miR-433 is aberrantly expressed in myeloproliferative neoplasms and suppresses hematopoietic cell growth and differentiation.

BCR-ABL-negative myeloproliferative neoplasms (MPNs) are most frequently characterized by the JAK2V617F gain-of-function mutation, but several studies showed that JAK2V617F may not be the initiating event in MPN development, and recent publications indicate that additional alterations such as chromatin modification and microRNA (miRNA) deregulation may have an important role in MPN pathogenesis. Here we report that 61 miRNAs were significantly deregulated in CD34+ cells from MPN patients compared with controls (P<0.01). Global miRNA analysis also revealed that polycythemia vera (JAKV617F) and essential thrombocythemia (JAK2 wild type) patients have significantly different miRNA expression profiles from each other. Among the deregulated miRNAs, expression of miR-134, -214 and -433 was not affected by changes in JAK2 activity, suggesting that additional signaling pathways are responsible for the deregulation of these miRNAs in MPN. Despite its upregulation in MPN CD34+ and during normal erythropoiesis, both overexpression and knockdown studies suggest that miR-433 negatively regulates CD34+ proliferation and differentiation ex vivo. Its novel target GBP2 is downregulated during normal erythropoiesis and regulates proliferation and erythroid differentiation in TF-1 cells, indicating that miR-433 negatively regulates hematopoietic cell proliferation and erythropoiesis by directly targeting GBP2.

Analysis of genomic aberrations and gene expression profiling identifies novel lesions and pathways in myeloproliferative neoplasms.

Polycythemia vera (PV), essential thrombocythemia and primary myelofibrosis, are myeloproliferative neoplasms (MPNs) with distinct clinical features and are associated with the JAK2V617F mutation. To identify genomic anomalies involved in the pathogenesis of these disorders, we profiled 87 MPN patients using Affymetrix 250K single-nucleotide polymorphism (SNP) arrays. Aberrations affecting chr9 were the most frequently observed and included 9pLOH (n=16), trisomy 9 (n=6) and amplifications of 9p13.3-23.3 (n=1), 9q33.1-34.13 (n=1) and 9q34.13 (n=6). Patients with trisomy 9 were associated with elevated JAK2V617F mutant allele burden, suggesting that gain of chr9 represents an alternative mechanism for increasing JAK2V617F dosage. Gene expression profiling of patients with and without chr9 abnormalities (+9, 9pLOH), identified genes potentially involved in disease pathogenesis including JAK2, STAT5B and MAPK14. We also observed recurrent gains of 1p36.31-36.33 (n=6), 17q21.2-q21.31 (n=5) and 17q25.1-25.3 (n=5) and deletions affecting 18p11.31-11.32 (n=8). Combined SNP and gene expression analysis identified aberrations affecting components of a non-canonical PRC2 complex (EZH1, SUZ12 and JARID2) and genes comprising a 'HSC signature' (MLLT3, SMARCA2 and PBX1). We show that NFIB, which is amplified in 7/87 MPN patients and upregulated in PV CD34+ cells, protects cells from apoptosis induced by cytokine withdrawal.

Deletion 5q in myelodysplastic syndrome: a paradigm for the study of hemizygous deletions in cancer.

Hemizygous deletions are common molecular abnormalities in cancer. In some cases, these deletions highlight chromosomal loci containing tumor suppressor genes that undergo homozygous inactivation. In other cases, hemizygous deletions cause disease by allelic insufficiency for one or more genes. As the intact allele has no identifiable lesions, functional approaches are critical for the identification of pathogenic genes within large deletions. Hemizygous, interstitial deletion of chromosome 5q is the most common cytogenetic abnormality in myelodysplastic syndrome (MDS) and has been the focus of functional analysis. Some patients with this molecular lesion have the 5q- syndrome, a disorder with a highly consistent clinical phenotype. A systematic RNA interference screen to interrogate the function of each gene in the common deleted region (CDR) for the 5q- syndrome identified RPS14 as a critical haploinsufficiency disease gene for the erythroid failure, which is a characteristic of this syndrome. Genes located in an adjacent deleted region have also been implicated in MDS. The full clinical phenotype is likely caused by the integration of effects from allelic insufficiency for multiple genes. With the identification and characterization of these genes, the 5q deletion is becoming a model for understanding hemizygous chromosomal deletions in cancer.

Regulation of transcription by hypoxia requires a multiprotein complex that includes hypoxia-inducible factor 1, an adjacent transcription factor, and p300/CREB binding protein.

Molecular adaptation to hypoxia depends on the binding of hypoxia-inducible factor 1 (HIF-1) to cognate response elements in oxygen-regulated genes. In addition, adjacent sequences are required for hypoxia-inducible transcription. To investigate the mechanism of interaction between these cis-acting sequences, the multiprotein complex binding to the lactate dehydrogenase A (LDH-A) promoter was characterized. The involvement of HIF-1, CREB-1/ATF-1, and p300/CREB binding protein (CBP) was demonstrated by techniques documenting in vitro binding, in combination with transient transfections that test the in vivo functional importance of each protein. In both the LDH-A promoter and the erythropoietin 3' enhancer, formation of multiprotein complexes was analyzed by using biotinylated probes encompassing functionally critical cis-acting sequences. Strong binding of p300/CBP required interactions with multiple DNA binding proteins. Thus, the necessity of transcription factor binding sites adjacent to a HIF-1 site for hypoxically inducible transcription may be due to the requirement of p300 to interact with multiple transcription factors for high-affinity binding and activation of transcription. Since it has been found to interact with a wide range of transcription factors, p300 is likely to play a similar role in other genes, mediating interactions between DNA binding proteins, thereby activating stimulus-specific and tissue-specific gene transcription.

Drosophila melanogaster SL2 cells contain a hypoxically inducible DNA binding complex which recognises mammalian HIF-binding sites.

Nuclear extracts from Drosophila SL2 cells were found to contain a hypoxically inducible complex capable of binding to hypoxia response elements from mammalian genes. This complex (HIF-D) resembled mammalian hypoxia inducible factor (HIF-1) in DNA sequence specificity, abrogation of induction by cycloheximide, induction by desferrioxamine and redox sensitivity of DNA binding. However, HIF-D was not induced by cobalt and was less sensitive to phosphatase than HIF-1. Endogenous phosphoglycerate kinase mRNA in SL2 cells showed similar inducible characteristics to HIF-D. These findings are evidence that the mammalian HIF-1 dependent system of oxygen regulated gene expression has a functional homologue in Drosophila.

Isoenzyme-specific regulation of genes involved in energy metabolism by hypoxia: similarities with the regulation of erythropoietin.

Recent studies have indicated that regulatory mechanisms underlying the oxygen-dependent expression of the haematopoietic growth factor erythropoietin are widely operative in non-erythropoietin-producing cells and are involved in the regulation of other genes. An important characteristic of this system is that the inducible response to hypoxia is mimicked by exposure to particular transition metals such as cobaltous ions, and by iron chelation. We have investigated the extent of operation of this system in the regulation of a range of genes concerned with energy metabolism. The effects of hypoxia (1% oxygen), cobaltous ions and desferrioxamine on gene expression in tissue-culture cells was studied using RNase protection assays. Hypoxia induced the expression of glucose transporters in an isoform-specific manner; GLUT-1 and GLUT-3 were induced by hypoxia, whereas expression of GLUT-2 was decreased. Isoenzyme-specific regulation by hypoxia was also observed for genes encoding phosphofructokinase, aldolase and lactate dehydrogenase. For all of these genes, responses to cobaltous ions and desferrioxamine correlated in both direction and magnitude with the response to hypoxia. In contrast, a reduction in mitochondrial transcripts was observed in hypoxia, but these changes were not mimicked by either cobaltous ions or desferrioxamine. These findings indicate that similarities with erythropoietin regulation extend to the oxygen-dependent regulation of genes encoding glucose transporters and glycolytic enzymes but not to the regulation of mitochondrial transcripts, and they show that in glucose metabolism regulation by this system is isoenzyme- or isoform-specific.

Hypoxia and mitochondrial inhibitors regulate expression of glucose transporter-1 via distinct Cis-acting sequences.

Studies of gene regulation by oxygen have recently defined the existence of a widely operative system that responds to hypoxia but not mitochondrial inhibitors and involves the induction of a DNA-binding complex termed hypoxia-inducible factor 1. This system has been implicated in the regulation of erythropoietin, certain angiogenic growth factors, and particular glycolytic isoenzymes. The glucose transporter Glut-1 is induced by both hypoxia and mitochondrial inhibitors, implying the operation of a different mechanism of oxygen sensing. To explore that possibility, we analyzed the cisacting sequences that convey these responses. An enhancer lying 5' to the mouse Glut-1 gene was found to convey responses both to hypoxia and to the mitochondrial inhibitors, azide and rotenone. However, detailed analysis of this enhancer demonstrated that distinct elements responded to hypoxia and the mitochondrial inhibitors. The response to hypoxia was mediated by sequences that contained a functionally critical, although atypical, hypoxia-inducible factor 1 binding site, whereas sequences lying approximately 100 nucleotides 5' to this site, which contained a critical serum response element, conveyed responses to the mitochondrial inhibitors. Thus, rather than reflecting an entirely different mechanism of oxygen sensing, regulation of Glut-1 gene expression by hypoxia and mitochondrial inhibitors arises from the function of two different sensing systems. One of these responds to hypoxia alone and resembles that involved in erythropoietin regulation, while the other responds to mitochondrial inhibitors and involves activation of a serum response element.

Diphenylene iodonium inhibits the induction of erythropoietin and other mammalian genes by hypoxia. Implications for the mechanism of oxygen sensing.

Recent studies on the induction of erythropoietin gene expression by hypoxia have indicated that erythropoietin forms part of a widely operative system of gene regulation by oxygen. Similar responses to hypoxia, cobaltous ions and desferrioxamine have indicated that the action of these agents is closely connected with the mechanism of oxygen sensing. To consider further the mechanisms underlying these responses, the effect of iodonium compounds was tested on five genes which show oxygen-regulated expression; erythropoietin, vascular endothelial growth factor (VEGF), lactate dehydrogenase-A (LDH-A), glucose transporter-1 (GLUT-1) and placental growth factor (PLGF). In each case, the response to hypoxia was specifically inhibited by low doses of diphenylene iodonium (Ph1I+). This occurred irrespective of whether the hypoxic response was induction of gene expression (erythropoietin, vascular endothelial growth factor, lactate dehydrogenase-A, glucose transporter-1) or inhibition of gene expression (PLGF). In contrast, the induction of gene expression by cobaltous ions or desferrioxamine was not inhibited by Ph2I+. The differential action of Ph2I+ on the response to hypoxia versus the response to cobaltous ions or desferrioxamine must reflect a difference in the mechanism of action of these stimuli, which will require accommodation in any model of the oxygen-sensing mechanism. Based on the known properties of Ph2I+, the implication of these findings is that the mechanism of oxygen sensing most probably involves the operation of a flavoprotein oxidoreductase.

Hypoxic regulation of lactate dehydrogenase A. Interaction between hypoxia-inducible factor 1 and cAMP response elements.

The oxygen-regulated control system responsible for the induction of erythropoietin (Epo) by hypoxia is present in most (if not all) cells and operates on other genes, including those involved in energy metabolism. To understand the organization of cis-acting sequences that are responsible for oxygen-regulated gene expression, we have studied the 5' flanking region of the mouse gene encoding the hypoxically inducible enzyme lactate dehydrogenase A (LDH). Deletional and mutational analysis of the function of mouse LDH-reporter fusion gene constructs in transient transfection assays defined three domains, between -41 and -84 base pairs upstream of the transcription initiation site, which were crucial for oxygen-regulated expression. The most important of these, although not capable of driving hypoxic induction in isolation, had the consensus of a hypoxia-inducible factor 1 (HIF-1) site, and cross-competed for the binding of HIF-1 with functionally active Epo and phosphoglycerate kinase-1 sequences. The second domain was positioned close to the HIF-1 site, in an analogous position to one of the critical regions in the Epo 3' hypoxic enhancer. The third domain had the motif of a cAMP response element (CRE). Activation of cAMP by forskolin had no effect on the level of LDH mRNA in normoxia, but produced a magnified response to hypoxia that was dependent upon the integrity of the CRE, indicating an interaction between inducible factors binding the HIF-1 and CRE sites.

Regulation of angiogenic growth factor expression by hypoxia, transition metals, and chelating agents.

Recent work has indicated that oxygen-sensing mechanism(s) resembling those controlling erythropoietin production operate in many non-erythropoietin-producing cells. To pursue the implication that such a system might control other genes, we studied oxygen-regulated expression of mRNAs for vascular endothelial growth factor, platelet-derived growth factor (PDGF) A and B chains, placental growth factor (PLGF), and transforming growth factor in four different cell lines and compared the characteristics with those of erythropoietin regulation. Oxygen-regulated expression was demonstrated for each gene in at least one cell type. However, the response to hypoxia (1% oxygen) varied markedly, ranging from a 13-fold increase (PDGF-B in Hep G2 cells) to a 2-fold decrease (PLGF in the trophoblastic line BeWo). For each gene/cell combination, both the magnitude and direction of the response to hypoxia were mimicked by exposure to cobaltous ions or two different iron-chelating agents, desferrioxamine and hydroxypyridinones. These similarities with established characteristics of erythropoietin regulation indicate that a similar mechanism of oxygen sensing is operating on a variety of vascular growth factors, and they suggest that chelatable iron is closely involved in the mechanism.

Oxygen-regulated control elements in the phosphoglycerate kinase 1 and lactate dehydrogenase A genes: similarities with the erythropoietin 3' enhancer.

Production of the glycoprotein hormone erythropoietin (Epo) in response to hypoxic stimuli is almost entirely restricted to particular cells within liver and kidney, yet the transcriptional enhancer lying 3' to the Epo gene shows activity inducible by hypoxia after transfection into a wide variety of cultured cells. The implication of this finding is that many cells which do not produce Epo contain a similar, if not identical, oxygen-regulated control system, suggesting that the same system is involved in the regulation of other genes. We report that the human phosphoglycerate kinase 1 and mouse lactate dehydrogenase A genes are induced by hypoxia with characteristics which resemble induction of the Epo gene. In each case expression is induced by cobalt, but not by cyanide, and hypoxic induction is blocked by the protein-synthesis inhibitor cycloheximide. We show that the relevant cis-acting control sequences are located in the 5' flanking regions of the two genes, and we define an 18-bp element in the 5' flanking sequence of the phosphoglycerate kinase 1 gene which is both necessary and sufficient for the hypoxic response, and which has sequence and protein-binding similarities to the hypoxia-inducible factor 1 binding site within the Epo 3' enhancer.

Characterisation of functional domains within the mouse erythropoietin 3' enhancer conveying oxygen-regulated responses in different cell lines.

We have analysed sequences within the mouse erythropoietin enhancer which are required for oxygen regulated operation in the erythropoietin producing cell line, HepG2, and in two non-erythropoietin producing cell lines; the lung fibroblastoid cell line a23, and mouse erythroleukaemia (MEL) cells. At least three critical sites were demonstrated within a 96 nucleotide sequence. Oxygen regulated operation was dependent on sites within the first 26 nucleotides. Sequences lying 3' to this region modulated enhancer function but did not themselves convey oxygen regulated operation. In HepG2 cells these 3' sequences co-operated to permit operation of the inducible element at a distance from a promoter, but in MEL cells 3' sequences repressed activity of the inducible element. Though operation of this 3' sequence differed according to the cell type, oxygen regulated operation was dependent on the same two critical sites in the 5' region in both erythropoietin producing and non-erythropoietin producing cells. These findings support the existence of a widespread oxygen sensing system in mammalian cells which is similar to that operating in specific cells to regulate erythropoietin production, and they indicate that the system activates factors with similar DNA sequence specificity in different cells.