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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 proliferation and differentiation of early (BFU-E) and late (CFU-E) erythroid progenitor cells by extracellular signals including erythropoietin, glucocorticoids, and oxygen. 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. 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 does require 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. Johan Flygare hypothesizes that if the molecular pathways that induce SE are understood it will be possible to develop erythropoiesis stimulating agents that will complement or replace Epo treatment in anemic patients. 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. He first established two FACS-based methods to separate and purify BFU-E and CFU-E cells from mouse fetal liver. He demonstrated that GCs induce self-renewal of BFU-E cells, and not of CFU-E cells or erythroblasts. GCs thereby protect BFU-E cells from exhaustion, and in parallel increase the number of CFU-E cells formed from each BFU-E >10-fold. He further demonstrated that GCs do not inhibit erythropoietin-dependent terminal differentiation of freshly isolated erythroid CFU-E progenitors.
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 HIF1ƒÑ may enhance or replace the effect of glucocorticoids on BFU-E self-renewal. Indeed, HIF1ƒnƒÑ 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, hope to answer many questions by mapping exactly where in the chromatin the activated GCR binds by ChIP-Seq, which binding partners it has, and how transcription is repressed and/or activated at these sites in BFU-E cells. Further insight into the mechanism of GC stimulation of SE will come from ongoing work by an MIT undergraduate student Evelyn Wang and research associate Gregory Hyde, who are determining exactly which functional domains of the glucocorticoid receptor are necessary to stimulate SE; to this end they are expressing specific mutant forms of the GR in BFU-Es purified from GR knock-out mice and studying their subsequent proliferation and differentiation in culture.
Johan is currently establishing his own group at the Lund Stem Cell Center in Sweden. In Sweden he will continue to collaborate with David Root at the Broad institute in a 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 is working on identification of genes essential for glucocorticoid mediated BFU-E self-renewal. His current research focuses 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. In summary, 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.
Mechanistic study of activation of normal and pathogenic Janus kinase 2 and their association 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.
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 using 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). She has since, in collaboration with members of Rick Young’s laboratory, moved onto sequencing of 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 focus 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 activated initially by signal transduction pathways downstream of the EpoR, but active in precursors no longer dependent on erythropoietin.
The second tier of this transcriptional network was evaluated by comprehensive mRNA expression profiling during erythroid differentiation: by isolation of mRNA from purified erythroid precursors in successive differentiation stages followed by hybridization to DNA microarrays and eventual confirmation of expression of selected genes by qRT-PCR. In collaboration with Bill Wong, this expression profiling has been confirmed and expanded by second-generation high throughput sequencing (RNA-seq). The results indicate that major changes in gene regulation occur during early erythroblast differentiation, concomitant with induction of Ter119 expression, an erythroid-specific cell surface protein, globin mRNAs, and other factors involved in hemoglobin production. Upregulated genes include many 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 TNF-alpha production, NADP metabolism, NF-kappaB binding, actin binding, ubiquitin protein ligation, and non-erythroid specific functions such as immune responses and phagocytosis.
Along with her technical assistant, Karly Burke, Shilpa studied the effects of a specific kinase, Hipk2, which modulates the function of other transcription factors and cofactors and chromatin-modifying enzymes. Hipk2 is highly induced during primary mouse fetal liver erythropoiesis and specific knockdown of Hipk2 inhibits terminal erythroid cell proliferation – probably explained by cell cycle arrest as well as increased apoptosis – and terminal enucleation as well as the reduced accumulation of hemoglobin. Hipk2 knockdown reduces the expression of some genes involved in proliferation and apoptosis as well as important, erythroid-specific genes involved in hemoglobin biosynthesis, but does not affect the induction of several erythroid-specific transcription factors. This suggests that Hipk2 plays a significant role in terminal fetal liver erythroid differentiation and may regulate hemoglobin expression through noncanonical regulatory pathways.
Recently, along with her technical assistant Jennifer Eng, Shilpa has begun to uncover an unusual regulator of erythroid development, specifically chromatin condensation and enucleation, the nuclear export protein, Xpo7. Xpo7 is highly erythroid specific, abundant, and regulated by some of the master erythroid regulators. It is unusual for an export protein in that it does not require a specific nuclear export signal as do all other export proteins. Interestingly, all other nuclear export protein transcripts are repressed during terminal erythropoiesis except for Xpo7. 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 (by confocal immunofluorescence microscopy). Xpo7-KD nuclei also retained almost all nuclear proteins while normal extruded nuclei had very little protein, as judged both by silver stained gels and mass spectrometry, suggesting that perhaps Xpo7 is a nonspecific nuclear export protein that removes all nuclear protein from the erythroid nucleus in order to allow chromatin to condense.
mRNA-seq analysis accurately quantifies the absolute abundance of individual genes and also the fold changes at different developmental changes. Some of the abundantly expressed genes, in particular transcription factors such as GATA1, Sp2, FOG1 and LMO2, show less than 2 fold induction during erythroid differentiation, yet they are critical for erythropoiesis. Another class of highly expressed genes shows more than 10 fold induction during erythropoiesis, including hbb-b1, hbb-b2, Alas2, Band3, Darc, and Tmod1. Examination of abundant induced genes, which were not previously implicated in erythroid development, identified a number of novel stress hormone related receptors, transcription factors, serine/threonine kinases, non-coding RNAs and splice variants. Together two UROPs, Paula Trepman and Katherine Luo, Bill is determining the functions or many of these new proteins, focusing on the mechanism by which several splice variants are formed and the functions of the different isoforms of several of these erythroid- induced proteins. Their functions are being studied by knocking down their expression in fetal liver erythroid CFU-E progenitors using shRNAs and chemical inhibitors and examining erythroid proliferation and differentiation in culture.
Epigenetic modifications during erythroid development
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 Ter119+ mouse fetal liver cells focusing on histone modifiers such as H3K4 di- and trimethylation, H3K4 dimethylation, H3K9 and H4K16 acetylation, H3K27 trimethylation and also RNA polymerase II. H3K4 tri- and dimethylation, H3K9 and H4K16 acetylation, along with RNA polymerase II binding, are generally associated with actively expressed transcripts such as Band3 and LMO2. However, some of the highly transcribed genes such as ferritin and Jag1 are only marked with H3K4 methylation, but not acetylated. Another interesting class of ‘trivalent’ genes is marked by H3K4 and H3K27 trimethylation and also H3K9 acetylation. Bill and Shilpa discovered that 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. Only the level of the elongation mark, H3K79me2, was most correlated with the direction of expression of highly induced and highly repressed genes. Their recent publication reflects how histone modifications undergo dynamic changes during terminal erythroid differentiation even in rapidly condensing chromatin.
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 is investigating the mechanism of mammalian erythroid cell enucleation. This and many of our other studies on EpoR signal transduction make use of the system Jing Zhang developed several years ago; purified fetal liver erythroid progenitors (mainly CFU-Es) are plated on fibronectin- coated dishes and cultured in the presence of Epo; they undergo normal terminal proliferation, differentiation, and enucleation that can be followed on a single cell level by FACS.
Since actin filaments have been shown to be critical for enucleation, Peng determined the role of different Rho GTPases, the master regulators of actin nucleation, on enucleation. Peng showed that deregulation of Rac GTPase during the late stages of erythropoiesis completely blocked enucleation of cultured mouse fetal erythroblasts without affecting normal proliferation and differentiation. The contractile actin ring formed on the plasma membrane of late-stage erythroblasts at the boundary between the cytoplasm and nucleus of enucleating cells was disrupted when Rac GTPase was inhibited in late stages of erythropoiesis. Peng further demonstrated that Rac GTPase activity is mediated by the downstream target protein, mDia2, a formin required for nucleation of unbranched actin filaments. These results reveal important roles for Rac GTPase and mDia2 in enucleation of mammalian erythroblasts.
In collaboration with Tzutzuy Ramirez and Junxia Wang, fellows with Maki Murata Hori of the Temasek Life Sciences Laboratory, Singapore, and Senthil Jayapal, Peng is investigating 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, enucleation requires establishment of cell polarization that is regulated by microtubule-dependent local activation of phosphoinositide 3-kinase (PI(3)K). When the nucleus becomes displaced to one side of the cell, and actin becomes restricted to the other side. The PI(3)K products PtdIns(3,4)P2-PtdIns(3,4,5)P3 become highly localized at the cytoplasmic side of the plasma membrane. PI(3)K inhibition caused impaired cell polarization, leading to a severe delay in enucleation. Depolymerization of microtubules reduced PI(3)K activity, resulting in impaired cell polarization and enucleation. They propose that enucleation is regulated by microtubules and PI (3)K signaling in a manner mechanistically similar to directed cell locomotion.
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 further showed that mDia2 is acetylated in vivo and his current aim is to determine whether HDAC6 can deacetylate mDia2 and in so doing activate this formin and thus promote red cell enucleation. In parallel he is investigating the roles of HDACs in inactivating gene transcription and condensing chromatin prior to enucleation. At the same time, Peng is also interested in the roles of mDia1 and mDia2 on hematopoietic stem cell homing and migration. He is currently generating an mDia2 knockout mouse model that he will use to study the roles of mDia2 in hematopoiesis. He will continue to work on these projects in his new position as Assistant Professor of Pathology at the Northwestern University Medical School.
Myc, Chromatin condensation, histone acetylation, and enucleation in late stage erythroblasts Using the in-vitro erythroid culture system developed in the Lodish lab, Senthil Raja Jayapal, who is a joint graduate student in the Lodish lab and in the labs of Bing Lim at the Genome Institute of Singapore and Philipp Kaldis at Institute of Molecular and Cell Biology in Singapore, is investigating the role of cell cycle proteins and epigenetic modifications of histones in late stage erythroid maturation. In order to study the relationship between proliferation and differentiation programs during terminal erythroid maturation, he initially chose to focus on c-myc, which directs proliferation in many cell types and is down regulated during terminal differentiation when cells withdraw from cell cycle. The protein levels of c-myc are reduced dramatically during late stage erythroid maturation, coinciding with cell cycle arrest in G1-phase and enucleation, suggesting possible roles for c-myc in one or both of these processes. Surprisingly, ectopic c-myc expression had a dose dependent effect on terminal erythroid maturation. Ectopic expression of c-myc at physiological levels did not affect erythroid differentiation or cell cycle shutdown, but specifically blocked erythroid nuclear condensation and enucleation. When over-expressed at levels much higher than physiological, c-myc blocked erythroid differentiation completely and the cells continued to proliferate in culture with an early erythroblast morphology. These studies revealed important roles for c-myc in erythroid cells independent of its cell cycle regulatory functions.
Since histone deacetylation has been associated with erythroid nuclear condensation and enucleation, he compared the changes in acetylation status of histones H3 and H4 in erythroid cells with physiological levels of ectopic myc expression that are specifically blocked in enucleation, relative to untreated wild type erythroblasts. c-myc ectopic expression prevented deacetylation at several lysine residues that are normally deacetylated during erythroid maturation. By transcriptional profiling, one specific histone acetyl transferase (HAT) was shown to be upregulated by ectopic myc expression. The level of this HAT, like that of c-myc, normally decreases dramatically during late stage erythroid maturation. Chromatin immunoprecipitation assays demonstrated binding of c-myc to the promoter region of this HAT, indicating that it is a direct myc target in erythroid cells. Over-expression of this HAT inhibits nuclear condensation and enucleation specifically without affecting other aspects of terminal erythroid differentiation, and prevents histone deacetylation similar to ectopic c-myc expression. These data support a model where histone deacetylation associated with down regulation of c-myc and this HAT is essential for chromatin condensation and enucleation in mammalian erythroid cells. Currently, he is investigating the roles of other HATs and HDACs in terminal erythroid differentiation.
Human genetics reveals new genes important for normal erythropoiesis In work being performed with Eric Lander and colleagues at the Broad Institute, Vijay Sankaran along with laboratory colleagues Leif Ludwig, Jenn Eng, and Evelyn Wang 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.
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 microRNAs 15a and 16-1 appeared as a top candidates as mediators of this phenotype. Indeed, increased expression of these microRNAs in primary human erythroid progenitor cells resulted in elevated fetal and embryonic hemoglobin gene expression. Moreover, they showed that a direct target of these microRNAs, MYB, plays an important role in silencing the fetal and embryonic hemoglobin genes. Thus they 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 using a variety of approaches in primary mouse and human erythroid progenitor cells. In addition, Jenn, Leif, and Vijay are following up on alternative strategies for targeting MYB and are attempting to better understand the mechanism of action by which this transcription factor is able to regulate fetal hemoglobin levels. Finally, ongoing work is aimed at understanding the mechanistic basis for alterations in hemoglobin expression in the context of other rare human syndromes.
Using complex trait genetics, Vijay, Jenn, and Leif 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. The studies involve close collaborations with several groups in Boston and Cambridge, as well as internationally.
To gain further insight into important regulators of erythropoiesis, Vijay has been using Mendelian genetic approaches to identify genes mutated in rare human diseases with perturbations in erythropoiesis. 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.
| 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.
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 examined the expression of lncRNAs during erythropoiesis and identified one erythroid-specific lncRNA with potent anti-apoptotic activity. Inhibition of this lncRNA blocks erythroid differentiation and promotes apoptosis. Ectopic expression of this lncRNA prevents erythroid progenitor cells from apoptosis induced by erythropoietin deprivation. This lncRNA represses expression of Pycard, a pro-apoptotic gene, 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. Together with UROP student, Ken Lin, Wenqian is determining the molecular mechanism by which this lncRNA functions, and is performing functional and mechanistic characterization of several additional regulated lncRNAs in erythropoiesis.
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.
In addition to microRNA- mediated posttranscriptional regulation, Lingbo Zhang is also interested in the identification of protein and gene based regulatory networks governing erythropoiesis through computational biology approaches. In contrast to wet lab experiments, such as Chip-seq, Lingbo is trying to decipher this regulatory network by public databases, such as Gene Expression Omnibus (GEO), and data mining, followed by rigorous experimental validation.
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. Minh, together with Huangming Xie, Beiyan Zhou, and Moon Um in my lab, first obtained the expression profile of microRNAs during neuronal differentiation of the human neuroblastoma cell line SH-SY5Y; six microRNAs were significantly upregulated during differentiation induced by all-trans-retinoic acid and brain-derived neurotrophic factor. She then demonstrated that ectopic expression of either miR-124a or miR-125b increased the percentage of differentiated SH-SY5Y cells with neurite outgrowth. miR-125b is also upregulated during differentiation of human neural progenitor ReNcell VM cells, and Minh showed that miR-125b ectopic expression significantly promoted neurite outgrowth of these cells. To identify the targets of miR-125b regulation, Minh profiled the global changes in gene expression following miR-125b ectopic expression in SH-SY5Y cells. More than 50% of the downregulated mRNAs contain the seed match sequence of miR-125b; transcripts with stronger seed matches were repressed to a greater extent. Importantly, TargetScan 5.1 predicted 188 of the downregulated transcripts to be direct targets of miR-125b. Pathway analysis suggests that a subset of miR-125b-repressed targets antagonize neuronal genes in several neurogenic pathways, thereby mediating the positive effect of miR-125b on neuronal differentiation. Minh further confirmed the binding of miR-125b to the microRNA response elements of nine selected mRNA targets and validated the binding specificity for three targets. Together, these data reveal for the first time the important role of miR-125b in human neuronal differentiation.
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 either ablation of endogenous p53 function or by ectopic expression of miR-125b. Interestingly, miR-125b is downregulated when zebra fish embryos are treated with gamma-irradiation or camptothecin, corresponding to the rapid increase in p53 protein in response to DNA damage. Ectopic expression of miR-125b suppresses both the increase of p53 and stress-induced apoptosis.
Minh and Shyh-Chang then used both gain- and loss-of-function screens for miR-125b targets in humans, mice and zebrafish, 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.
MicroRNAs that modulate myeloid development and leukemias 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. Cell culture experiments demonstrate that miR-125b per se is able to block the myeloid differentiation of human cell lines under various stimulations. 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.
To check if the overexpression of miR-125b blocks myeloid differentiation and/or causes leukemias in vivo, Marina used a retroviral construct encoding miR-125b to infect enriched hematopoietic stem/ progenitor cell populations. These cells were then 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.
To better understand the role of miR-125b in hematopoiesis Marina is trying to identify miR-125b targets using two experimental in vitro models: NB4 (human promyelocytic cell line) and 32Dcl3 (mouse promyelocytic cell line). NB4 and 32Dcl3 can be induced to differentiate to granulocytic cells with retinoic acid and G-CSF respectively. She showed that the overexpression of miR-125b in these cell lines blocks granulocytic differentiation, reduces apoptosis, and induces proliferation. To identify miR-125b targets involved in these processes, she is analyzing the total cellular gene expression pattern by both RQ-PCR and mRNA-seq, comparing cell lines expressing or not miR-125b. During the last two years, Diu Nguyen (a visiting MS student), Lauren Shield (UROP student) and Cynthia Chen (UROP student) focused on the identification of miR-125b’s targets by using mRNA-seq on NB4 and 32Dcl3 cell lines expressing or not miR-125b. By using a computational approach, they selected genes downregulated by miR-125b overexpression and containing a potential binding site for miR-125b in their 3’UTRs. By using reporter assay, they validated some putative miR-125b target segments in these mRNAs. Western blot and in vitro assays should allow us to validate new miR-125b targets.
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.
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.
IntroductionHematopoietic 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.
Novel growth factors for hematopoietic stem cells No single known growth factor or combination of growth factors supported HSC expansion in culture. Furthermore existing lines of “supportive stromal cells” did not support expansion of HSCs; at best they maintained the level of HSCs over time, presumably due to a steady state between generation of new HSCs by division and differentiation of “old” stem cells. Thus, several years ago Chengcheng (Alec) Zhang turned to mouse fetal liver since the number of fetal HSCs normally increased markedly between embryonic Day 14 and Day 21. Chengcheng hypothesized that unknown growth proteins are produced by as- yet unidentified populations of fetal liver cells that stimulate the expansion of fetal liver HSCs. He identified Embryonic Day 15 fetal liver CD3+ Ter119- cells as a novel cell population that supports a net expansion of HSC numbers in culture. By transcriptional profiling of these cells and several others that do not support HSC expansion, Chengcheng uncovered several novel growth factors that, together, supported an unprecedented extent of ex vivo expansion of bone marrow HSCs: insulin - like growth factor 2 (IGF - 2) and Angiopoietin-like 2 and 3. A serum- free medium containing only low levels of stem cell factor (SCF), thrombopoietin (TPO), IGF-2, FGF-1, and Angiopoietin-like 2 or 3 stimulated a 24-30-fold expansion of HSCs following 10 days of culture of highly enriched mouse stem cells.
Next we found a ~20-fold expansion of human cord blood-derived CD133+ HSCs in a 10-day culture with a similar defined medium containing SCF, IGFBP2, TPO, FGF-1, and Angiopoietin-like 5, as measured by transplantation into NOD- scid immune- defective mice. In collaboration with Dr. Adam Drake and colleagues in Professor Jianzhu Chen’s lab at the MIT Koch Institute for Integrative Cancer Research, we used NOD-scid Il2rg-/- (NSG) mice that support long-term human HSC engraftment as recipients, and have definitively assessed the presence of HSCs in the expanded cell population. We showed that the SCID repopulating activity resides in the CD34+ CD133+ fraction of expanded cells. The expanded cells also mediate long-term hematopoiesis and serial reconstitution in NSG mice. Furthermore, the expanded CD34+ CD133+ cells efficiently reconstitute not only neonate but also adult NSG recipients, generating human blood cell populations similar to those reported in neonate recipients reconstituted with uncultured human HSCs. These findings suggest that these growth factors, Angiopoietin like 5, insulin growth factor binding protein 2, stem cell factor, thrombopoietin, and fibroblast growth factor 1, in the defined medium support the expansion of long-term human HSCs. The ability to expand human HSCs in vitro should facilitate clinical application of HSCs and large-scale construction of humanized mice from the same source for research applications. We are also collaborating William Hwang at the Singapore General Hospital to carry out preclinical and eventually clinical studies on ex vivo cord blood HSC expansion.
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+ potential 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 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. Song is current analyzing the ability of several growth factors secreted by these stromal cells to support HSC expansion as well as testing the ability of these cells to expand HSCs in ex vivo culture.
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 TNF -a even though there is no homology at the primary sequence level. Like TNF -a 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. Moreover, in adipocytes insulin affects the subcellular localization of the acyl-CoA synthetase FATP1. Thus she also tested if insulin activates AMPK in adipocytes, and if so through a similar mechanism. Qingqing examined these hypotheses by measuring the AMP/ATP ratio and AMPK activation upon adiponectin and insulin stimulation, and after knocking down acyl-CoA synthetases in adipocytes. She showed 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.
The transcriptional regulatory role of adiponectin in 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 and compared the expression profiles with those of wild-type mice. She confirmed the changes in expression of critical genes by real-time PCR. Qingqing has identified several important pathways that are severely downmodulated by the absence of adiponectin, and currently she is using several computational and experimental techniques to identify the transcription factors responsible for changed expression of these groups of genes. The goal of her study is to identify the specific transcription factors involved in regulating glucose and lipid metabolism, as they are the potential therapeutic targets in the treatments of obesity induced diseases such as diabetes and cardiovascular diseases.
Insulin resistance 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. Using the 3T3-L1 adipogenesis model we have used extensively, she adopted the mRNA-seq technique to study how the transcriptional outcomes of diverse insulin resistance models differ from each other and how they compare to in vivo models of insulin resistance. She confirmed a previous observation made by a former post-doc, Hong Ruan, that TNFalpha induced insulin resistance by suppressing many adipocyte important genes (eg Pparg, Adipoq) and inducing many preadipocyte genes. She is investigating if this phenomenon is also present in other insulin resistance models. Among all the in vitro models that she set up, she has found a certain model that most able to mimic the changes that we observe in vivo. In order to understand gene regulation, she is also using the DNA-hypersensitivity cum sequencing technique to investigate chromatin changes and transcriptional factor binding that lead to the diverse transcriptional outcomes.
Heide Christine Patterson, a new post-doc in the laboratory and a pathologist at Brigham and Women’s Hospital, is investigating whether a kinase important for signal transduction in immune cells also mediates activation of pathways critical for cellular insulin resistance in adipocytes in response to the same stimuli used by Alice Lo and whether this kinase controls glucose homeostasis in vivo.
| E. miRs and lincRNAs that regulate differentiation and function of white and brown adipose cells. |
MicroRNAs in fat cell development and obesity Huangming Xie examined the role of miRNAs in adipogenesis using several adipocyte cell culture differentiation systems. He profiled miRNA expression during in vitro adipogenesis of the preadipocyte 3T3-L1 cells using miRNA microarrays and validated by RT-PCR eight miRNAs that are significantly upregulated and four that are downregulated. Similar changes in miRNA expression were observed by comparison of mature primary adipocytes and enriched primary preadipocytes. He also profiled miRNA expression in purified mature adipocytes and compared miRNA profiles in epididymal adipocytes from normal and leptin deficient or diet-induced obese mice. Importantly, miRNAs that were induced during adipogenesis were downregulated in adipocytes from both types of obese mice and vice versa. These changes are likely associated with the chronic inflammatory environment in obese adipose tissue as they were mimicked by TNFa treatment of differentiated adipocytes. Ectopic expression of two adipocyte-enriched miRNAs in preadipocytes accelerated adipogenesis, as measured both by the upregulation of many adipocyte-important genes including adiponectin and the key transcription factor PPARg, and by an increase in triglyceride accumulation at an early stage of adipogenesis.
Lei Sun, Huangming 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, 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 Runx1t1 (runt-related transcription factor 1; translocated to 1) expression whereas myogenic markers were significantly induced. 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 Ppara. Taken together, these results underlie the importance of tissue enriched miRNAs in regulating lineage specification between brown fat and muscle, and also suggest that certain miRNAs may have therapeutic potential in inducing expression of brown fat-specific genes.
LincRNAs in fat cell development and function In addition, 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. They profiled the transcriptome of primary adipocytes, pre-adipocytes and cultured adipocytes and identified 481 lincRNAs that are specifically regulated during adipogenesis. Many lincRNAs are adipose-enriched, strongly induced during adipogenesis and bound at their promoters by key adipocyte transcription factors such as PPARg and CEBPa. RNAi-mediated loss of function screens identified functional lincRNAs with varying impacts on adipogenesis. They adapted an information-theoretic metric to score cellular phenotypes by quantifying the global “transcriptome shift” between precursor and adipocyte cell states. This analysis honed in on one lincRNA required for proper adipogenesis and that shares numerous copies of a conserved non-coding RNA sequence motif.
| F. Regulated cleavage and release of the extracellular domain of transmembrane precursors of several secreted growth factors. |
Protease cleavage and release of the extracellular domain (ECD, "ectodomain shedding") of a multitude of transmembrane proteins has been linked to the activation of many signaling pathways including the MAPK pathway. Cleavage of the ECD is mostly carried out by metalloproteases (MMPs) of the ADAM family (A Disintegrin And Metalloprotease). ECD cleavage is often followed by and is a prerequisite for intramembranous cleavage of the intracellular domain (ICD) of the same protein by γ-secretase; some of the cleaved ICDs translocate to the nucleus, where they may regulate gene transcription. Membrane-spanning pro-hormone ligands of the epidermal growth factor receptor (HER) family are well-studied examples of proteins that undergo ectodomain shedding and are physiologically important in many cellular contexts in organisms from Drosophila to mammals. But how the ectodomain cleavage machinery is regulated is largely unknown, as only a few specific stimuli that induce ectodomain shedding have been identified. As example, prolonged activation of the cardiac β-adrenergic receptor leads to HB-EGF-cleavage, release of soluble HB-EGF, and development of cardiac hypertrophy. Andreas Herrlich showed that another HER-ligand, neuregulin1β (NRG1β), is cleaved by an MMP in response to hypertonic stress and subsequently activates EGF- family receptors in an autocrine fashion. This signaling step leads to MAPK activation followed by enhanced expression of genes encoding water channels (aquaporins). Regulation of ectodomain cleavage could occur at two (or more) levels - at the level of the MMP or via covalent modifications of the target protein, such as phosphorylation or ubiquitination on the cytosolic face.
Andreas, with the assistance of Antonio D’Aiello, a technician, and Michelle Dang, Melissa Ko and Efrain Cermeno, three MIT undergraduates, is cloning novel genes that regulate ectodomain shedding using a high-throughput lentiviral shRNA gene knock-down strategy. They can detect cleavage of all chosen HER-ligands either by hypertonic stress, phorbol ester addition, or stimulation with lysophosphatidic acid in a FACS-based assay using mouse or human cell lines stably expressing one of the chosen pro-hormone ligands. The ligands are tagged at the extracellular domain with one of several epitope tags; at their cytosol-facing C- termini the proteins have been fused with EGFP. The extracellular epitope of the transmembrane pro-hormone ligand is detected with a fluorochrome-coupled (red) anti-epitope antibody, while the intracellular domain of the EGFP- fusion is detected by green fluorescence. Stimulation of cleavage results in a decrease of the red to green fluorescence ratio, while inhibition of basal or induced cleavage is reflected by an increase in this ratio. Andreas¡¯ initial studies with this system showed that, when expressed in mouse lung epithelial cells, ectodomain cleavage of these three EGF ligands is specifically triggered by different stimuli and involves different PKC isoenzymes.
Studies utilizing inhibitors of protein kinase C isoenzymes or metalloproteinase inhibition by batimastat showed that different regulatory signals are used by different stimuli and EGF substrates, suggesting differential effects that act on the substrate, the metalloproteinase, or both. Andreas, Antonio, Michelle, Melissa and Efrain have used this assay system for a 96 well plate high-throughput shRNA gene knockdown screen, testing the effect of shRNA constructs targeting about 95% of all known mammalian kinases and phosphatases on TPA-induced ectodomain cleavage of TGF-alpha. By stimulating cleavage to only about 50% of total possible cleavage they were able to detect both decreases (inhibitors of cleavage) and increases (activators of cleavage) in the red: green fluorescent ratio in the same screen. Several candidate genes have been identified and they are being tested in the context of the other physiological cleavage stimuli, hypertonic stress and GPCR stimulation. Further experiments to test the physiological significance of the candidate genes will be carried out in breast cancer and kidney disease cell culture model systems, both of which have relevant connections to EGF ligand cleavage.
Additionally, Andreas and Michelle have generated both wild type and ADAM knockout mouse embryonic fibroblasts cell lines expressing the three different EGF reporter ligands. Compared with their previous studies in mouse lung epithelial (MLE) cells, induced cleavage of HB-EGF, NRG and TGF alpha is regulated significantly different in the two cell types. Taken together, these results suggest that a significant part of the regulation of metalloprotease-mediated EGF ligand cleavage occurs on the substrate and not on the metalloprotease level and is regulated differently in different cell types.
Andreas will assume his new position as Assistant Professor at Harvard Medical School in October 2011 and will take this project into his own lab.
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: 3 Oct 2011