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Lodish Lab Research Summary
Research in my lab focuses on several important areas at the interface between molecular cell biology and medicine:
Red blood cell development, especially the regulation of self- renewal, proliferation and differentiation of early (BFU-E) and late (CFU-E) erythroid progenitor cells by extracellular signals including erythropoietin, glucocorticoids, and oxygen. We are identifying many novel genes that are important for terminal stages of erythropoiesis, including chromatin condensation and enucleation, and uncovering their mechanism of action. One goal is the development of new therapies for erythropoietin- resistant anemias.
microRNAs (miRs) and long non-coding RNAs (lincRNAs) that regulate erythroid and myeloid progenitor cells. We are identifying their mRNA and protein targets, and defining their roles in several hematopoietic cancers.
Hematopoietic stem cells. Identifying the stromal cells in the fetal liver and bone marrow that support stem cell self- renewal in vivo, and identifying novel growth factors made by these cells that support stem cell expansion in culture. We are beginning clinical trials to expand cord blood stem cells using our recently- identified growth factors.
Adipocyte biology. Defining the mechanisms of insulin resistance and the functions of adiponectin, a hormone we cloned that is made exclusively by fat cells and that increases fatty acid and glucose metabolism by muscle and liver.
E.miRs and lincRNAs that regulate differentiation and function of white and brown adipose cells.
What ties all of these projects together is their focus on the basic cell and molecular biology of genes and proteins important for human physiology and disease.
Introduction Erythropoietin (Epo) is the principal regulator of red blood cell production; Epo is produced by the kidney in response to low oxygen pressure in the blood. Epo binds to Epo receptors on the surface of committed erythroid CFU-E progenitors, blocking apoptosis (programmed cell death), their usual fate, and triggering them to undergo a program of 4 – 5 terminal erythroid cell divisions and differentiation. We showed that the first two cell divisions, concomitant with differentiation from CFU-Es to late basophilic erythroblasts, are highly Epo-dependent; differentiation beyond this stage, involving chromatin condensation, ~1-2 terminal cell divisions, and enucleation, is no longer dependent on Epo but is enhanced by adhesion of the cells to a fibronectin matrix. Following condensation of chromatin and subsequent enucleation reticulocytes (immature red cells) are released into the blood.
The earliest committed progenitor, termed the burst- forming unit erythroid (BFU-E), can divide and generate additional BFU-Es (that is, undergo partial self- renewal) as well as generate later Epo- dependent CFU-E progenitors. Several cytokines and hormones are known to support BFU-E proliferation and formation of CFU-Es, including stem cell factor (SCF, the ligand for the c-kit protein tyrosine receptor) as well as IL-3, IL-6, and IGF-1. However, regulation of BFU-E proliferation and differentiation during basal and stress conditions is not well understood. We decided to focus on this important area based on the clinical observation that many bone marrow failure patients are helped by glucocorticoids (GCs) rather than Epo treatment. These patients already have very high Epo levels in the blood, but do not have sufficient Epo-responsive CFU-E cells in the bone marrow.Mechanisms of stress erythropoiesis
In situations of severe loss of red blood cells mammals and birds respond by a process known as stress erythropoiesis (SE), in which there is increased formation of erythroid progenitors. Glucocorticoids (GCs) are known to be very potent enhancers of SE. This stimulatory effect of GCs on SE is utilized in the therapeutic regimen of Diamond-Blackfan Anemia (DBA), an erythropoietin-resistant congenital red cell aplasia. While an Epo-dependent balance of late red cell precursor survival normally maintains red cell homeostasis, Johan’s findings indicate that the physiology of SE involves a stimulation of earlier erythroid progenitors, which when activated are able to rescue red cell production in conditions such as DBA, where erythropoietin has little effect.
Johan showed that glucocorticoids stimulate self-renewal of early Epo-independent progenitor cells (burst-forming units erythroid or BFU-Es), over time increase production of colony-forming units erythroid (CFU-E) erythroid progenitors from the BFU-E cells, and enhance terminal erythroid differentiation. GCs do not affect CFU-E cells or erythroblasts. In mRNA-seq experiments, he found that glucocorticoids induced expression of ~86 genes more than 2- fold in BFU-E cells. Computational analyses indicated that, of all transcription factors, binding sites for hypoxia-induced factor 1 alpha (HIF1α) were most enriched in the promoter regions of these genes, suggesting that activation of HIF1α may enhance or replace the effect of glucocorticoids on BFU-E self-renewal. Indeed, HIF1α activation by the prolyl hydroxylase inhibitor (PHI) DMOG synergized with glucocorticoids and enhanced production of CFU-Es and later erythroblasts over 170-fold. PHI-induced stimulation of BFU-E progenitors thus represents a conceptually new therapeutic window for treating Epo-resistant anemia. Johan proposes a physiological model of stress erythropoiesis where increased levels of GCs and reduced oxygen help maintain the earliest erythroid progenitors, increase CFU-E output, and at the same time stimulate terminal differentiation, thus promoting both a rapid and long-lasting increase in red blood cell production.
Since the main action of the activated GCR is to interact with chromatin and regulate transcription Johan and his technical assistant Violeta Rayon Estrada together with a graduate student, Lingbo Zhang, used ChIP-Seq on BFU-E cells to map the locations of the activated GCR along the genome, determine which binding partners it has, and how transcription is repressed and/or activated at these sites in BFU-E cells. Johan recently established his own group at the Lund Stem Cell Center in Sweden, where he will search for genes, molecular pathways and compounds that modify the red cell progenitor defect in Diamond Blackfan anemia. The aim of this work is to develop novel treatments for this disorder.
Proteins required for glucocorticoid- triggered self renewal of BFU-E erythroid progenitors
Lingbo Zhang, together with his technical assistant Lina Prak and MIT undergraduate student Juliann Shih, is working on identification of genes essential for glucocorticoid mediated BFU-E self-renewal. His current research has focused on functional characterization of one gene that is indispensible for BFU-E self-renewal. This gene is normally downregulated during differentiation from BFU-E stage to CFU-E stage. In our in vitro primary “BFU-Es” culture system, glucocorticoid addition blocked this downregulation and knockdown of this gene by shRNAs completely disrupted glucocorticoid mediated BFU-E self-renewal, but without any effects on cell division rates or cell survival. In our GR Chip-Seq dataset, the activated glucocorticoid receptor binds to a genomic region several kb upstream of the transcription start site of this gene, and a luciferase reporter assay demonstrated that this region is indeed glucocorticoid inducible. These data suggest this gene as a direct transcriptional target of GR. Now Lingbo is trying to understand how this gene is involved in BFU-E self-renewal regulation. Additionally, Lingbo and Lina are establishing a high-throughput screening strategy using libraries of shRNAs to identify other genes essential for glucocorticoid mediated BFU-E self-renewal. We trust that this work will uncover the molecular mechanism how glucocorticoids control BFU-E self-renewal and how glucocorticoid treatment benefits Diamond-Blackfan Anemia and potentially other EPO unresponsive anemias.
Hypoxia and stress erythropoiesis
Hypoxia - inadequate oxygen supply to cells and tissues - is a strong regulator of gene expression in both eukaryotic and prokaryotic cells; that hypoxia results in an increased number of red blood cells in humans and animals was observed over 100 years ago. Synthesis of Epo in the kidney is induced by hypoxia and mediated by HIF1α; additionally HIF1α synergizes with glucocorticoids (GCs) to stimulate red blood cell formation by promoting self-renewal of erythroid burst-forming unit progenitors (BFU-Es). Strikingly, the promoter/enhancers of many genes induced in BFU-Es by GCs are enriched for HIF1α binding sites, supporting the notion that HIF1α is likely to play an essential role in promoting self-renewal of BFU-Es. Moreover, stabilizing HIF1α by the prolyl hydroxylase inhibitor (PHI) dimethyloxaloylglycine (DMOG) alone results in enhanced self-renewal of BFU-Es and an increased number of red blood cells. However, the mechanism by which HIF1α regulating the self-renewal of BFU-Es is unknown. Xiaofei Gao, a new postdoc, is interested in identifying and characterizing genes regulated by HIF1α essential for BFU-E self-renewal. He will use mRNA sequencing and computational approaches to identify HIF1α-up-regulated “core genes”, whose expression correlates best with BFU-E self-renewal upon treatment with different PHIs with or without GCs. He will also characterize the roles of these genes in BFU-E self-renewal by multiple means including an in vitro fetal liver BFU-E cell culture system established in our laboratory, and also gene-altered mice. We expect this research to benefit future drug development for the treatment of bone marrow failure anemias, including Diamond-Blackfan anemia (DBA). Moreover, considering the importance of HIF1α and HIF1α–regulated genes in the survival and self-renewal of normal stem cells, cancer cells, and cancer stem cells, these experiments will expand our knowledge of how HIF1α is participating in these cellular processes.Mechanistic study of activation of normal and pathogenic Janus kinase 2 and their associations with the erythropoietin receptor
A point mutation in the Janus kinase 2 (JAK2) pseudo-kinase domain, V617F, is found in most patients with Polycythemia Vera and those with other myeloproliferative disorders. This mutation enables cytokine-independent activation of JAK2 in cells that express a homodimeric cytokine receptor such as the erythropoietin receptor (EpoR). The activation mechanisms of both normal and pathogenic JAK2 are poorly understood. Jiahai Shi is studying the interaction between JAK2 and the EpoR cytoplasmic domain by X-ray crystallography. In particular he will determine the binding interface between the EpoR BOX 1 motif and JAK2 FERM domain. This interface would be a novel and specific drug target against JAK2-V617F positive myeloproliferative disorders. This work is being done in collaboration with Prof. Thomas Schwartz of the MIT Biology Department.
Production of large numbers of enucleated human erythroblasts in culture.
Sherry Lee and Jiahai Shi set out to optimize our system of in vitro culture of murine fetal liver Epo- responsive CFU-E progenitors to generate the maximum numbers of enucleated erythroid cells, i.e. reticulocytes. Careful examination of media constituents led to the discovery that the ferro- transferrin concentration indeed limits terminal erythropoiesis, and supplementation with as much as 500 µg/ml ferrotransferrin greatly enhance production of reticulocytes. Fetal bovine serum was essential, and ferro transferrin could be partially but not completely replaced by a water- soluble iron chelating agent.
Sherry then turned her attention to erythroid culture of mobilized human blood CD34+ erythroid progenitor cells; these cells could easily be cultured under conditions such that nucleated hemoglobin- containing erythroid cells were formed but little enucleation occurred. Sherry developed a four- stage culture system yielding an approximately 90,000-fold expansion after a 19- day culture. At the end of the culture over 46% of the cells had undergone enucleation and all of the cells were hemoglobinized; each enucleated reticulocyte contained ~30 pg hemoglobin, similar to the amount in normal human red blood cells. Some serum is essential for enucleation. Her experiments indicate that timely supply and withdrawal of cytokines required for each developmental stage is important for erythroid differentiation and synchronizing the cell population in culture. This culture system enables many studies on terminal differentiation of human erythrocytes.
Engineered erythrocytes generated by sortase-mediated modification (“sortagging”) of erythroid membrane proteins.
For several reasons red blood cells are an attractive vehicle for delivering proteins and small molecules and for targeting to specific tissues: They lack a nucleus, and therefore, there will no concerns about remnants of genes that could alter normal cell physiology, and they have a lifespan of around 120 days in the blood stream. In collaboration with Dr. Lenka Kundrat in Prof. Hidde Ploegh’s laboratory at the Whitehead Institute, Jiahai Shi and Sherry Lee are generating specific types of genetically engineered erythrocytes (eRBCs) in culture, and will use the sortase technique to covalently link proteins or small molecules to these eRBCs which then can be used for multiple types of applications. The sortase technique for cell and protein engineering was developed in the Ploegh laboratory. Sortases are transpeptidases derived from bacteria – they are involved in cell wall biogenesis – that normally link together two proteins that contain at their ends particular short amino acid sequences. While the sortases from different bacteria recognize different sequences, the most commonly used sortase cleaves between the T and G residues in the flexible motif: LPXTG, generating a thioacyl enzyme intermediate at the C-terminus of the threonine residue. This is resolved by nucleophilic attack in a reaction that involves an N- terminal glycine-initiated peptide or probe, (G)n-Y, where n =1 - 5. Therefore, the sortase-based approach (sortagging) covalently links the peptide-based motifs, X-LPXTG and (G)n-Y, resulting in a new structure: X-LPXT(G)n-Y, where X or Y could be any protein, peptide, high molecular weight polymer, or small molecule. There are many ways to use these sortase- and genetically modified erythrocytes produced in culture; initially we will focus on linking fibrinolytic proteins to red cells that could be used therapeutically. The engineered red cells with fibrinolytic protein, like plasminogen activators (PA), should have a long half-life in the circulation. These cells will aggregate around a nascent blood clot, as would normal erythrocytes, and increase the local PA concentration to dissolve the blood clot. This therapeutic should be very useful in preventing thrombosis in patients having a high risk for life-threatening thrombosis like coronary thrombosis. Importantly, there are many other potential uses that we will explore as time permits, including novel therapeutics, stabilizing otherwise unstable red cells, novel immune modulators and vaccines, and novel imaging modalities.
Transcriptional control of gene expression during terminal erythroid differentiation
Shilpa Hattangadi’s project involves determining the transcriptional regulatory networks governing the important changes in gene expression that occur during terminal proliferation and differentiation of erythroid precursors. She began by profiling comprehensive mRNA expression patterns during erythroid differentiation: isolation of mRNA from purified erythroid precursors in successive differentiation stages followed by hybridization to DNA microarrays and confirmation of expression of selected genes by qRT-PCR. She then went on to perform chromatin immunoprecipitation with antibodies specific for various erythroid- important transcription factors (ChIP), followed by hybridization of the recovered DNA to a promoter DNA microarray (ChIP-chip). In collaboration with members of Rick Young’s laboratory, she sequenced the resulting DNA fragments (ChIP-Seq). This protocol enables her to determine all of the genes that have critical erythroid-important transcription factors bound to their promoter/ enhancer segments. Initial studies focused on transcriptional activation by Stat5, Klf1, and Foxo3, but other factors will soon be investigated. Shilpa's long-term goal is to understand how the complex pattern of gene expression during terminal erythroid differentiation is regulated by transcription factors that are initially activated by signal transduction pathways downstream of the EpoR, but continue to remain active in precursors no longer dependent on erythropoietin.
In collaboration with Bill Wong, this expression profiling was confirmed and expanded by second-generation high throughput sequencing (RNA-seq). Their results indicate that major changes in gene regulation occur during early erythroblast differentiation, concomitant with induction of Ter119 (an erythroid-specific glycophorin), globin mRNAs, and other proteins involved in hemoglobin production. Many upregulated genes fall into expected categories such as those involved in hemoglobin metabolism, heme and porphyrin ring metabolism, cell and nuclear membrane structure, iron homeostasis, negative regulators of cell cycle, oxygen transport, and metabolism of oxygen and reactive oxygen species. Genes that were significantly downregulated included those involved in TNFα production, NADP metabolism, NF-kappaB binding, actin binding, ubiquitin protein ligation, and non-erythroid specific functions such as immune responses and phagocytosis.
Gene induction and repression during terminal erythropoiesis are mediated by distinct epigenetic changes
Chromatin modifications, such as histone modifications, are critical to maintain a stable pattern of either gene activation or repression in cell fate specification and terminal differentiation. Bill and Shilpa performed ChIP-seq on differentiated Ter119+ mouse fetal liver erythroblasts and their Ter119- progenitors, focusing on histone modifiers such as H3K4 di- and trimethylation, H3K9 and H4K16 acetylation, H3K27 trimethylation and also RNA polymerase II binding. Among genes induced during this erythroid developmental transition, such as Band3 and LMO2, there was an increase in the occupancy of Pol II, the activation marks H3K4me2, H3K4me3, H3K9Ac and H4K16Ac, and the elongation methylation mark H3K79me2. However, some of the highly transcribed genes such as ferritin and Jag1 are only marked with H3K4 methylation, but not acetylated. In contrast, genes that were repressed during differentiation showed relative decreases in H3K79me2 levels yet had levels of Pol II binding and active histone marks similar to those in erythroid progenitors. Bill and Shilpa discovered that in progenitors the active marks were present on both highly induced and highly repressed genes but increase significantly on induced genes before they are expressed, while repressive marks are present at relatively equal levels on repressed and induced genes. Even RNA Pol II was bound to promoters of repressed genes, but found to increase both at the promoter and along the gene body of induced genes, suggesting that proximal promoter pausing prevented elongation of repressed genes, as Pol II was still present at their promoters. They also found that relative changes in histone modification levels—in particular, H3K79me2 and H4K16ac—were most predictive of gene expression patterns. Their results suggest that in terminal erythropoiesis both promoter and elongation-associated marks contribute to the induction of erythroid genes, while gene repression is marked by changes in histone modifications mediating Pol II elongation. Their data maps the epigenetic landscape of terminal erythropoiesis and suggests that control of transcription elongation regulates gene expression during terminal erythroid differentiation.Chromatin condensation and enucleation in late stage erythroblasts
Mammalian erythroid cells undergo enucleation during a late stage of differentiation, a process that does not occur in other vertebrates. This process has critical physiological and evolutional significance for the morphogenesis and hemoglobin enrichment of mature mammalian red blood cells. Although enucleation has been known for decades, the mechanisms that regulate this process remain obscure. Peng Ji began studying enucleation in our lab, and identified key roles for Rac GTPase and fir the formin (actin nucleating protein) mDia2 in the final step of erythroblast enucleation – the formation of the contractile actin ring on the plasma membrane of late-stage erythroblasts at the boundary between the cytoplasm and nucleus of enucleating cells.
In collaboration with Tzutzuy Ramirez and Junxia Wang, fellows with Maki Murata Hori of the Temasek Life Sciences Laboratory, Singapore, Peng investigated the the roles of many cytoskeletal and other proteins in nuclear migration and enucleation of these cells, in part using video microscopy of cells expressing fluorescent- tagged proteins. Initial results show that, unlike conventional cytokinesis, the nucleus is squeezed out by formation of a bleb-like protrusion from a limited area of the erythroblast cell cortex; the bleb increases in size by dynamic contractions of asymmetrically distributed actomyosin. Importantly, they showed that enucleation requires establishment of cell polarization that is regulated by microtubule-dependent local activation of phosphoinositide 3-kinase (PI(3)K), displacing the nucleus to one side of the cell, and restricting actin to the other side. Dynamic actin- mediated cytoplasmic contractions generate pressure that pushes the viscoelastic nucleus through a narrow constriction in the cell surface, forming a bud. The PI3K products PtdIns(3,4)P2 and PtdIns(3,4,5)P3 are highly localized at the cytoplasmic side of the plasma membrane. PI3K inhibition caused impaired cell polarization, leading to a severe delay in enucleation. Also depolymerization of microtubules reduced PI3K activity, resulting in impaired cell polarization and enucleation.
Peng also focused on the role of histone deacetylases (HDACs) in chromatin and nuclear condensation and enucleation of late erythroid cells. He showed that inhibition of HDACs by Trichostatin A (TSA) or Valproic acid (VPA) prior to the start of enucleation blocks chromatin condensation, contractile actin ring formation, and enucleation. He further demonstrated that HDAC1, HDAC2, HDAC3 and HDAC5 are highly expressed in mouse fetal erythroblasts. shRNA down-regulation of HDAC2, but not the other HDACs, phenotypically mimicked TSA and VPA treated cells with significant inhibition of chromatin condensation and enucleation. Importantly, knockdown of HDAC2 does not affect erythroblast proliferation, differentiation, or apoptosis. These results identify HDAC2 as an important regulator in mediating chromatin condensation and enucleation in the final stages of mammalian erythropoiesis. Peng is continuing to work on these and related projects in his new position as Assistant Professor of Pathology at the Northwestern University Medical School.
A nuclear export protein essential for enucleation
Recently, along with her undergraduate student, Austin Gromatzky, Shilpa Hattangadi has begun to uncover the function of an unusual regulator of erythroid chromatin condensation and enucleation, the nuclear export protein, Xpo7. Xpo7 is highly erythroid specific and induced markedly during terminal differentiation; its expression is regulated by some of the master erythroid transcriptional regulators. It is unusual for a nuclear export protein in that it does not require a specific nuclear export signal, as do all other exportins.. Interestingly, except for Xpo7 all other nuclear exportins transcripts are repressed during terminal erythropoiesis. Shilpa studied the function of Xpo7 by shRNA knockdown and discovered that erythroblast nuclei from Xpo7-kd cells were less condensed and larger than control nuclei, as judged by confocal immunofluorescence microscopy. Enucleation was blocked, and Xpo7-KD nuclei retained almost all nuclear proteins while normal extruded nuclei had very little protein, as judged both by silver stained gels and mass spectrometry. This suggested that Xpo7 is a nonspecific nuclear export protein that removes all nuclear proteins from the erythroid nucleus in order to allow chromatin to condense. Along with Austin, she is using immunoprecipitation and yeast-2-hybrid methods to uncover the erythroid-specific cargos of Xpo7. She will continue her work as Assistant Professor in the Departments of Pediatrics and Pathology at Yale University School of Medicine beginning in the fall of 2012
The pre-mRNA splicing factor Muscleblind-like 1(Mbnl1) is required for terminal erythroid differentiation
The scope and role of regulated exon use in pre-mRNAs during erythroid development is poorly understood. Using their mRNA- seq data sets from erythroid progenitors and mature Ter-119+ erythroblasts, Bill Wing, working with Albert W. Cheng in Prof. Chris Burge’s lab and UROPs Katherine Luo and Paula Trepman, identified hundreds of differentiation-associated isoform changes during terminal erythropoiesis. During differentiation there were major changes in eight classes of alternative isoform expression events involving alternative splicing, alternative 3’ end cleavage and polyadenylation, and/or alternative promoter usage; these included skipped exons (SE), mutually exclusive exons (MXEs), alternative 5′ and 3′ splice sites (A5SS and A3SS), alternative first exons (AFE), alternative last exons (ALE), tandem 3′ untranslated regions (tandem 3′ UTRs), and retained introns (RI). They focused on alternative exon use during differentiation; many of these changes in usage coincided with induction of ~400 erythroid-important genes as well as repression of about 6000 early- stage genes, suggesting that both large-scale transcriptional and post-transcriptional programs are critical to ensure proper erythroid differentiation.
Segments in pre mRNAs surrounding regulated exons were enriched in motifs corresponding to the splicing factor, muscleblind-like1 (Mbnl1). Knockdown of Mbnl1 in cultured murine fetal liver erythroid progenitors resulted in a strong block in erythroid differentiation and disrupted the developmentally regulated exon skipping of several pre mRNAs, including Ndel1 mRNA, which they showed is a direct target of Mbnl1 and required for enucleation. These findings reveal an unanticipated scope of the alternative splicing program and the importance of Mbnl1 during erythroid differentiation.
The Genetics of Human Erythropoiesis
In work being performed with Eric Lander and colleagues at the Broad Institute, Vijay Sankaran along with laboratory colleagues Leif Ludwig and Jenn Eng are 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. Additionally, in close collaboration with laboratory colleague Sherry Lee, this group is using the insight from such approaches to refine current culture conditions of human erythroid cells.
Elevated levels of fetal hemoglobin can ameliorate the major disorders of beta-hemoglobin, sickle cell disease and beta-thalassemia. Vijay and his colleagues had 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, Leif and Vijay are defining the physiological function of these microRNAs and their targets using a variety of approaches in primary mouse and human erythroid progenitor cells. Ongoing work is aimed at understanding the mechanistic basis for alterations in hemoglobin expression in the context of other rare human syndromes and clinical conditions.
Using complex trait genetics, Vijay, Leif, and Jenn have 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, Vijay and Leif 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 human and mouse primary erythroid 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.
Leif, Vijay, and Jenn are going on to use an integrative genomic approach using a combination of GWAS results, RNA profiling from erythroid cells, chromatin modification mapping, and RNAi screening to more globally define new regulators of erythropoiesis. The studies involve close collaborations with several groups in Boston and Cambridge, as well as internationally.
Finally, to gain further insight into important regulators of erythropoiesis, Vijay, Leif, and Jenn have 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, Vijay and close collaborator Hanna Gazda recently defined the first non-ribosomal protein gene involved in Diamond-Blackfan anemia, GATA1. Ongoing studies are defining the mechanisms by which this and other mutations affect human erythropoiesis. Additionally Vijay, Jenn, along with laboratory colleague Sherry Lee are taking advantage of insights from common and rare human mutations to refine in vitro culture conditions of human erythroid cells. This research promises to refine the current approaches that may yield more effective strategies for producing erythrocytes in vitro from human hematopoietic progenitor, induced pluripotency, and embryonic stem cells.
| B. microRNAs (miRs) and long non-coding RNAs (lincRNAs) that regulate hematopoiesis |
Introduction
MicroRNAs (miRNAs) are small endogenous ~22-nt non-coding RNAs that base pair to sites within target mRNAs, triggering either a block in translation or mRNA degradation or both. The expression of miRNAs is often tissue-specific or developmental-specific. As shown by the Bartel laboratory and others, humans have several hundred genes that encode miRNAs, an abundance corresponding to almost three percent of protein-coding genes; computational and experimental analyses suggest that miRNAs may regulate expression of ~30% of human and mouse genes. Based on the evolutionary conservation of many miRNAs among different animal lineages, it is reasonable to suspect that some mammalian miRNAs have important conserved functions in cellular development and function. Indeed, the post-transcriptional programs controlled by specific miRNAs affect diverse biological processes, including development, cell differentiation, apoptosis, immune responses, metabolism and many diseases including various cancers, cardiovascular disease, viral infection and neurodegenerative diseases. Long non-coding RNAs (lncRNAs), transcripts longer than 200nt, constitute a significant fraction of the mammalian transcriptome. While many lincRNAs are differentially expressed under both normal and pathological conditions, the biological functions of most of these transcripts still remain uncharacterized.
Long non-coding RNAs (lncRNAs) are transcripts longer than 200nt that do not encode proteins. Many are capped, polyadenylated and often spliced, and presumably transcribed by RNA Polymerase 2 (Pol2). LncRNAs constitute a significant fraction of the mammalian transcriptome. Compared to mRNAs, lncRNAs tend to be shorter and less well conserved at the primary sequence level. Expression of lncRNAs is often restricted to specific tissues and developmental stages, suggesting that many may regulate cell fate specification .A few dozen intergenic lncRNAs (lincRNAs) have been functionally characterized in mammals, and they have been associated with important developmental processes such as apoptosis, proliferation and lineage commitment However, the biological functions of most of these genes and their potential roles in disease still remain uncharacterized.
An erythroid-specific long non-coding RNA prevents apoptosis of erythroid progenitors and promotes terminal proliferation.
Erythropoiesis is regulated at multiple levels by different factors to ensure the proper generation of red blood cells in response to various physiological and pathological stimuli. Although the regulation of erythropoiesis by transcription factors and microRNAs is becoming well understood, the modulation of red blood cell development by lncRNAs is still unknown. LncRNAs can regulate gene expression via multiple mechanisms and many lncRNAs are differentially expressed in many developmental and pathological processes, suggesting that they play important biological roles.
Wenqian Hu identified one erythroid-specific lncRNA, LincRNA-EPS, with potent anti-apoptotic activity. Expression of LincRNA-EPS is largely confined to terminally differentiating erythroid cells and its expression is induced in CFU-E progenitors by Epo. Inhibition of this lncRNA blocks erythroid differentiation and promotes apoptosis. Ectopic expression of this lncRNA in CFU-E progenitors prevents erythroid progenitor cells from the apoptosis that is normally induced by erythropoietin deprivation. This lncRNA represses expression of several proapoptotic genes including the one encoding Pycard, an activator of caspases, explaining in part the inhibition of programmed cell death. These findings reveal a novel layer of regulation of cell differentiation and apoptosis by a lncRNA. Currently Wenqian with Juan R. Alvarez-Dominguez are identifying and cloning the human LincRNA-EPS ortholog and characterizing its putative antiapoptotic functions.
To obtain a comprehensive view of how lncRNAs contribute to erythropoiesis, Wenqian Hu, performed high depth RNA-sequencing on both Poly(A)+ RNAs and Poly(A)- RNAs from erythroid progenitor cells and terminal differentiating erythroblasts. Using computational methods, Juan R. Alvarez-Dominguez, Wenqian Hu, and Bingbing Yuan are cataloguing, annotating, and characterizing the polyadenylated and non-polyadenylated lncRNAs identified through this approach. Also, in collaboration with Carlos Gonzalez, a Visiting Professor from the University of Puerto Rico and his student Jose Gonzalez, Wenqian Hu and Juan R. Alvarez-Dominguez are performing loss-of-function and gain-of-function studies on some of the identified lncRNAs in terminal proliferation and erythroid differentiation.
MicroRNAs that modulate erythropoiesis
Lingbo Zhang is interested in microRNA -mediated regulation of erythropoiesis. Currently, one aspect of his research focuses on the identification and functional characterization of functionally important microRNAs that regulate erythroid terminal differentiation, including enucleation. Using Johan Flygare’s RNA-seq deep sequencing data, he found that the majority of microRNAs present in CFU-E erythroid progenitors are downregulated during terminal erythroid differentiation. Taking advantage of our in vitro erythrocyte progenitor culture and differentiation system, Lingbo used retrovirus infection to overexpress many erythroid lineage – enriched microRNAs in mouse fetal liver erythroid progenitors, followed by FACS analysis after two days of culture.
Of the predominant developmentally down-regulated miRNAs, ectopic overexpression only of miR-191 blocked erythroid enucleation but had minor effects on proliferation or erythroid differentiation. Lingbo further identified two developmentally upregulated genes, Riok3 and Mxi1, as direct targets of miR-191. More importantly, he found that the upregulation of Riok3 and Mxi1 are required for chromatin condensation and enucleation. Either overexpression of miR-191 or knockdown of Riok3 or Mxi1 impaired the normal downregulation of histone acetyltransferase Gcn5 (whose downregulation is required for histone deacetylation and chromatin condensation). Thus normal down-regulation of miR-191 is essential for erythroid chromatin condensation and enucleation by allowing up-regulation of Riok3 and Mxi1 and downregulation of Gcn5. Since our understanding of erythropoiesis regulation is still limited, this is a good example to illustrate how we may be able to uncover novel protein coding genes regulating erythropoiesis through identification of microRNA target genes. In all, these discoveries will shed light on post-transcriptional regulation of erythropoiesis.
miR-125b, a microRNA important for regulation of the p53 pathway
Minh Le, a former graduate student working jointly with our lab and that of Bing Lim in the Genome Institute of Singapore, has elucidated the role of miR-125b in neurogenesis. miR-125 is a homolog of lin-4, which is important for developmental timing in C. elegans. The expression of miR-125b is upregulated during embryogenesis and enriched in the nervous system of vertebrate species. Furthermore, Minh, together with Shyh-Chang Ng and Cathleen The, demonstrated that miR-125b is indispensable for zebra fish embryogenesis, particularly for the survival of neural cells during development. She identified p53, a key tumor suppressor, as a bona fide target of miR-125b in both zebra fish and humans. miR-125b-mediated downregulation of p53 is strictly dependent on the binding of miR-125b to a microRNA-response element in the 3’ UTR of p53 mRNA. Overexpression of miR-125b represses the endogenous level of p53 protein and suppresses apoptosis in human neuroblastoma cells and human lung fibroblast cells. By contrast, knockdown of miR-125b elevates the level of p53 protein and induces apoptosis in human lung fibroblasts and in the zebra fish brain. In zebra fish this phenotype can be rescued significantly by ablation of endogenous p53 function.
Minh and Shyh-Chang then used both gain- and loss-of-function screens for miR-125b targets in humans, mice and zebra fish, and validated these targets with the luciferase assay and a novel miRNA pull-down assay. They demonstrated that miR-125b directly represses 20 novel targets in the p53 network. These targets include both apoptosis regulators like Bak1, Igfbp3, Itch, Puma, Prkra, Tp53inp1, Tp53, Zac1,and also cell-cycle regulators like cyclin C, Cdc25c, Cdkn2c, Edn1, Ppp1ca, Sel1l,in the p53 network. They found that although each miRNA-target pair was seldom conserved, miR-125b regulation of the p53 pathway is conserved at the network-level. Their results led us to propose that miR-125b buffers and fine-tunes p53 network activity by regulating the dose of both proliferative and apoptotic regulators, with implications for tissue stem cell homeostasis and oncogenesis.
miR-125b modulates myeloid development and causes lukemias
Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are frequently associated with chromosomal translocations; most involve oncogenes or transcription factors that are up regulated or that form part of chimeric genes. The t(2;11)(p21;q23) translocation is observed in cases of MDS and AML and in her previous laboratory in Toulouse Marina Bousquet showed that this translocation triggers upregulation of miR-125b. This was the first description of microRNA deregulation by a chromosomal translocation, and implied that AML and MDS carrying the t(2;11) translocation represent a new clinico-pathological entity. Since lin-4, the miR-125b ortholog in Caenorhaditis elegans, isimplicated in several developmental process, she hypothesized that deregulation of miR-125b expression would impair human and mouse haematopoiesis.
Marina used a retroviral construct encoding miR-125b to infect enriched hematopoietic stem/ progenitor cell population, which were injected into lethally irradiated recipient mice. At 16 weeks all mice transplanted with fetal liver cells ectopically expressing miR-125b showed an increase in white blood cell count, in particular neutrophils and monocytes, associated with a macrocytic anemia, suggesting an important role for miR-125b early in hematopoiesis. Among these mice, half died within 12 to 29 weeks post-transplantation of B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, or a myeloproliferative neoplasm. Furthermore, co-expression of miR-125b and the BCR-ABL fusion oncogene in transplanted cells accelerated the development of leukemia, compared to control mice expressing only BCR-ABL, suggesting that miR-125b confers a proliferative advantage to the leukemic cells. Thus, she showed that overexpression of miR-125b was sufficient both to shorten the latency of BCR-ABL–induced leukemia and to independently induce leukemia in a mouse model.
Marina next showed that miR-125b blocks myeloid differentiation and transforms myeloid cell lines by repressing multiple mRNA targets. To investigate the consequences of miR-125b overexpression on myeloid differentiation, apoptosis and proliferation, she used the NB4 and HL60 human promyelocytic cell lines and the 32Dclone3 murine promyelocytic cell line; she showed that miR-125b overexpression blocks apoptosis, myeloid differentiation and enhances proliferation in both species. To test whether miR-125b is able to transform myeloid cells, she used the non-tumorigenic and IL-3 dependent 32Dclone3 cell line overexpressing miR-125b, in xenograft experiments in Nude mice and in IL-3 deprivation conditions. Importantly, miR-125b was able to transform the 32Dclone3 cell line by conferring growth independence to IL-3; xenograft experiments showed that these cells form tumors in Nude mice. To identify new miR-125b targets, she compared the transcriptome of cell lines overexpressing or not miR-125b by RNA-sequencing. Using RNA-sequencing and RQ-PCR experiment, she identified multiple miR-125b targets. ABTB1, an anti-proliferative factor, was one new direct target of miR-125b and she confirmed that CBFB, a transcription factor involved in hematopoiesis, is also targeted by miR-125b. In these cells MiR-125b restricts apoptosis by down regulating genes involved in the p53 pathway including BAK1 and TP53INP1. This study demonstrates that in a myeloid context, miR-125b is an oncomiR. miR-125b blocks myeloid differentiation in part by targeting CBFB, blocks apoptosis through downregulation of multiple genes involved in the p53 pathway, and confers a proliferative advantage to human and mouse myeloid cell lines in part by targeting ABTB1.
Dr. Marina Bousquet is continuing this work in the Cancer Research Center of Toulouse, France, focusing on how overexpression of miR-125b causes a macrocytic anemia, promotes proliferation of myeloid cells, and blocks granulocyte and monocyte differentiation.
As noted above, recent work from our lab indicates that mir-125b is a novel bona fide negative regulator of p53 in human and zebra fish. One hypothesis is that miR-125b downregulation of p53 in some specific hematopoietic cell facilitates development of leukemic cells. However, p53 is not a conserved target among all vertebrate species and in particular the binding site for miR-125b is not conserved in mouse p53 mRNA. We hypothesize that even if the miR-125b binding site is not conserved in mouse p53, the p53 pathway is regulated by miR-125b in both human and mouse.
Introduction
Hematopoietic stem cells (HSCs) are defined by their ability to self-renew and to differentiate into all blood cell types – erythroid, myeloid, and lymphoid cells. These very rare cells – about 1:10,000 in fetal liver and bone marrow - form the basis of bone marrow transplantation for treatment of leukemia and other cancers, and are also a promising cell target for developing gene therapies for treating a broad variety of human diseases. However, development of these important clinical applications of HSCs are greatly hampered by the lack of understanding of the extracellular and intracellular signals that govern their fates and the difficulty in ex vivo expansion of these cells. We quantitate these cells by bone marrow transplantation, monitoring long- term repopulation of the hematopoietic compartment of lethally irradiated mice. This assay requires several months to complete.
Supportive stromal cells for hematopoietic stem cells
Hematopoietic stem cell environments or niches are very important in determination of HSC self-renewal and differentiation; fibroblasts, endothelial cells, and osteoblasts have been postulated as important constituents and regulators of HSC niches in the bone marrow. Song Chou is characterizing the stromal cells that support HSC expansion in fetal liver. Because fetal liver contains various types of cells at different developmental stages, he developed a novel strategy to enrich the potential stromal cells for HSC expansion. SCF is fabricated as a transmembrane plasma membrane protein that normally binds to its receptor, c-Kit, on the surface of adjacent cells. Since all HSCs in fetal liver express c-Kit, these stromal cells may be located in close proximity to HSCs and interact with HSCs through SCF. In addition, SCF is also essential for HSC expansion ex vivo. Song purified SCF+ stromal cells from fetal liver. He showed that these also express high levels of DLK, another membrane-bound cytokine that is involved in the maintenance and self-renewal of HSCs. Using flow cytometry, he showed that ~1-2% of total fetal liver cells are SCF+DLK+ and that the vast majority of SCF+ cells are also DLK+. He purified SCF+DLK+ cells by flow cytometry and found that the mRNA levels not only of SCF, but also of several other HSC expansion factors, including IGF2, Angptl3 and TPO, are highly enriched in these cells relative to SCF-DLK+ and SCF-DLK- cells. Furthermore, these SCF+DLK+ cells are highly enriched for expression of CXCL12, a chemo-attractant for HSCs. CXCL12 is secreted by stromal cells in bone marrow and regulates trafficking of HSCs. Thus SCF+DLK1+ cells are the principal cells in fetal liver that synthesize several cytokines that support HSC maintenance, expansion, and trafficking.
DLK has been characterized as a specific marker for fetal hepatic stem and progenitors, and thus it is likely that the SCF+DLK+ stromal cells are actually hepatic cells. Song discovered that SCF+DLK+ cells also highly enriched for Albumin (ALB) and alpha-fetoprotein (AFP) mRNAs, two specific markers for hepatic progenitors in fetal liver relative, to SCF-DLK+ and SCF-DLK- cells, suggesting these SCF+DLK+ cells are indeed hepatic progenitors.
To examine the homogeneity of these SCF+DLK+ cells and to confirm these cells are indeed hepatic cells, Song performed immunocytochemistry experiments with total fetal liver cells. By staining the fetal liver cells with antibodies against SCF and ALB simultaneously, Song discovered that the vast majority (>93%) of cells positive for SCF are also positive for ALB, proving that the SCF+ stromal cells are indeed hepatic cells. Similarly, more than 93% of SCF+ cells are also DLK+ and Angptl3+. Thus the SCF+DLK+ cells are a highly homogenous population expressing markers for hepatic cells as well as factors for HSC expansion. Interestingly, only 34% of SCF+ cells are positive for CXCL12. However about 80% of CXCL12+ cells in fetal liver are SCF+, indicating that CXCL12+ cells form a large a subpopulation of SCF+DLK+ cells. These CXCL12+ SCF+DLK+ cells might have greater chance of establishing close cell-cell contacts with HSCs and thus stimulating their expansion. Song is currently trying to identify other novel signaling molecules secreted by these SCF+DLK+ stromal cells that support HSC expansion.
A co-culture system that can expand HSCs
The most direct way to prove that fetal hepatic progenitors are bona fide supportive cells for HSC expansion is to establish a co-culture assay that expands HSCs ex vivo. Initially Song co-cultured sorted SCF+DLK+ cells with SLAM+ (CD150+CD48-CD41-) fetal liver HSCs for five days. Although SCF+DLK+ cells were able to maintain fetal liver HSCs numbers in short-term ex vivo culture, as judged by transplantation experiments, there was no net expansion of HSCs. Thus to increase the numbers of and the survival rate of hepatic progenitors, Song used magnetic beads to purify DLK+ cells from the fetal liver. Song chose to culture DLK+ cells in serum-containing medium supplemented with 50 ng/ml SCF, 20 ng/ml TPO, and 50 ng/ml FLT3L (STF medium) to support expansion of HSCs. Sorted SLAM+ bone marrow HSCs from CD45.1 mice were co-cultured with DLK+ cells from CD45.2 mice with conditioned medium from stromal cells for one week, HSCs co-cultured with DLK+ cells showed a clear increase of donor derived peripheral nucleated blood cells relative to uncultured SLAM cells at both one month and four months after transplantation, indicating significant expansion of HSCs.
To examine whether HSCs can be expanded by DLK+ cells beyond a one-week culture, Song extended the co-culture experiment to three weeks; At the end of week three, one SLAM+ cell co-cultured with DLK+ stromal cells produced nearly three million progeny - an over 200 fold increase over HSCs cultured with cytokines alone and ~ 100 fold higher than those cultured in conditioned medium. Co-culture with DLK+ cells for three weeks resulted in a minimum of a 20-fold increase in HSC numbers. Direct cell-cell contact is required for HSC expansion in these long term cultures, and a similar HSC expansion (~10 fold) was achieved in co-cultures using a serum-free, low cytokine- containing medium. Song showed that HSCs expanded in such culture condition maintain their long-term repopulating ability and can reconstitute all blood lineages equally. This result establishes that long-term co-culture with fetal hepatic progenitors is capable of expanding bona fide long-term self-renewing HSCs.
In summary, this is the first time that co-culture with either cell lines or primary cultured cells showed a significant expansion of HSCs. This co-culture system closely mimics HSC and progenitor expansion in the fetal liver in vivo and is an excellent model system to study HSC expansion ex vivo.
Adiponectin and its paralogs:
In 1995 we cloned adiponectin, originally called Acrp30, as a novel adipocyte- specific secreted protein hormone. Adiponectin addition potently elevates fat and glucose catabolism by muscle, enhances glycogen accumulation in muscle, and inhibits gluconeogenesis in liver. Mutations in the adiponectin gene are linked to development of adult- onset diabetes and the levels of adiponectin in serum are reduced in obese and diabetic patients and mice. Circulating adiponectin levels negatively correlate with human plasma triglyceride and fasting insulin levels and several clinical studies showed that persons with low adiponectin levels are more likely to develop type II diabetes mellitus and cardiovascular disease. This data suggests that adiponectin is a potential determinant of insulin sensitivity.
Adiponectin has four domains: a cleaved amino-terminal signal sequence, a region without homology to known proteins, a collagen-like region, and a globular segment at the carboxy-terminus. The three-dimensional structure of the globular domain is strikingly similar to that of TNFα even though there is no homology at the primary sequence level. Like TNFα the globular domain forms homotrimers, and intermolecular disulfide bonds generate hexameric and high molecular weight Adiponectin species.
In collaboration with the Ruderman laboratory at B. U. Medical School, we showed several years ago that treatment of rat striated muscle with trimeric adiponectin led to phosphorylation and activation of AMP-activated protein kinase (AMPK), an enzyme that when activated causes increases in muscle fatty acid oxidation, glucose uptake and oxidation, and insulin sensitivity. Adiponectin- mediated activation of AMPK caused phosphorylation and thus diminished activity of acetyl CoA carboxylase, a corresponding decrease in the concentration of malonyl CoA, and a corresponding increase in long- chain fatty acid oxidation. In addition, adiponectin caused an increase in glucose uptake by muscle. Both in vivo and in muscle culture adiponectin most likely exerts its actions on muscle fatty acid oxidation by inactivating ACC, via activation of AMPK and perhaps other signal transduction proteins.
Activation of AMP kinase by adiponectin and insulin
Adiponectin activates AMP-activated protein kinase (AMPK) in adipocytes but the underlying mechanism remained unclear. Qingqing Liu tested the hypothesis that AMP, generated in activating fatty acids to their CoA derivatives in a reaction catalyzed by acyl-CoA synthetases, is involved in AMPK activation by adiponectin. To this end she measured the AMP/ATP ratio and AMPK activation upon adiponectin and insulin stimulation, and after knocking down acyl-CoA synthetases in adipocytes. She confirmed that adiponectin activation of AMPK is accompanied by a ~ 2-fold increase in the cellular AMP/ATP ratio. Moreover, FATP1 and Acsl1, the two major acyl-CoA synthetase isoforms in adipocytes, are essential for AMPK activation by adiponectin. Qingqing also showed that after 40 min. insulin activated AMPK in adipocytes, which was coupled with a 5-fold increase in the cellular AMP/ATP ratio. Knockdown studies show that FATP1 and Acsl1 are required for these processes, as well as for stimulation of long chain fatty acid uptake by adiponectin and insulin. These studies demonstrate that a change in cellular energy state is associated with AMPK activation by both adiponectin and insulin, which requires the activity of FATP1 and Acsl1.
Adiponectin regulates expression of hepatic genes critical for glucose and lipid metabolism
Qingqing’s current project is focused on the transcriptional regulation of global glucose and lipid metabolism by adiponectin; she wants to determine the important signaling and metabolic pathways affected by the deficiency of adiponectin and, more particularly, to identify the specific transcription factors involved in these regulations. To this end she used second- generation technologies to sequence total mRNA isolated from the liver of adiponectin knockout mice fed a chow diet and compared the expression profiles with those of wild-type mice. She confirmed the changes in expression of critical genes by real-time PCR. Compared to WT mice, adiponectin KO mice exhibited decreased mRNA expression of rate-limiting enzymes in several important glucose and lipid metabolic pathways including glycolysis, TCA cycle, fatty-acid activation and synthesis, triglyceride synthesis and cholesterol synthesis. In addition, binding of the transcription factor Hnf4α to DNAs encoding several key metabolic enzymes was reduced in KO mice, suggesting that adiponectin might regulate hepatic gene expression via Hnf4α. Phenotypically, adiponectin KO mice possessed smaller epididymal fat pads and showed reduced body weights comparing to WT mice. When fed a high fat diet, adiponectin KO mice showed significantly reduced lipid accumulation in the livers. These lipogenic defects are consistent with the downregulation of lipogenic genes in the KO mice.
Comprehensive analysis of four in vitro insulin resistance models and their physiological relevance to adipose insulin resistance resulting from diet-induced obesity
Kin Yui Alice Lo is a joint graduate student also in Ernest Fraenkel's lab in the Biological Engineering Department at MIT. She is adopting a systems approach to understand multiple forms of insulin resistance in adipocytes at the transcriptional level due to various physiological and pathological insults. Adipose insulin resistance can be induced in cultured adipocytes by a variety of treatments, including TNFα, hypoxia, the corticosteroid dexamethasone, and high insulin, but it is unknown what aspects of in vivo adipose insulin resistance are captured by the different in vitro models. She used global RNA-sequencing (RNA-Seq) to investigate the gene expression changes of these four different in vitro insulin resistance models and she analyzed these changes in parallel with those from three independent microarray studies, done by others, of white adipose tissue from diet-induced obese (DIO) mice, examining the transcriptional effects on a genome-wide level and with a focus on a set of 1,319 adipogenesis-related genes. She confirmed a previous observation made by a former post-doc, Hong Ruan, that TNFα induced insulin resistance by suppressing many adipocyte important genes (eg Pparγ, Adiponectin) and inducing many preadipocyte genes. Importantly, she observed that while no in vitro model could faithfully mimic all the transcriptional changes occurring in DIO mouse adipose tissue, both TNFα and hypoxia treatments capture the downregulation of various metabolic pathways, such as glucose, lipid, and branched-chain amino acid metabolic pathways. Using genome-wide DNAse I hypersensitivity cum sequencing analysis (DHS-Seq), she further examined the transcriptional regulation of TNFα-induced insulin resistance. She discovered that, in addition to NF-κB, C/EBPβ is also likely to mediate the gene induction upon TNFα treatment. She concluded that different in vitro models capture different features of DIO adipose insulin resistance.
A novel kinase that mediates signaling by oxidative stress and in vivo insulin resistance
| E. miRs and lincRNAs that regulate differentiation and function of white and brown adipose cells. |
MicroRNAs in fat cell development and obesity
Lei Sun, Huangming Xie, and Ryan Alexander are 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 demonstrate that brown adipocytes arise in vivo from a Myf5-positive, bipotential myoblastic progenitor by the action of the Prdm16 (PR domain containing 16) transcription factor. 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, in adipogenic conditions, miR-193b induced myoblasts to differentiate into brown adipocytes. MiR-193b-365 was upregulated by Prdm16 partially through the action of the transcription factor PPARγ. Taken together, these results underlie the importance of tissue enriched miRNAs 193b-365 in regulating lineage specification between brown fat and muscle, and also suggest that these or other miRNAs may have therapeutic potential in inducing expression of brown fat-specific genes.
LincRNAs in fat cell development and function.
Many protein coding genes, mRNAs, and microRNAs have been implicated in regulating adipocyte development; however, the global expression patterns and functional contributions of long intergenic noncoding RNA (lincRNA) during adipogenesis have not been explored. Lei and Ryan, collaborating with John Rinn’s group at the Broad Institute, is examining the roles of large intergenic non-coding RNAs (lincRNAs) in adipogenesis. To begin, they have 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 transcription factors such as PPARγ and CEBPα. RNAi-mediated loss of function screens identified 9 functional lincRNAs with varying impact on adipogenesis. They further focused on an X-linked lincRNA required for proper adipogenesis; both the human and mouse orthologs of this lincRNA contain numerous copies of a conserved RNA sequence motif. Collectively, they have identified numerous lincRNAs that comprise a critical transcriptional regulatory layer that is functionally required for proper differentiation of both brown and white adipocytes.
Harvey F. Lodish, Ph.D.
Member, Whitehead Institute
Professor of Biology, MIT
Professor of Bioengineering, MIT
Phone: 617.258.5216
Fax: 617.258.6768
lodish@wi.mit.edu
last updated: 20 Aug. 2012