A. Red blood cell development
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
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 treatment of Diamond-Blackfan Anemia (DBA), an erythropoietin-resistant congenital red cell aplasia, but severe side effects limit its usefulness.
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. 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.
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.
B. Red blood cells as vehicles for the introduction of novel therapeutics, immunomodulatory agents, and diagnostic imaging probes into the human body.
Red blood cells possess many unique characteristics that make them attractive candidates for in vivo delivery of natural and synthetic payloads. They have a long circulatory half-life (~120 days in humans and ~50 days in mice), and old or damaged RBCs are removed and degraded by cells of the reticuloendothelial system. They are biocompatible and have a large surface area of ~ 140 µm2 with a favorable surface to volume ratio. Importantly, they contain no DNA; any genes introduced into red cell precursors will no longer be present in the enucleated red cells introduced into a recipient.
A large DARPA- supported project, in collaboration with Prof. Hidde Ploegh, involves the generation in culture of both murine and human red blood cells that have on their surface monoclonal antibodies that inactivate a variety of toxic substances, or receptors that can bind and remove unwanted materials from the blood. Gene- modified red cells can be targeted to specific sites in the vasculature where they can deliver drugs or reagents or serve as imaging modalities.
Engineered red blood cells that bind and neutralize toxic proteins
Single domain antibodies (VHH) are the antigen- binding domains of the unique functional heavy- chain- only camelid antibodies. These VHHs have equivalent binding activities to their cognate antigens compared to conventional IgGs and these VHHs are more stable and easier to make. VHHs that can target botulinum neurotoxin (BoNT) have been generated and studied intensively by our collaborator Dr. Charles B. Shoemaker of Tufts University Veterinary School; these can neutralize botulinum neurotoxins in cell culture and in vivo. Nai-Jia Huang and Nova Pishesha generated genes encoding four chimeric proteins, fusing the cDNAs encoding a chimeric VHH for BoNT/A or the VHH for BoNT/B, with either the N- terminus of glycophorin A or the C- terminus of Kell. Both types of red cells expressing the VHH BoNT/A chimeras effectively neutralized BoNT/A in an in vitro nerve- protection assay; in this assay primary neuronal cells are incubated with the toxin and the cleavage of the SNAP-25 protein by the toxin is quantified. They calculated that it should be possible to protect mice from a 1 LD100 toxin dose by administering as few as 2000 engineered red cell – VNA conjugates.
More recently they generated and tested the in vitro neutralization ability of VHH BoNT/A -engineered human red blood cells.. As few as 5 x106 human red cells expressing GPA-VNA/A and 106 cells expressing Kell-VNA/A effectively neutralized the botulinum toxin.
In collaboration with the Shoemaker laboratory Nai-Jia and Nova transplanted murine hematopoietic stem/progenitor cells expressing the VHH BoNT/A glycophorin and the Kell - VHH BoNT/A chimeric proteins into irradiated CD1 mice to produce normal red blood cells bearing these chimeras. These mice, together with control transplanted mice, were challenged with BoNT/A. All control mice died after challenge with 10 LD50 of BoNT/A. In contrast, all mice bearing red cells expressing either GPA-VNA/A or Kell-VNA/A survived successive challenges, at one - week intervals, of up to 1,000 LD50 BoNT/A treatment.
They are currently testing the ability of their engineered human red blood cells to neutralize botulinum toxin A in immune-compromised mouse strains, and also quantifying the in vivo efficacy, potency, and persistence of both mouse and human red cells, generated in culture and expressing the VHH BoNT/A – glycophorin chimeric protein. In collaboration with the Shoemaker lab they are testing whether similarly generated red cells can neutralize other bacterial toxins and also several pathogenic viruses.
Engineered red blood cells that induce immune tolerance
Nova Pishesha, in a collaborative project with Hidde Ploegh’s laboratory, is currently exploring the possibility of applying the aforementioned methods to generate red cells that have on their surface any of several covalently linked foreign or proteins. She aims to use these red cells to induce immune tolerance rather than an immune reaction, offering promise for novel treatments of several autoimmune disorders. Her current work has shown that administration of red blood cells that carry a relevant peptide epitope indeed leads to the drastic reduction in the number of transferred T cells that can specifically recognize this epitope. Building on the encouraging results of this T cell adoptive transfer model, she is adapting the system to several animal models of autoimmune diseases.
C. Non-coding RNAs that regulate development
Long non-coding RNAs (lncRNAs) are transcripts longer than 200nt that do not function through encoded protein products. Many are capped, polyadenylated, and often spliced, and 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 development and disease still remain uncharacterized.
An erythroid-specific long non-coding RNA prevents apoptosis of erythroid progenitors and promotes terminal proliferation.
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. Wenqian, together with Juan R. Alvarez-Dominguez, have identified and cloned the human LincRNA-EPS ortholog, and are now characterizing its putative antiapoptotic functions. In addition, Wenqian has generated a LincRNA-EPS knockout mouse and is characterizing several interesting in vivo phenotypes.
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, generated and analyzed >1 billion RNA-seq reads of both Poly(A)+ and Poly(A)- RNA from mouse fetal liver erythroid progenitor cells and terminal differentiating erythroblasts. Using de novo transcript reconstruction, they identified 655 lncRNA genes including not only intergenic, antisense, and intronic RNAs but also transcripts of pseudogenes and enhancer loci. Over 100 of these genes were previously unrecognized and are highly erythroid specific. They then combined genome-wide surveys of expression levels, chromatin states, and transcription factor occupancy in these 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 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.
They then focused on differentiation-induced lncRNAs, including novel erythroid-specific lncRNAs conserved in humans that are nuclear-localized. They selected 12 erythroid-specific lncRNAs that, like lincRNA-EPS, are greatly induced during erythroid terminal differentiation and are targeted at their promoters by the key erythroid transcription factors GATA1, TAL1 and KLF1. Remarkably, shRNA-mediated loss-of-function assays revealed that all 12 are essential for this developmental process. Juan is now focusing on how one of them, alncRNA-EC7, contributes to red blood cell development and physiology. alncRNA-EC7 is transcribed from an erythroid-specific enhancer upstream of the gene encoding Band 3, a major erythrocyte membrane protein, and is specifically needed for Band3 gene activation. Strikingly, EC7 is also essential for establishing the red cell gene expression program during terminal differentiation. Thus, diverse types of erythroid lineage-specific lncRNAs participate in the regulatory circuitry underlying red blood cell development.
In mammals, definitive erythropoiesis occurs sequentially in the fetal liver followed by the bone marrow. To explore the differences in definitive erythropoiesis in these two distinct niches, Wenqian performed transcriptomic profiling of terminal differentiating erythroblasts isolated from these two organs. In collaboration with Juan R. Alvarez-Dominguez, Wenqian is annotating and characterizing differentially expressed lncRNAs in these two hematopoietic organs and characterizing their functions in erythropoiesis. Much of this work is continuing in Wenqian’s own laboratory in the Mayo Clinic.
Jide Ezike, a new technical assistant who works closely with Sherry and Xiaofei, is computationally analyzing public RNA-seq datasets to identify lncRNAs that are expressed in BFU-E cells and that may be important for BFU-E self-renewal. He will then interrogate this data set against past RNA-seq data sets generated by our laboratory. Jide will then conduct experiments to determine whether any of these candidate lncRNAs are important for BFU-E self-renewal promoted by corticosteroids and PPARα agonists, and proceed to uncover the molecular mechanisms.
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 RNAs (lincRNAs) 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 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 one of them, Firre, an X-linked lincRNA required for proper adipogenesis. Firre is exclusively nuclear and interacts with the nuclear matrix factor hnRNP-U through numerous copies of an RNA sequence motif conserved between human and mouse, an association that is required to mediate trans-chromosomal interactions between loci encoding known adipogenic factors. Thus, numerous lincRNAs comprise a critical transcriptional regulatory layer that is functionally required for proper differentiation of both brown and white adipocytes.
Marko Knoll has chosen 9 functional lincRNAs to determine the mechanism by which they influence the adipocyte developmental program. All 9 are upregulated during adipogenesis in vitro. Using a candidate approach Marko immunoprecipitated hnRNPU, Suz12, Ezh2 and CoREST proteins and tested if these 9 lincRNAs were potential interaction partners; three potential candidates were identified, besides linc FIRRE, that are associated with hnRNPU. All three are strictly cytoplasmic lncRNAs, raising the question whether lincRNAs shuttle between the nucleus and cytoplasm because hnRNPU is strictly nuclear. Using RNA immunoprecipitation of hnRNPU in preadipocytes and differentiated mature adipocytes followed by deep sequencing, Marko aims to identify possible interaction partners. Further, using the CRISPR/Cas system, Marko aims to knock out or mutate these lincRNAs in the preadipocytic 3T3-L1 cell line.
Lei Sun is now continuing work on other lncRNAs in his own laboratory in the Duke- NUS Medical School in Singapore. Together with Juan R. Alvarez-Dominguez, they have used RNA-seq to reconstruct de novo transcriptomes across different fat depots, identifying ~1,500 adipose lncRNAs, including 127 brown fat-restricted lncRNAs that are often targeted by key transcriptional regulators PPARγ, C/EBPα, and C/EBPβ. They showed that one +of them, lnc-BATE1, is required for establishment and maintenance of BAT identity and thermogenic capacity. lnc-BATE1 inhibition impairs concurrent activation of brown fat and repression of white fat genes and is partially rescued by exogenous lnc-BATE1 with mutated siRNA-targeting sites, demonstrating a function of this lnc RNA in trans. Like Firre, lnc-BATE1 binds heterogeneous nuclear ribonucleoprotein U and both are required for brown adipogenesis. They have thus established lnc-BATE1 as a prototypical fat depot-selective lncRNA regulator of BAT development and physiology.
Mengxi Jiang, a new postdoctoral fellow, is investigating the role of lncRNAs in the development of two types of white adipose tissue – visceral (around internal organs) and subcutaneous fat (underneath the skin) – that differ in metabolic functions and are regulated by shared yet distinct transcriptional cascades. It is excess visceral fat that is the major risk factor for Type II diabetes. Our previous RNA-seq analysis identified several visceral, subcutaneous, and BAT- specific noncoding RNAs. Her study aims to understand the role and mechanism of selected non-coding RNAs in regulating the function and development of these two types of WAT by both gain of function and loss of function studies. She will analyze these by several molecular and cell biology techniques, and in genetically modified mice.
MicroRNAs in brown fat cell development and obesity
Marko Knoll, building on work of former lab members Lei Sun, Huangming Xie, and Ryan Alexander, is investigating the role of miRNAs in brown fat adipogenesis. 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 demonstrated 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. Lei and colleagues 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 ectopic miR-193b expression 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 the work by Ryan Alexander. Ryan showed that 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 in a gene. Marko will use the CRISPR/Cas approach to generate mice that have a mutated seed sequence in miR-203 and analyze the effect of miR-203 seed region mutation on the development of white and brown adipose tissue.
D. Adipocyte biology and insulin resistance
A kinase important for immune cells in the development of adipocytes
Marko Knoll, together with Heide Christine Patterson, 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 under the control of the adiponectin promoter. Identified as a top hit in a screen against the kinome, inhibition of this kinase and/or its deletion diminished whereas enhanced expression and activity increased β – agonist- triggered thermogenesis; this was measured my induced expression of the mitochondrial uncoupling protein Ucp1 in brown adipocytes in vitro. Further, this kinase promoted protein kinase B (Akt) activation, mitochondrial respiration, and expression of genes critically required for mitochondrial biogenesis and function in response to induction of thermogenesis. Kinase deletion in adipose tissue resulted in partial lethality of the mice that was rescued by thermoneutrality, as well as impaired expansion of brown preadipocytes, decreased brown fat mass, and impaired cold-induced browning. The only brown and beige adipocytes that developed were those that escaped kinase deletion. On a high fat diet, these mice displayed severe glucose intolerance and insulin resistance associated with obesity and decreased energy expenditure. These results establish this kinase as an essential mediator of brown fat formation and function, suggesting that pharmacological modulation of kinase activity and its targets has the potential to treat diabetes.
A 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 Marko Knoll, a post-doc, are investigating whether a kinase important for signal transduction in immune cells also mediates activation of oxidative stress induced pathways. They are determining in what subcellular compartment the signaling occurs and what components regulate activation of this kinase, as well as its in vivo relevance for function of brown adipocytes. They are using a combination of approaches that include genetic and pharmacological manipulation of primary B cells, fibroblasts, and adipocytes as well as bioinformatics and in vivo functional assays using conditional gene targeting in mice.
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 20:748-753 (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 124:1931 - 1940 (2014).
Hacisuleyman, E., L. Goff, C. Trapnell, A. Williams, J.Henao-Mejia, L. Sun, P. McClanahan, D. Hendrickson, M. Sauvageau, D. Kelley, M. Morse, J. Engreitz, E. Lander, M. Guttman, H. Lodish, R. Flavell, A. Raj, and J. Rinn Topological Organization of Multi-chromosomal Domains by linc-Firre Nature Structure Molecular Biol. 21:198 - 206 (2014).
Kim, H-J., H. Cho, R. Alexander, H. Patterson, M. Gu, K. Lo, C. Huang, D. Xu, V. Goh, L. Nguyen, X. Chai, C. Huang, J-P. Kovalik, S. Ghosh, M. Trajkovski, D. Silver, H. Lodish, and L. Sun MicroRNAs are required for the feature maintenance and differentiation of brown adipocytes Diabetes 63: 4045- 4056 (2014) 10.2337/db14-0466.
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Chen, C., and H. Lodish Global analysis of induced transcription factors and cofactors identifies Tfdp2 as an essential coregulator during terminal erythropoiesis. Exp. Hematol 42: 464-476 e5 (2014).
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Shi, J., L. Kundrat, N. Pishesha, A. Bilate, C. Theile, T. Maruyama, S. Dougan, H. Ploegh, and H. Lodish. Engineered red cells as carriers for systemic delivery of a wide array of functional probes. PNAS 111: 10131 - 10136 (2014).
Cheng, A., J. Shi, P. Wong, K. Luo, P. Trepman, E. Wang, H. Choi, C. Burge, and H. Lodish Muscleblind-like 1 (Mbnl1) regulates pre-mRNA alternative splicing during terminal erythropoiesis Blood 124: 598 - 610 (2014).
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Yien, R. Robledo, I. Schultz, N. Takahashi-Makise, B. Gwynn, D. Bauer, A. Dass, G. Yi, L. Li, G. Hildick-Smith, J. Cooney, E. Pierce, K. Mohler, T. Dailey, N. Miyata, P. Kingsley, S. Hattangadi, H. Huang, W. Chen, E. Keenan, D. Shah, T. Schlaeger, S. Orkin, A. Cantor, J. Palis, C. Koehler, H. Lodish, D. Ward, H. Dailey, J. Phillips, L. Peters, and Barry H. Paw TMEM14C is required for mitochondrial heme metabolism. J. Clin. Investigation 124: 4294 - 4304 (2014).
Lodish, H., Accommodating Family Life: Mentoring future female faculty members. Trends Cell Biol. 25:109 - 111 (2015).
Ludwig, L., H. Cho, A. Wakabayashi, J. Eng, J. Ullrich, 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 Am. J. Hematol. 90:386–391 (2015).
Knoll, M., H. Lodish, and L. Sun Long non-coding RNAs as regulators of the endocrine system. Nature Rev. Endocrinol. 11, 151–160 (2015)
Alvarez-Dominguez, J., Z. Bai, D. Xu, B. Yuan, K. Lo, M. Yoon, Y. Lim, M. Knoll, N. Slavov, S. Chen, P. Chen, H. Lodish, and L.Sun De novo reconstruction of adipose tissue transcriptomes reveals novel long non-coding RNA Regulators of Brown Adipocyte Development Cell Metab. 21, 764–776 (2015).
Lee, H-Y, X. Gao, M. Barrasa, H. Li, R. Elmes, and H. Lodish PPARα and glucocorticoid receptor synergize to promote erythroid progenitor self-renewal Nature 522, 474–477 (2015).
Shi, J.,B. Yuan, W. Hu, H. Choi, and H. Lodish JAK2 V617F stimulates proliferation of erythropoietin- dependent erythroid progenitors and delays their differentiation by activating non-erythroid signaling pathways. Journal of Biological Chemistry. submitted (2015).
H. Patterson, C. Gerbeth, P. Thiru, F. Voegtle, M. Knoll, A. Shahsafaei, K. Samocha, C. Huang, M. Harden, R. Song, C. Chen. J. Kao, J. Shi, W. Salmon, Y. Shaul, M. Stokes, J. Silva, G. Bell, D. MacArthur, J. Ruland, C. Meisinger, and H. Lodish. A Respiratory Chain Controlled Signal Transduction Cascade in the Mitochondrial Intermembrane Space Mediates H2O2 Signaling. PNAS submitted (2015)
Chu, P., X, Fan, Z. Li, S. Bari, J. Ang, J. Huang, A Prasath, H. Lodish. C. Zhang, G. Chiu, S. Lim, W. Hwang, Mesenchymal Stromal Cell Co-culture Enhances Viability of Thawed Human Cord Blood through Rescue from Early Apoptosis in a Contact-Dependent Process. Submitted. (2015)
Chung, J., D. Bauer, A. Ghamari, C. Nizzi, K. Deck, P. Kingsley, Y. Yien, N. Huston, C. Chen, I. Schultz, A.. Dalton, J. Wittig, J. Palis, S. Orkin, H. Lodish, R. Eisenstein,A. Cantor, and B. Paw The mTORC1/4E-BP pathway coordinates hemoglobin production with L-leucine availability. Science Signaling Vol 8 Issue 372 ra34 (2015).
Gao, X., H-Y Lee, W. Li, R. Platt, M. Barrasa, R. Elmes, M. Rosenfeld, and H. Lodish Thyroid hormone receptor beta and NCOA4 regulate terminal erythrocyte differentiation. Submitted (2015)
Li, H., J. Shi, J. C. Eng, and H. Lodish Efficient CRISPR-Cas9 mediated gene disruption in primary erythroid progenitor cells. Submitted (2015)