky, and H. Lodish. Long Noncoding RNAs during Normal and Malignant Hematopoiesis. Progress in Hematology in the press (2014).
Cheng, A., P. Wong, K. Luo, P. Trepman, J. Shi, E. Wang, C. Burge, and H. Lodish Muscleblind-like 1 (Mbnl1) is required for terminal murine erythroid differentiation Blood submitted (2014).
Shi, J., L. Kundrat, N. Pishesha, A. Bilate, C. Theile, H. Ploegh, and H. Lodish. Engineered red cells as vehicles for systemic delivery of a wide array of functional probes. PNAS submitted (2014).