We are characterizing many novel genes that are important for terminal stages of erythropoiesis, including gene induction and repression, chromatin condensation, and enucleation. A major focus is identifying genes and extracellular signals that regulate the self- renewal, proliferation, and differentiation of early (BFU-E) erythroid progenitor cells; extracellular signals include activators of the glucocorticoid and PPAR receptors and oxygen. This work has led to the characterization of several molecules, including two that are FDA-approved drugs for other indications, that show great promise as therapeutics for bone marrow failure disorders and erythropoietin- resistant anemias. This research involves extensive computational analyses of large datasets of gene expression profiles and chromatin modifications generated from cells at different stages of human and mouse red cell development.
Introduction
Erythropoietin (Epo), the principal regulator of red blood cell production, 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 default 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. During this terminal differentiation about 400 erythroid- important genes are induced; over many years we have identified many novel transcription factors and cofactors, splicing protein, chromatin modifying proteins, and enzymes in metabolic pathways including heme biosynthesis, that are essential for red cell formation.
An earlier committed erythroid progenitor, termed the burst- forming unit erythroid (BFU-E), can divide and generate additional BFU-Es (that is, undergo self- renewal) or diviide and 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, prior to our research regulation of BFU-E proliferation, self-renewal, and differentiation during basal and stress conditions was not well understood.
We are focusing much effort in this important area because of the clinical observation that many patients with bone marrow failure disorders such as Diamond-Blackfan anemia are helped by glucocorticoids (GCs) rather than by 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 to support life.
Culture systems that supports normal expansion and terminal differentiation of human hematopoietic stem/progenitor cells
There are many published cell culture systems for expanding human hematopoietic stem and progenitor cells such that they generate hemoglobin- containing nucleated red cell progenitors. But invariably these fail to undergo normal terminal differentiation, condense their nuclei, and expel the nucleus from the cell. Sherry Lee recently developed a 21-day four- stage culture system that supports synchronized erythroid expansion, terminal differentiation, and enucleation of mobilized human peripheral blood CD34+ stem and progenitor cells. There is a 15,000 to 30,000 fold cell expansion, equal to 14 to 15 cell doublings. At the end of the culture all of the cells were hemoglobinized and over 45% had undergone enucleation. Each enucleated reticulocyte contained ~30 pg hemoglobin, similar to the amount in each human red blood cell. The hemoglobin composition in these cells was the same as in adult human red cells, and the enucleated reticulocytes averaged 6.7 pg hemoglobin, similar to the amount in normal red blood cells and reticulocytes. More recently Novalia Pishesha, Nai-Jia Huang, and Jiahai Shi have improved this system, using a lower serum concentration supplemented with human plasma. Differentiation is highly synchronized and cell expansion is over 70,000 fold. Over 90% of the cells undergo enucleation and the resulting reticulocytes contain the normal amount of 30 pg hemoglobin per cell. These enucleated cells survive for several days when transfused into immune- compromised mice that accept human cell transplants.
These culture systems enable many studies on terminal differentiation of human erythrocytes. For instance, using lentivirus vectors we have succeeded in expressing one or in some cases two foreign genes in well over 90% of the erythroid cells generated in culture. Similarly, we have used lentiviral vectors expressing shRNAs to knock down expression at will of any erythroid gene. This culture system combined with these manipulations allow unprecedented and focused genetic manipulation of red cells and their major proteins, and allow us to do many experiments on drugs and genes and their effects on red cell formation.
Corticosteroids, hypoxia and stress erythropoiesis
These culture systems enable many studies on terminal differentiation of human erythrocytes. For instance, using lentivirus vectors we have succeeded in expressing one or in some cases two foreign genes in well over 90% of the erythroid cells generated in culture. Similarly, we have used lentiviral vectors expressing shRNAs to knock down expression at will of any erythroid gene. This culture system combined with these manipulations allow unprecedented and focused genetic manipulation of red cells and their major proteins, and allow us to do many experiments on drugs and genes and their effects on red cell formation.
Findings by a former postdoc, Johan Flygare, indicate that the physiology of SE involves a stimulation of the earlier BFU-E 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 enhancing the numbers of terminally differentiated red cells. GCs do not affect CFU-E cells or erythroblasts. In mRNA-seq experiments on BFU-E cells, he found that glucocorticoids induced expression of ~86 genes more than 2- fold. 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. Johan recently established his own research group at the Lund Stem Cell Center in Sweden.
More recently two current postdoctoral fellows, Sherry Lee and Xiaofei Gao, showed that two clinically-tested specific inhibitors of the prolyl hydroxylase that regulates HIF1α activation also synergize with corticosteroids to stimulate both human and mouse BFU-E self renewal and at orders of magnitude lower concentrations than DMOG.
More recently two current postdoctoral fellows, Sherry Lee and Xiaofei Gao, showed that two clinically-tested specific inhibitors of the prolyl hydroxylase that regulates HIF1α activation also synergize with corticosteroids to stimulate both human and mouse BFU-E self renewal and at orders of magnitude lower concentrations than DMOG. We propose and are testing a physiological model of stress erythropoiesis where increased levels of GCs –systemic stress hormones - and reduced oxygen – local stress - help stimulate self-renewal of the earliest erythroid BFU-E 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. Also, PHI-induced stimulation of BFU-E progenitors represents a conceptually new therapeutic window for treating Epo-resistant anemias.
Identifying potential drugs for Epo-resistant anemias that stimulate BFU-E self-renewal and increase production of red blood cells.
Based on our previous study showing that glucocorticoids specifically stimulate self-renewal of BFU-Es and over time increase the production of terminally differentiated red cells, Sherry Lee and Xiaofei Gao tested whether known pharmaceuticals that are either agonists or antagonists of other nuclear receptors affect BFU-E self-renewal and could potentially be used as new therapeutics for anemias that are not treatable by Epo. Using first the mouse fetal liver BFU-E culture system we developed and then our new ex vivo human CD34+ erythroid culture system, they found that two clinically-tested agonists of the peroxisome proliferator-activated receptor alpha (PPARα), fenofibrate and GW7647, synergize with glucocorticoids to promote BFU-E self-renewal and over time greatly increase red cell production. Genome-wide gene expression analyses both in control and corticosteroid- treated mouse BFU-E cells showed that PPARα occupies many chromatin sites that are in close proximity to those occupied by the glucocorticoid receptor (GR), indicating that the GR and PPARα function cooperatively to regulate gene expression. In particular the GR and PPARα together activate PPARα gene expression, leading to a feed-forward circuit enhancing BFU-E self-renewal.
While PPARα-/- mice show no hematological difference from wild-type mice in both normal and phenylhydrazine (PHZ)-induced stress erythropoiesis, PPARα agonists facilitate recovery of wild-type mice, but not PPARα-/- mice, from PHZ-induced acute hemolytic anemia. The mutant "Nan" (neonatal anemia) mouse has a single amino acid substitution in the erythroid- important transcription factor EKLF (KLF), which abrogates the DNA-binding capacity of EKLF to certain target genes. Heterozygotes (Nan/+) survive with a life-long, intermediate- to- severe hemolytic anemia, displaying many features of hereditary spherocytosis. With the assistance of Russell Elmes, Xiaofei and Sherry showed that both fenofibrate and GW7647 stimulated red cell formation in these mice, raising the level of red cells to almost normal. Both also caused an increase in the numbers of splenic BFU-E progenitors, suggesting that as expected these increase erythroid output via promoting BFU-E self-renewal.
Understanding erythroid self-renewal and fate commitment using single-cell RNA sequencing
All erythroid and megakaryocytic lineage cells are produced by bipotential Megakaryocyte-Erythroid Progenitors (MEPs). Current protocols for isolating these cells have shown that only a small fraction - ~30% of colonies produced by these cells are actually the mixed erythroid-megakaryocyte colonies expected of a bipotential progenitor; most other colonies are entirely composed of megakaryocytes or erythroid cells indicative of unipotential progenitors. A key step, then, is to identify the cell surface markers that can be used to isolate the bipotential cells. Anirudh Natarajan is using single-cell RNA-seq and computational approaches to address this problem by identifying cell surface proteins likely to be unique to the bipotential progenitors. Following this, he will capture the transcriptomes of these MEP cells as they self-renew or differentiate in culture to erythroid or megakaryocyte progenitors. This will help us understand how lineage restriction from bipotential to unipotent progenitors is established. In addition, he will identify candidate regulators of these processes. Experiments perturbing the expression of these genes will identify novel regulators of fate commitment in these bipotential progenitors.
He is also using single-cell RNA sequencing to understand the transition of erythroid cells as they differentiate from self-renewing BFU-Es to CFU-Es. In addition, he will investigate how the transcriptome changes during self-renewal under stimulation by drugs including dexamethasone and prolyl hydroxylase inhibitors. These experiments will help us understand the nature of BFU-E self-renewal and help identify regulators of the fate commitment process.
Pathogenic mutant JAK2 V617F stimulates proliferation of erythropoietin- dependent erythroid progenitors and delays their differentiation by activating non-erythroid signaling pathways
JAK2 is a protein tyrosine kinase activated by the Epo receptor and several other cytokine receptors. JAK2-V617F is a mutant activated JAK2 kinase found in most polycythemia vera (PV) patients and those with other myeloproliferative disorders. Several years ago we showed that the 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. JAK2-V617F 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. Expression of genes promoting DNA replication continues in these JAK2-V617- expressing cells for 2 divisions longer than normal. 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 enucleated cells with an mRNA composition very similar to that of normal mouse 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. He showed that purified fetal liver Epo- dependent progenitors express many cytokine receptors additional to the EpoR as well as Stat1 and Stat3 in addition to Stat5, the only STAT normally activated by the Epo receptor and JAK2. JAK2-V617F triggers activation of Stat1 and Stat3, and inhibition of Stat1 by a drug blocks Jak2 V617F mediated erythropoiesis, but does not affect normal erythropoiesis. This abnormal activation of Stat1 and Stat3 leads to transient induction of many genes not normally activated in terminally differentiating erythroid cells and that are characteristic of other hematopoietic lineages. He hypothesizes that these non-erythroid-signaling pathways delay terminal erythroid differentiation and permit extended numbers of 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.
Transcriptional control of gene expression during terminal erythroid differentiation: Thyroid hormone receptor beta and NCOA4
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 whose expression is up-regulated during terminal differentiation.
An effect of thyroid hormone on erythropoiesis has been known for more than a century but the molecular mechanism(s) by which thyroid hormone affects red cell formation have been elusive. Recently Sherry and Xiaofei demonstrated an essential role of thyroid hormone during terminal human erythroid cell differentiation; specific depletion of thyroid hormone from the culture medium completely blocked mouse and human erythroid differentiation. Genome wide analysis showed that thyroid hormone receptor β (TRβ) occupies many gene loci encoding transcripts abundant during terminal erythropoiesis, including globin genes, and cooperates with GATA-1 and RNA polymerase II (Pol II) to regulate their expression. TRβ agonists stimulated red cell formation in Nan/+ mice, raising the level of red cells to normal; these agonists also accelerated erythroblast differentiation from the BFU-E stage in vitro, likely by reducing BFU-E self renewal.
To identify factors that cooperate with TRβ during human erythroid terminal differentiation, they conducted RNA-Seq in human reticulocytes and identified nuclear coactivator 4 (NCOA4) as a critical regulator of terminal differentiation. Furthermore, Ncoa4-/- mice are anemic in both the embryonic and perinatal periods and fail to respond to thyroid hormone by enhanced erythropoiesis. Genome wide analysis suggested that thyroid hormone promotes NCOA4 recruitment to chromatin regions that are in proximity to Pol II and are highly associated with transcripts abundant during terminal differentiation. Additionally, knocking down NCOA4 interrupted the terminal differentiation in both our human CD34 ex vivo differentiation system and in cultured mouse fetal liver cells. Collectively, their results reveal the molecular mechanism of thyroid hormone function in accelerating terminal red blood cell formation and are potentially useful to treat certain anemias.
Transcriptional divergence and conservation of human and mouse erythropoiesis
Mouse models have been used extensively for decades and have been instrumental in improving our understanding of mammalian erythropoiesis. Nevertheless, there are several examples of variation between human and mouse erythropoiesis. In collaboration with Vijay G. Sankaran, a recent postdoc and currently an Assistant Professor at Boston Children's Hospital, Nova Pishesha performed a comparative global gene expression study using publicly available data from morphologically identical stage-matched sorted populations of human and mouse erythroid precursors from early to late erythroblasts. Surprisingly, they found that, at a global level, there is a significant extent of divergence between the species, both at comparable stages and in the transitions between stages. This was especially the case for the 500 most highly expressed genes during development, save for some major transcriptional regulators of erythropoiesis and major erythroid-important proteins. This suggests that the response of multiple developmentally regulated genes to key erythroid transcriptional regulators represents an important modification that has occurred in the course of mammalian evolution. They further developed this compendium of data as a systematic framework that is very practical and useful to understand and study conservation and divergence between human and mouse erythropoiesis as well as to help translate findings from mouse models to potential therapies for human disease.
Translational control of red cell development
Wenqian Hu is investigating how mRNA-binding proteins regulate erythroid terminal differentiation. He characterized one such protein, Cpeb4, that is required for terminal erythropoiesis. Specifically, he found that Cpeb4 is dramatically induced during erythroid terminal differentiation by the two erythroid-important transcription factors, GATA1 and Tal1. Knocking down Cpeb4 inhibits this cell differentiation process. Interestingly, Cpeb4 interacts with eIF3, a general translation initiation factor, to repress the translation of a large set of mRNAs in terminal differentiating erythroblasts, including its own mRNA. Thus, transcriptional induction synchronizes with translational repression to maintain Cpeb4 protein within a specific range during terminal erythropoiesis; this precise control of gene expression is required for normal cell differentiation. Wenqian is establishing his own independent laboratory as an Assistant Professor at the Mayo Clinic where he will continue these lines of investigations. Specifically, in collaboration with Juan Alvarez-Dominguez, he identified a group of mRNA-binding proteins that are specific to erythroid cells and that are dramatically induced during terminal erythropoiesis. Currently, Wenqian is characterizing whether and how these mRNA-binding proteins regulate terminal erythroid differentiation.
Genes important for red cell formation identified by genetic analyses of human erythropoiesis
Vijay Sankaran, laboratory colleagues Leif Ludwig, Jenn Eng and Hyunjii Cho, along with colleagues at the Broad Institute, have been dissecting the genetic architecture of human erythropoiesis,. This work is being performed using a combination of complex trait genetics, Mendelian genetics, and analysis of rare human syndromes.
Cyclins that regulate proliferation of red cell progenitors and red cell size
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. Similar findings have identified cyclin A2 as a novel regulator of red blood cell size. by regulating the passage through cytokinesis during the final cell division of terminal erythropoiesis. These studies provide new insight into cell cycle regulation during terminal erythropoiesis and more generally illustrates the value of functional GWAS follow-up to gain mechanistic insight into hematopoiesis. Ongoing studies are aimed at broadening these approaches to other loci across the genome. Leif successfully defended his PhD thesis at the Free University of Berlin and is now continuing his medical studies.
Diamond-Blackfan anemia
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 Dr. Hanna Gazda, this group has recently defined the first non-ribosomal protein gene involved in Diamond-Blackfan anemia, GATA1. Diamond-Blackfan anemia is more commonly caused by heterozygous deletions or loss of function mutations in one of 16 ribosomal protein genes, but it was not clear how mutations in ubiquitously expressed ribosomal protein genes could result in an erythroid-specific defect. Further work by Leif and Vijay showed that translation of GATA1 mRNA is impaired in the setting of ribosomal haploinsufficiency. Since GATA1 is essential for erythropoiesis, this provided compelling evidence about the specificity of the defect observed in patients with Diamond-Blackfan anemia with ribosomal gene mutations. When studying the transcriptional signature in stage-matched sorted cells obtained directly from patients with RPS19 mutations, GATA1 target genes were significantly downregulated and GATA1 overexpression could partially rescue defects in primary cells from patients with Diamond-Blackfan anemia. These observations illuminate the central role of GATA1 in the pathogenesis of Diamond-Blackfan anemia. 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 Dr. 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.
Congenital Dyserythropoietic Anemia (CDA)
Insight into the rare disease Congenital Dyserythropoietic Anemia (CDA) is further elucidating our understandings of erythropoiesis. Unlike Diamond-Blackfan Anemia where there is a lack of erythroid progenitors, patients with CDA have bone marrow hyperplasia, but a derailment in later erythropoiesis causes a decreased output of mature red blood cells. In recent years the genetic cause of the most common CDA subtype, CDAII, was discovered to be caused by a mutation in SEC23b, implicating it in a vesicle transport defect. Recently two siblings with an unusual form of CDA were mapped by Vijay Sankaran to a different genetic locus. In collaboration with Vijay Sankaran’s lab at Boston Children’s Hospital, Jenn Eng is performing experiments to follow-up on exome sequencing on the effected siblings and their unaffected parents in order to gain a better understanding of what is causing their CDA and what clinical implications this may have for understanding late erythropoiesis.
Regulation of fetal globin expression
Elevated levels of fetal hemoglobin can ameliorate the major disorders of beta-hemoglobin diseases, sickle cell disease and beta-thalassemia in particular. 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.
Cyclins that regulate proliferation of red cell progenitors and red cell size
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. Similar findings have identified cyclin A2 as a novel regulator of red blood cell size. Ongoing studies are aimed at broadening these approaches to other loci across the genome.
Diamond-Blackfan anemia
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 Dr. Hanna Gazda, this group has recently defined the first non-ribosomal protein gene involved in Diamond-Blackfan anemia, GATA1. Diamond-Blackfan anemia is more commonly caused by heterozygous deletions or loss of function mutations in one of 12 ribosomal protein genes, but it was not clear how mutations in ubiquitously expressed ribosomal protein genes could result in an erythroid-specific defect. Further work showed that translation of GATA1 mRNA is impaired in the setting of ribosomal haploinsufficiency. Since GATA1 is essential for erythropoiesis, this provided compelling evidence about the specificity of the defect observed in patients with Diamond-Blackfan anemia with ribosomal gene mutations. When studying the transcriptional signature in stage-matched sorted cells obtained directly from patients with RPS19 mutations, GATA1 target genes were significantly downregulated and GATA1 overexpression can partially rescue defects in primary cells from patients with Diamond-Blackfan anemia. These observations illuminate the central role of GATA1 in the pathogenesis of Diamond-Blackfan anemia. 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 Dr. 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.
Congenital Dyserythropoietic Anemia (CDA)
Insight into the rare disease Congenital Dyserythropoietic Anemia (CDA) is further elucidating our understandings of erythropoiesis. Unlike Diamond-Blackfan Anemia where there is a lack of erythroid progenitors, patients with CDA have bone marrow hyperplasia, but a derailment in later erythropoiesis causes a decreased output of mature red blood cells. In recent years the genetic cause of the most common CDA subtype, CDAII, was discovered to be caused by a mutation in SEC23b, implicating it in a vesicle transport defect. Recently two siblings with an unusual form of CDA were mapped by Vijay Sankaran to a different genetic locus. In collaboration with Vijay Sankaran's lab at Boston Children's Hospital, Jenn Eng is performing experiments to follow-up on exome sequencing on the effected siblings and their unaffected parents in order to gain a better understanding of what is causing their CDA and what clinical implications this may have for understanding late erythropoiesis.
Histones to the cytosol: Exportin 7 is essential for erythroid nuclear condensation and enucleation.
Together 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 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 discovered that erythroblast nuclei from Xpo7- knockdown cells were less condensed and larger than control nuclei, as judged by confocal immunofluorescence microscopy. Enucleation was blocked, and Xpo7- knockdown 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.
Dynamics of the nuclear lamina during terminal erythropoiesis
Chromatin condensation during terminal differentiation is accompanied by proportional shrinkage in the size of the nucleus. The nuclear lamina is composed of the fibrous proteins nuclear lamin A/C and nuclear lamin B, and forms a dense fibrillar network on the inside the nuclear envelope that provides structural support to the nucleus. Jiahai Shi and undergraduate Heejo Choi showed that the expression of the three lamins initially increased and then decreased markedly during terminal erythroid development, and that the dynamic expressions of Lamin A/C and Lamin B controls the thickness of the nuclear lamina. Unlike the gradual decrease in nuclear size, the nuclear lamina increases dramatically in the early stages of terminal erythropoiesis, followed by a sudden decrease at the end, as revealed both by western blotting and immunofluorescence confocal microscopy. These results suggest that enhanced expression of lamin A/C may be important for erythroid terminal differentiation. Jiahai is continuing to explore the functional role of the up-regulation of nuclear lamina in terminal erythropoiesis in his own laboratory as an Assistant Professor at the City University in Hong Kong.
Modeling disorders of erythropoiesis in primary human and mouse erythroid cells
Ongoing work by postdoctoral fellow Hojun Li, is attempting to create models of anemia through genome editing in primary erythroid progenitor cells. Hojun Li, Jiahai Shi, and Jenn Eng have developed a method for high efficiency genome editing in mouse fetal liver erythroid progenitor cells using a retroviral vector to deliver the components of the CRISPR-Cas9 nuclease system. They have been able to demonstrate that over 50% of the cells in a population of fetal liver erythroid progenitors can be successfully transduced by this vector and that gene knockout results in loss of protein expression. This is the highest to-date reported efficiency of CRISPR-Cas9 mediated gene knockout in isolated primary cells, They have been able to replicate the phenotype of the impaired hemoglobin switch in Bcl11a knockout mice by using CRISPR-Cas9 to disrupt the Bcl11a gene, and have gone on to show developmental phenotypes for knockout of other genes that cannot be knocked down successfully in fetal liver culture. In particular they used this method to demonstrate a novel requirement for Lamins A and C in erythroid differentiation Hojun is working with Jenn to adapt CRISPR-Cas9 genome editing to our human erythroid culture system in order to introduce known mutations associated with human anemias for the purpose of modeling the effects of these mutations on human erythroid development.
Membrane protein sorting during enucleation
Certain membrane proteins are selectively retained on the red blood cell membrane and others in the erythroblast are lost; this selection process mainly occurs during enucleation when nuclei are expelled, surrounded by a segment of the erythroblast plasma membrane, and separated from the remaining reticulocyte. As examples, Glycophorin A, protein 4.1 and Kell protein are retained on the surface of the reticulocytes whereas erythroblast macrophage protein and transferrin receptor (Tfr) are extruded with the nuclei. Proper sorting of membrane proteins at the end of erythroid differentiation is essential for generating normal red blood cells but the regulation and mechanisms of protein sorting before and during enucleation are poorly understood. Nai-Jia Huang is beginning to investigate the mechanisms of protein selection during enucleation. Understanding these mechanisms might provide a new way to express proteins on red blood membranes without modifying endogenous proteins.