Prof Claus Nerlov

Research Area: Developmental and Stem Cell Biology
Technology Exchange: Cell sorting, Flow cytometry, Mouse models and Transgenesis
Scientific Themes: Haematology and Molecular, Cell & Systems Biology
Keywords: Hematopoiesis, Lineage commitment, Transcription, Leukemia and Cancer
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Wild type bone marrow (left) and bone marrow cells transformed by different combinations of CEBPA mutations (Cancer Cell 16: 390-400 (2009))

Wild type bone marrow (left) and bone marrow cells transformed by different combinations of CEBPA mu ...

The hematopoietic stem cell is capable of maintaining the output of >10 cellular lineage for the entire lifespan of the organism. This is made possible by the presence of structures (niches) dedicated to long-term maintenance of the multi-potent stem cell state, as well as mechanisms for the generation and subsequent lineage specification of stem cell progeny lacking long-term self-renewal capacity.

We use genetic methods, combined with genome-wide gene expression and chromatin profiling, to address the complexity of the hematopoietic stem cell population, the niches that maintain them, and the changes hematopoietic stem cells and niches undergo during aging. We investigate the regulators (transcription factors, signaling molecules) that control the lineage commitment of multi-potent hematopoietic progenitors, as well as the cellular pathways that they specify. Finally, we model how mutations affecting the normal transcriptional control of myelopoiesis result in acute myeloid leukemia, and address how the leukemic stem cells responsible for the disease are maintained.

The final goal is to understand the molecular basis for and spatial organization of normal, aging and malignant hematopoiesis, and to use this knowledge to devise cell based and molecular therapies that can be used to treat hematopoietic insufficiencies and malignancies.

Name Department Institution Country
Prof Sten Eirik Jacobsen Nuffield Division of Clinical Laboratory Sciences University of Oxford United Kingdom
Prof Paresh Vyas MRCP FRCP FRCPath Nuffield Division of Clinical Laboratory Sciences University of Oxford United Kingdom
Prof Thomas Milne Nuffield Division of Clinical Laboratory Sciences University of Oxford United Kingdom
Dr Liliana Minichiello Department of Pharmacology University of Oxford United Kingdom
Professor Giulio Superti-Furga Research Center for Molecular Medicine Austria
Prof Adam Mead MRCP FRCPath Nuffield Division of Clinical Laboratory Sciences University of Oxford United Kingdom

Pietras EM, Mirantes-Barbeito C, Fong S, Loeffler D, Kovtonyuk LV, Zhang S, Lakshminarasimhan R, Chin CP et al. 2016. Chronic interleukin-1 exposure drives haematopoietic stem cells towards precocious myeloid differentiation at the expense of self-renewal. Nat Cell Biol, 18 (6), pp. 607-618. Read abstract | Read more

Haematopoietic stem cells (HSCs) maintain lifelong blood production and increase blood cell numbers in response to chronic and acute injury. However, the mechanism(s) by which inflammatory insults are communicated to HSCs and their consequences for HSC activity remain largely unknown. Here, we demonstrate that interleukin-1 (IL-1), which functions as a key pro-inflammatory 'emergency' signal, directly accelerates cell division and myeloid differentiation of HSCs through precocious activation of a PU.1-dependent gene program. Although this effect is essential for rapid myeloid recovery following acute injury to the bone marrow, chronic IL-1 exposure restricts HSC lineage output, severely erodes HSC self-renewal capacity, and primes IL-1-exposed HSCs to fail massive replicative challenges such as transplantation. Importantly, these damaging effects are transient and fully reversible on IL-1 withdrawal. Our results identify a critical regulatory circuit that tailors HSC responses to acute needs, and is likely to underlie deregulated blood homeostasis in chronic inflammation conditions. Hide abstract

Grebien F, Vedadi M, Getlik M, Giambruno R, Grover A, Avellino R, Skucha A, Vittori S et al. 2015. Pharmacological targeting of the Wdr5-MLL interaction in C/EBPα N-terminal leukemia. Nat Chem Biol, 11 (8), pp. 571-578. Read abstract | Read more

The CEBPA gene is mutated in 9% of patients with acute myeloid leukemia (AML). Selective expression of a short (30-kDa) CCAAT-enhancer binding protein-α (C/EBPα) translational isoform, termed p30, represents the most common type of CEBPA mutation in AML. The molecular mechanisms underlying p30-mediated transformation remain incompletely understood. We show that C/EBPα p30, but not the normal p42 isoform, preferentially interacts with Wdr5, a key component of SET/MLL (SET-domain/mixed-lineage leukemia) histone-methyltransferase complexes. Accordingly, p30-bound genomic regions were enriched for MLL-dependent H3K4me3 marks. The p30-dependent increase in self-renewal and inhibition of myeloid differentiation required Wdr5, as downregulation of the latter inhibited proliferation and restored differentiation in p30-dependent AML models. OICR-9429 is a new small-molecule antagonist of the Wdr5-MLL interaction. This compound selectively inhibited proliferation and induced differentiation in p30-expressing human AML cells. Our data reveal the mechanism of p30-dependent transformation and establish the essential p30 cofactor Wdr5 as a therapeutic target in CEBPA-mutant AML. Hide abstract

Tawana K, Wang J, Renneville A, Bödör C, Hills R, Loveday C, Savic A, Van Delft FW et al. 2015. Disease evolution and outcomes in familial AML with germline CEBPA mutations. Blood, 126 (10), pp. 1214-1223. Read abstract | Read more

In-depth molecular investigation of familial leukemia has been limited by the rarity of recognized cases. This study examines the genetic events initiating leukemia and details the clinical progression of disease across multiple families harboring germ-line CEBPA mutations. Clinical data were collected from 10 CEBPA-mutated families, representing 24 members with acute myeloid leukemia (AML). Whole-exome (WES) and deep sequencing were performed to genetically profile tumors and define patterns of clonal evolution. Germline CEBPA mutations clustered within the N-terminal and were highly penetrant, with AML presenting at a median age of 24.5 years (range, 1.75-46 years). In all diagnostic tumors tested (n = 18), double CEBPA mutations (CEBPAdm) were detected, with acquired (somatic) mutations preferentially targeting the C-terminal. Somatic CEBPA mutations were unstable throughout the disease course, with different mutations identified at recurrence. Deep sequencing of diagnostic and relapse paired samples confirmed that relapse-associated CEBPA mutations were absent at diagnosis, suggesting recurrence was triggered by novel, independent clones. Integrated WES and deep sequencing subsequently revealed an entirely new complement of mutations at relapse, verifying the presentation of a de novo leukemic episode. The cumulative incidence of relapse in familial AML was 56% at 10 years (n = 11), and 3 patients experienced ≥3 disease episodes over a period of 17 to 20 years. Durable responses to secondary therapies were observed, with prolonged median survival after relapse (8 years) and long-term overall survival (10-year overall survival, 67%). Our data reveal that familial CEBPA-mutated AML exhibits a unique model of disease progression, associated with favorable long-term outcomes. Hide abstract

Woll PS, Kjällquist U, Chowdhury O, Doolittle H, Wedge DC, Thongjuea S, Erlandsson R, Ngara M et al. 2014. Myelodysplastic syndromes are propagated by rare and distinct human cancer stem cells in vivo Cancer Cell, 25 (6), pp. 794-808. Read abstract | Read more

Evidence for distinct human cancer stem cells (CSCs) remains contentious and the degree to which differentcancer cells contribute to propagating malignancies in patients remains unexplored. In low- to intermediate-risk myelodysplastic syndromes (MDS), we establish the existence of rare multipotent MDS stem cells (MDS-SCs), and their hierarchical relationship to lineage-restricted MDS progenitors. All identified somatically acquired genetic lesions were backtracked to distinct MDS-SCs, establishing their distinct MDS-propagating function invivo. In isolated del(5q)-MDS, acquisition of del(5q) preceded diverse recurrent driver mutations. Sequential analysis in del(5q)-MDS revealed genetic evolution in MDS-SCs and MDS-progenitors prior to leukemic transformation. These findings provide definitive evidence for rare human MDS-SCs invivo, with extensive implications for the targeting of the cells required and sufficient for MDS-propagation. © 2014 Elsevier Inc. Hide abstract

Grover A, Mancini E, Moore S, Mead AJ, Atkinson D, Rasmussen KD, O'Carroll D, Jacobsen SE, Nerlov C. 2014. Erythropoietin guides multipotent hematopoietic progenitor cells toward an erythroid fate. J Exp Med, 211 (2), pp. 181-188. Read abstract | Read more

The erythroid stress cytokine erythropoietin (Epo) supports the development of committed erythroid progenitors, but its ability to act on upstream, multipotent cells remains to be established. We observe that high systemic levels of Epo reprogram the transcriptomes of multi- and bipotent hematopoietic stem/progenitor cells in vivo. This induces erythroid lineage bias at all lineage bifurcations known to exist between hematopoietic stem cells (HSCs) and committed erythroid progenitors, leading to increased erythroid and decreased myeloid HSC output. Epo, therefore, has a lineage instructive role in vivo, through suppression of non-erythroid fate options, demonstrating the ability of a cytokine to systematically bias successive lineage choices in favor of the generation of a specific cell type. Hide abstract

Zhang H, Alberich-Jorda M, Amabile G, Yang H, Staber PB, Di Ruscio A, Welner RS, Ebralidze A et al. 2013. Sox4 is a key oncogenic target in C/EBPα mutant acute myeloid leukemia. Cancer Cell, 24 (5), pp. 575-588. Read abstract | Read more

Mutation or epigenetic silencing of the transcription factor C/EBPα is observed in ∼10% of patients with acute myeloid leukemia (AML). In both cases, a common global gene expression profile is observed, but downstream targets relevant for leukemogenesis are not known. Here, we identify Sox4 as a direct target of C/EBPα whereby its expression is inversely correlated with C/EBPα activity. Downregulation of Sox4 abrogated increased self-renewal of leukemic cells and restored their differentiation. Gene expression profiles of leukemia-initiating cells (LICs) from both Sox4 overexpression and murine C/EBPα mutant AML models clustered together but differed from other types of AML. Our data demonstrate that Sox4 overexpression resulting from C/EBPα inactivation contributes to the development of leukemia with a distinct LIC phenotype. Hide abstract

Sanjuan-Pla A, Macaulay IC, Jensen CT, Woll PS, Luis TC, Mead A, Moore S, Carella C et al. 2013. Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy. Nature, 502 (7470), pp. 232-236. Read abstract | Read more

The blood system is maintained by a small pool of haematopoietic stem cells (HSCs), which are required and sufficient for replenishing all human blood cell lineages at millions of cells per second throughout life. Megakaryocytes in the bone marrow are responsible for the continuous production of platelets in the blood, crucial for preventing bleeding--a common and life-threatening side effect of many cancer therapies--and major efforts are focused at identifying the most suitable cellular and molecular targets to enhance platelet production after bone marrow transplantation or chemotherapy. Although it has become clear that distinct HSC subsets exist that are stably biased towards the generation of lymphoid or myeloid blood cells, we are yet to learn whether other types of lineage-biased HSC exist or understand their inter-relationships and how differently lineage-biased HSCs are generated and maintained. The functional relevance of notable phenotypic and molecular similarities between megakaryocytes and bone marrow cells with an HSC cell-surface phenotype remains unclear. Here we identify and prospectively isolate a molecularly and functionally distinct mouse HSC subset primed for platelet-specific gene expression, with enhanced propensity for short- and long-term reconstitution of platelets. Maintenance of platelet-biased HSCs crucially depends on thrombopoietin, the primary extrinsic regulator of platelet development. Platelet-primed HSCs also frequently have a long-term myeloid lineage bias, can self-renew and give rise to lymphoid-biased HSCs. These findings show that HSC subtypes can be organized into a cellular hierarchy, with platelet-primed HSCs at the apex. They also demonstrate that molecular and functional priming for platelet development initiates already in a distinct HSC population. The identification of a platelet-primed HSC population should enable the rational design of therapies enhancing platelet output. Hide abstract

Kaveri D, Kastner P, Dembélé D, Nerlov C, Chan S, Kirstetter P. 2013. β-Catenin activation synergizes with Pten loss and Myc overexpression in Notch-independent T-ALL. Blood, 122 (5), pp. 694-704. Read abstract | Read more

Wnt signaling is important for T-cell differentiation at the early CD4(-)CD8(-) stage and is subsequently downregulated with maturation. To assess the importance of this downregulation, we generated a mouse line (R26-βcat) in which high levels of active β-catenin are maintained throughout T-cell development. Young R26-βcat mice show a differentiation block at the CD4(+)CD8(+) double-positive (DP) stage. These DP cells exhibit impaired apoptosis upon irradiation or dexamethasone treatment. All R26-βcat mice develop T-cell leukemias at 5 to 6 months of age. R26-βcat leukemias remain dependent on β-catenin function but lack Notch pathway activation. They exhibit recurrent secondary genomic rearrangements that lead to Myc overexpression and loss of Pten activity. Because β-catenin activation and Myc translocations were previously found in murine T-cell acute lymphoblastic leukemias (T-ALLs) deficient for Pten, our results suggest that activation of the canonical Wnt pathway is associated with a subtype of Notch-independent T-ALLs that bear Myc gene rearrangements and Pten mutations. Hide abstract

Mead AJ, Kharazi S, Atkinson D, Macaulay I, Pecquet C, Loughran S, Lutteropp M, Woll P et al. 2013. FLT3-ITDs instruct a myeloid differentiation and transformation bias in lymphomyeloid multipotent progenitors. Cell Rep, 3 (6), pp. 1766-1776. Read abstract | Read more

Whether signals mediated via growth factor receptors (GFRs) might influence lineage fate in multipotent progenitors (MPPs) is unclear. We explored this issue in a mouse knockin model of gain-of-function Flt3-ITD mutation because FLT3-ITDs are paradoxically restricted to acute myeloid leukemia even though Flt3 primarily promotes lymphoid development during normal hematopoiesis. When expressed in MPPs, Flt3-ITD collaborated with Runx1 mutation to induce high-penetrance aggressive leukemias that were exclusively of the myeloid phenotype. Flt3-ITDs preferentially expanded MPPs with reduced lymphoid and increased myeloid transcriptional priming while compromising early B and T lymphopoiesis. Flt3-ITD-induced myeloid lineage bias involved upregulation of the transcription factor Pu.1, which is a direct target gene of Stat3, an aberrantly activated target of Flt3-ITDs, further establishing how lineage bias can be inflicted on MPPs through aberrant GFR signaling. Collectively, these findings provide new insights into how oncogenic mutations might subvert the normal process of lineage commitment and dictate the phenotype of resulting malignancies. Hide abstract

Buza-Vidas N, Cismasiu VB, Moore S, Mead AJ, Woll PS, Lutteropp M, Melchiori L, Luc S et al. 2012. Dicer is selectively important for the earliest stages of erythroid development. Blood, 120 (12), pp. 2412-2416. Read abstract | Read more

MicroRNAs (miRs) are involved in many aspects of normal and malignant hematopoiesis, including hematopoietic stem cell (HSC) self-renewal, proliferation, and terminal differentiation. However, a role for miRs in the generation of the earliest stages of lineage committed progenitors from HSCs has not been identified. Using Dicer inactivation, we show that the miR complex is not only essential for HSC maintenance but is specifically required for their erythroid programming and subsequent generation of committed erythroid progenitors. In bipotent pre-MegEs, loss of Dicer up-regulated transcription factors preferentially expressed in megakaryocyte progenitors (Gata2 and Zfpm1) and decreased expression of the erythroid-specific Klf1 transcription factor. These results show a specific requirement for Dicer in acquisition of erythroid lineage programming and potential in HSCs and their subsequent erythroid lineage differentiation, and in particular indicate a role for the miR complex in achieving proper balance of lineage-specific transcriptional regulators necessary for HSC multilineage potential to be maintained. Hide abstract

Bereshchenko O, Mancini E, Luciani L, Gambardella A, Riccardi C, Nerlov C. 2012. Pontin is essential for murine hematopoietic stem cell survival. Haematologica, 97 (9), pp. 1291-1294. Read abstract | Read more

Pontin is a highly conserved DNA helicase/ATPase which is a component of several macromolecular complexes with functions that include DNA repair, telomere maintenance and tumor suppression. While Pontin is known to be essential in yeast, fruit flies and frogs, its physiological role in mammalian organisms remains to be determined. We here find that Pontin is highly expressed in embryonic stem cells and hematopoietic tissues. Through germline inactivation of Ruvbl1, the gene encoding Pontin, we found it to be essential for early embryogenesis, as Ruvbl1 null embryos could not be recovered beyond the blastocyst stage where proliferation of the pluripotent inner cell mass was impaired. Conditional ablation of Ruvbl1 in hematopoietic tissues led to bone marrow failure. Competitive repopulation experiments showed that this included the loss of hematopoietic stem cells through apopotosis. Pontin is, therefore, essential for the function of both embryonic pluripotent cells and adult hematopoietic stem cells. Hide abstract

Luc S, Luis TC, Boukarabila H, Macaulay IC, Buza-Vidas N, Bouriez-Jones T, Lutteropp M, Woll PS et al. 2012. The earliest thymic T cell progenitors sustain B cell and myeloid lineage potential. Nat Immunol, 13 (4), pp. 412-419. Read abstract | Read more

The stepwise commitment from hematopoietic stem cells in the bone marrow to T lymphocyte-restricted progenitors in the thymus represents a paradigm for understanding the requirement for distinct extrinsic cues during different stages of lineage restriction from multipotent to lineage-restricted progenitors. However, the commitment stage at which progenitors migrate from the bone marrow to the thymus remains unclear. Here we provide functional and molecular evidence at the single-cell level that the earliest progenitors in the neonatal thymus had combined granulocyte-monocyte, T lymphocyte and B lymphocyte lineage potential but not megakaryocyte-erythroid lineage potential. These potentials were identical to those of candidate thymus-seeding progenitors in the bone marrow, which were closely related at the molecular level. Our findings establish the distinct lineage-restriction stage at which the T cell lineage-commitment process transits from the bone marrow to the remote thymus. Hide abstract

Reckzeh K, Bereshchenko O, Mead A, Rehn M, Kharazi S, Jacobsen S-E, Nerlov C, Cammenga J. 2012. Molecular and cellular effects of oncogene cooperation in a genetically accurate AML mouse model LEUKEMIA, 26 (7), pp. 1527-1536. Read abstract | Read more

Biallelic CEBPA mutations and FMS-like tyrosine kinase receptor 3 (FLT3) length mutations are frequently identified in human acute myeloid leukemia (AML) with normal cytogenetics. However, the molecular and cellular mechanisms of oncogene cooperation remain unclear because of a lack of disease models. We have generated an AML mouse model using knockin mouse strains to study cooperation of an internal tandem duplication (ITD) mutation in the Flt3 gene with commonly observed CCAAT/enhancer binding protein alpha (C/EBPα) mutations. This study provides evidence that FLT3 ITD cooperates in leukemogenesis by enhancing the generation of leukemia-initiating granulocyte-monocyte progenitors (GMPs) otherwise prevented by a block in differentiation and skewed lineage priming induced by biallelic C/EBPα mutations. These cellular changes are accompanied by an upregulation of hematopoietic stem cell and STAT5 target genes. By gene expression analysis in premalignant populations, we further show a role of FLT3 ITD in activating genes involved in survival/transformation and chemoresistance. Both multipotent progenitors and GMP cells contain the potential to induce AML similar to corresponding cells in human AML samples showing that this model resembles human disease. © 2012 Macmillan Publishers Limited. Hide abstract

Mancini E, Sanjuan-Pla A, Luciani L, Moore S, Grover A, Zay A, Rasmussen KD, Luc S et al. 2012. FOG-1 and GATA-1 act sequentially to specify definitive megakaryocytic and erythroid progenitors EMBO Journal, 31 (2), pp. 351-365. Read abstract | Read more

The transcription factors that control lineage specification of haematopoietic stem cells (HSCs) have been well described for the myeloid and lymphoid lineages, whereas transcriptional control of erythroid (E) and megakaryocytic (Mk) fate is less understood. We here use conditional removal of the GATA-1 and FOG-1 transcription factors to identify FOG-1 as required for the formation of all committed Mk-and E-lineage progenitors, whereas GATA-1 was observed to be specifically required for E-lineage commitment. FOG-1-deficient HSCs and preMegEs, the latter normally bipotent for the Mk and E lineages, underwent myeloid transcriptional reprogramming, and formed myeloid, but not erythroid and megakaryocytic cells in vitro. These results identify FOG-1 and GATA-1 as required for formation of bipotent Mk/E progenitors and their E-lineage commitment, respectively, and show that FOG-1 mediates transcriptional Mk/E programming of HSCs as well as their subsequent Mk/E-lineage commitment. Finally, C/EBPs and FOG-1 exhibited transcriptional cross-regulation in early myelo-erythroid progenitors making their functional antagonism a potential mechanism for separation of the myeloid and Mk/E lineages. © 2012 European Molecular Biology Organization | All Rights Reserved. Hide abstract

Kharazi S, Mead AJ, Mansour A, Hultquist A, Böiers C, Luc S, Buza-Vidas N, Ma Z et al. 2011. Impact of gene dosage, loss of wild-type allele, and FLT3 ligand on Flt3-ITD-induced myeloproliferation. Blood, 118 (13), pp. 3613-3621. Read abstract | Read more

Acquisition of homozygous activating growth factor receptor mutations might accelerate cancer progression through a simple gene-dosage effect. Internal tandem duplications (ITDs) of FLT3 occur in approximately 25% cases of acute myeloid leukemia and induce ligand-independent constitutive signaling. Homozygous FLT3-ITDs confer an adverse prognosis and are frequently detected at relapse. Using a mouse knockin model of Flt3-internal tandem duplication (Flt3-ITD)-induced myeloproliferation, we herein demonstrate that the enhanced myeloid phenotype and expansion of granulocyte-monocyte and primitive Lin(-)Sca1(+)c-Kit(+) progenitors in Flt3-ITD homozygous mice can in part be mediated through the loss of the second wild-type allele. Further, whereas autocrine FLT3 ligand production has been implicated in FLT3-ITD myeloid malignancies and resistance to FLT3 inhibitors, we demonstrate here that the mouse Flt3(ITD/ITD) myeloid phenotype is FLT3 ligand-independent. Hide abstract

Nerlov C. 2010. Transcriptional and translational control of C/EBPs: the case for "deep" genetics to understand physiological function. Bioessays, 32 (8), pp. 680-686. Read abstract | Read more

The complexity of organisms is not simply determined by the number of their genes, but to a large extent by how gene expression is controlled. In addition to transcriptional regulation, this involves several layers of post-transcriptional control, such as translational repression, microRNA-mediated mRNA degradation and translational inhibition, alternative splicing, and the regulated generation of functionally distinct gene products from a single mRNA through alternative use of translation initiation sites. Much progress has been made in describing the molecular basis for these gene regulatory mechanisms. However, it is now a major challenge to translate this knowledge into deeper understanding of the physiological processes, both normal and pathological, that they govern. Using the C/EBP family of transcription factors as an example, the present review describes recent genetic experiments addressing this general problem and discusses how the physiological importance of newly discovered regulatory mechanisms might be determined. Hide abstract

Bereshchenko O, Mancini E, Moore S, Bilbao D, Månsson R, Luc S, Grover A, Jacobsen SE, Bryder D, Nerlov C. 2009. Hematopoietic stem cell expansion precedes the generation of committed myeloid leukemia-initiating cells in C/EBPalpha mutant AML. Cancer Cell, 16 (5), pp. 390-400. Read abstract | Read more

We here use knockin mutagenesis in the mouse to model the spectrum of acquired CEBPA mutations in human acute myeloid leukemia. We find that C-terminal C/EBPalpha mutations increase the proliferation of long-term hematopoietic stem cells (LT-HSCs) in a cell-intrinsic manner and override normal HSC homeostasis, leading to expansion of premalignant HSCs. However, such mutations impair myeloid programming of HSCs and block myeloid lineage commitment when homozygous. In contrast, N-terminal C/EBPalpha mutations are silent with regards to HSC expansion, but allow the formation of committed myeloid progenitors, the templates for leukemia-initiating cells. The combination of N- and C-terminal C/EBPalpha mutations incorporates both features, accelerating disease development and explaining the clinical prevalence of this configuration of CEBPA mutations. Hide abstract

Bröske AM, Vockentanz L, Kharazi S, Huska MR, Mancini E, Scheller M, Kuhl C, Enns A et al. 2009. DNA methylation protects hematopoietic stem cell multipotency from myeloerythroid restriction. Nat Genet, 41 (11), pp. 1207-1215. Read abstract | Read more

DNA methylation is a dynamic epigenetic mark that undergoes extensive changes during differentiation of self-renewing stem cells. However, whether these changes are the cause or consequence of stem cell fate remains unknown. Here, we show that alternative functional programs of hematopoietic stem cells (HSCs) are governed by gradual differences in methylation levels. Constitutive methylation is essential for HSC self-renewal but dispensable for homing, cell cycle control and suppression of apoptosis. Notably, HSCs from mice with reduced DNA methyltransferase 1 activity cannot suppress key myeloerythroid regulators and thus can differentiate into myeloerythroid, but not lymphoid, progeny. A similar methylation dosage effect controls stem cell function in leukemia. These data identify DNA methylation as an essential epigenetic mechanism to protect stem cells from premature activation of predominant differentiation programs and suggest that methylation dynamics determine stem cell functions in tissue homeostasis and cancer. Hide abstract

Ruffell D, Mourkioti F, Gambardella A, Kirstetter P, Lopez RG, Rosenthal N, Nerlov C. 2009. A CREB-C/EBPbeta cascade induces M2 macrophage-specific gene expression and promotes muscle injury repair. Proc Natl Acad Sci U S A, 106 (41), pp. 17475-17480. Read abstract | Read more

Macrophages play an essential role in the resolution of tissue damage through removal of necrotic cells, thus paving the way for tissue regeneration. Macrophages also directly support the formation of new tissue to replace the injury, through their acquisition of an anti-inflammatory, or M2, phenotype, characterized by a gene expression program that includes IL-10, the IL-13 receptor, and arginase 1. We report that deletion of two CREB-binding sites from the Cebpb promoter abrogates Cebpb induction upon macrophage activation. This blocks the downstream induction of M2-specific Msr1, Il10, II13ra, and Arg-1 genes, whereas the inflammatory (M1) genes Il1, Il6, Tnfa, and Il12 are not affected. Mice carrying the mutated Cebpb promoter (betaDeltaCre) remove necrotic tissue from injured muscle, but exhibit severe defects in muscle fiber regeneration. Conditional deletion of the Cebpb gene in muscle cells does not affect regeneration, showing that the C/EBPbeta cascade leading to muscle repair is muscle-extrinsic. While betaDeltaCre macrophages efficiently infiltrate injured muscle they fail to upregulate Cebpb, leading to decreased Arg-1 expression. CREB-mediated induction of Cebpb expression is therefore required in infiltrating macrophages for upregulation of M2-specific genes and muscle regeneration, providing a direct genetic link between these two processes. Hide abstract

Lopez RG, Garcia-Silva S, Moore SJ, Bereshchenko O, Martinez-Cruz AB, Ermakova O, Kurz E, Paramio JM, Nerlov C. 2009. C/EBPalpha and beta couple interfollicular keratinocyte proliferation arrest to commitment and terminal differentiation. Nat Cell Biol, 11 (10), pp. 1181-1190. Read abstract | Read more

The transcriptional regulators that couple interfollicular basal keratinocyte proliferation arrest to commitment and differentiation are yet to be identified. Here we report that the basic region leucine zipper transcription factors C/EBPalpha and C/EBPbeta are co-expressed in basal keratinocytes, and are coordinately upregulated as keratinocytes exit the basal layer and undergo terminal differentiation. Mice lacking both C/EBPalpha and beta in the epidermis showed increased proliferation of basal keratinocytes and impaired commitment to differentiation. This led to ectopic expression of keratin 14 (K14) and DeltaNp63 in suprabasal cells, decreased expression of spinous and granular layer proteins, parakeratosis and defective epidermal water barrier function. Knock-in mutagenesis revealed that C/EBP-E2F interaction was required for control of interfollicular epidermis (IFE) keratinocyte proliferation, but not for induction of spinous and granular layer markers, whereas C/EBP DNA binding was required for DeltaNp63 downregulation and K1/K10 induction. Finally, loss of C/EBPalpha/beta induced stem cell gene expression signatures in the epidermis. C/EBPs, therefore, couple basal keratinocyte cell cycle exit to commitment to differentiation through E2F repression and DNA binding, respectively, and may act to restrict the epidermal stem cell compartment. Hide abstract

Nerlov C. 2009. Targeting a chronic problem: elimination of cancer stem cells in CML. EMBO J, 28 (3), pp. 167-168. | Read more

Weischenfeldt J, Damgaard I, Bryder D, Theilgaard-Mönch K, Thoren LA, Nielsen FC, Jacobsen SE, Nerlov C, Porse BT. 2008. NMD is essential for hematopoietic stem and progenitor cells and for eliminating by-products of programmed DNA rearrangements. Genes Dev, 22 (10), pp. 1381-1396. Read abstract | Read more

Nonsense-mediated mRNA decay (NMD) is a post-transcriptional surveillance process that eliminates mRNAs containing premature termination codons (PTCs). NMD has been hypothesized to impact on several aspects of cellular function; however, its importance in the context of a mammalian organism has not been addressed in detail. Here we use mouse genetics to demonstrate that hematopoietic-specific deletion of Upf2, a core NMD factor, led to the rapid, complete, and lasting cell-autonomous extinction of all hematopoietic stem and progenitor populations. In contrast, more differentiated cells were only mildly affected in Upf2-null mice, suggesting that NMD is mainly essential for proliferating cells. Furthermore, we show that UPF2 loss resulted in the accumulation of nonproductive rearrangement by-products from the Tcrb locus and that this, as opposed to the general loss of NMD, was particularly detrimental to developing T-cells. At the molecular level, gene expression analysis showed that Upf2 deletion led to a profound skewing toward up-regulated mRNAs, highly enriched in transcripts derived from processed pseudogenes, and that NMD impacts on regulated alternative splicing events. Collectively, our data demonstrate a unique requirement of NMD for organismal survival. Hide abstract

Hasemann MS, Damgaard I, Schuster MB, Theilgaard-Mönch K, Sørensen AB, Mrsic A, Krugers T, Ylstra B, Pedersen FS, Nerlov C, Porse BT. 2008. Mutation of C/EBPalpha predisposes to the development of myeloid leukemia in a retroviral insertional mutagenesis screen. Blood, 111 (8), pp. 4309-4321. Read abstract | Read more

The CCAAT enhancer binding protein alpha (C/EBPalpha) is an important myeloid tumor suppressor that is frequently mutated in human acute myeloid leukemia (AML). We have previously shown that mice homozygous for the E2F repression-deficient Cebpa(BRM2) allele develop nonfatal AML with long latency and incomplete penetrance, suggesting that accumulation of secondary mutations is necessary for disease progression. Here, we use SRS19-6-driven retroviral insertional mutagenesis to compare the phenotypes of leukemias arising in Cebpa(+/+), Cebpa(+/BRM2), and Cebpa(BRM2/BRM2) mice, with respect to disease type, latency of tumor development, and identity of the retroviral insertion sites (RISs). Both Cebpa(+/BRM2) and Cebpa(BRM2/BRM2) mice preferentially develop myeloid leukemias, but with differing latencies, thereby demonstrating the importance of gene dosage. Determination of RISs led to the identification of several novel candidate oncogenes, some of which may collaborate specifically with the E2F repression-deficient allele of Cebpa. Finally, we used an in silico pathway analysis approach to extract additional information from single RISs, leading to the identification of signaling pathways which were preferentially deregulated in a disease- and/or genotype-specific manner. Hide abstract

Kirstetter P, Schuster MB, Bereshchenko O, Moore S, Dvinge H, Kurz E, Theilgaard-Mönch K, Månsson R et al. 2008. Modeling of C/EBPalpha mutant acute myeloid leukemia reveals a common expression signature of committed myeloid leukemia-initiating cells. Cancer Cell, 13 (4), pp. 299-310. Read abstract | Read more

Mutations in the CEBPA gene are present in 7%-10% of human patients with acute myeloid leukemia (AML). However, no genetic models exist that demonstrate their etiological relevance. To mimic the most common mutations affecting CEBPA-that is, those leading to loss of the 42 kDa C/EBPalpha isoform (p42) while retaining the 30kDa isoform (p30)-we modified the mouse Cebpa locus to express only p30. p30 supported the formation of granulocyte-macrophage progenitors. However, p42 was required for control of myeloid progenitor proliferation, and p42-deficient mice developed AML with complete penetrance. p42-deficient leukemia could be transferred by a Mac1+c-Kit+ population that gave rise only to myeloid cells in recipient mice. Expression profiling of this population against normal Mac1+c-Kit+ progenitors revealed a signature shared with MLL-AF9-transformed AML. Hide abstract

Nerlov C. 2008. C/EBPs: recipients of extracellular signals through proteome modulation. Curr Opin Cell Biol, 20 (2), pp. 180-185. Read abstract | Read more

C/EBP transcription factors are involved in the interpretation of extracellular signaling through a variety of mechanisms. These include the signaling-induced nuclear accumulation of C/EBP-interacting transcription factors such as Foxo1 and SREBP-1, leading to the formation of complexes that may themselves be subject to regulation by signal-induced post-translational modification. Post-translational modification may also control the interaction between C/EBPs and chromatin modifiers, as exemplified by decreased HDAC1-C/EBPbeta interaction upon GCN5-mediated lysine acetylation, and the ability of sumoylation to inhibit C/EBPalpha-SWI/SNF interaction. Finally, interaction with Smad proteins, which are accumulated in the nucleus upon TGFbeta or BMP signaling, may lead to the formation of C/EBP-Smad complexes and activation of Smad-C/EBPbeta coregulated promoters, while at the same time inhibiting other C/EBP-dependent transcription. These observations underline the importance of understanding signaling regulated transcription in terms of the proteomic changes that are induced, and how these are interpreted in the relevant promoter contexts. Hide abstract

Nygren JM, Liuba K, Breitbach M, Stott S, Thorén L, Roell W, Geisen C, Sasse P et al. 2008. Myeloid and lymphoid contribution to non-haematopoietic lineages through irradiation-induced heterotypic cell fusion. Nat Cell Biol, 10 (5), pp. 584-592. Read abstract | Read more

Recent studies have suggested that regeneration of non-haematopoietic cell lineages can occur through heterotypic cell fusion with haematopoietic cells of the myeloid lineage. Here we show that lymphocytes also form heterotypic-fusion hybrids with cardiomyocytes, skeletal muscle, hepatocytes and Purkinje neurons. However, through lineage fate-mapping we demonstrate that such in vivo fusion of lymphoid and myeloid blood cells does not occur to an appreciable extent in steady-state adult tissues or during normal development. Rather, fusion of blood cells with different non-haematopoietic cell types is induced by organ-specific injuries or whole-body irradiation, which has been used in previous studies to condition recipients of bone marrow transplants. Our findings demonstrate that blood cells of the lymphoid and myeloid lineages contribute to various non-haematopoietic tissues by forming rare fusion hybrids, but almost exclusively in response to injuries or inflammation. Hide abstract

Nilsson L, Edén P, Olsson E, Månsson R, Astrand-Grundström I, Strömbeck B, Theilgaard-Mönch K, Anderson K et al. 2007. The molecular signature of MDS stem cells supports a stem-cell origin of 5q myelodysplastic syndromes. Blood, 110 (8), pp. 3005-3014. Read abstract | Read more

Global gene expression profiling of highly purified 5q-deleted CD34+CD38(-)Thy1+ cells in 5q- myelodysplastic syndromes (MDSs) supported that they might originate from and outcompete normal CD34+CD38(-)Thy1+ hematopoietic stem cells. Few but distinct differences in gene expression distinguished MDS and normal stem cells. Expression of BMI1, encoding a critical regulator of self-renewal, was up-regulated in 5q- stem cells. Whereas multiple previous MDS genetic screens failed to identify altered expression of the gene encoding the myeloid transcription factor CEBPA, stage-specific and extensive down-regulation of CEBPA was specifically observed in MDS progenitors. These studies establish the importance of molecular characterization of distinct stages of cancer stem and progenitor cells to enhance the resolution of stage-specific dysregulated gene expression. Hide abstract

Nerlov C. 2007. The C/EBP family of transcription factors: a paradigm for interaction between gene expression and proliferation control. Trends Cell Biol, 17 (7), pp. 318-324. Read abstract | Read more

In recent years, a link between the transcriptional regulators of lineage-specific gene expression and progenitor proliferation control has emerged. A main exponent of this phenomenon is the CCAAT/enhancer binding protein (C/EBP) family of basic region-leucine zipper proteins. These transcription factors control the differentiation of a range of cell types, and have key roles in regulating cellular proliferation through interaction with cell cycle proteins. More recently, their position at the crossroads between proliferation and differentiation has made them strong candidate regulators of tumorigenesis, and C/EBPs have been described as both tumor promoters and tumor suppressors. Hide abstract

Fujimoto T, Anderson K, Jacobsen SE, Nishikawa SI, Nerlov C. 2007. Cdk6 blocks myeloid differentiation by interfering with Runx1 DNA binding and Runx1-C/EBPalpha interaction. EMBO J, 26 (9), pp. 2361-2370. Read abstract | Read more

Interactions between the cell cycle machinery and transcription factors play a central role in coordinating terminal differentiation and proliferation arrest. We here show that cyclin-dependent kinase 6 (Cdk6) is specifically expressed in proliferating hematopoietic progenitor cells, and that Cdk6 inhibits transcriptional activation by Runx1, but not C/EBPalpha or PU.1. Cdk6 inhibits Runx1 activity by binding to the runt domain of Runx1, interfering with Runx1 DNA binding and Runx1-C/EBPalpha interaction. Cdk6 expression increased myeloid progenitor proliferation, and inhibited myeloid lineage-specific gene expression and terminal differentiation in vitro and in vivo. These effects of Cdk6 did not require Cdk6 kinase activity. Cdk6-mediated inhibition of granulocytic differentiation could be reversed by excess Runx1, consistent with Runx1 being the major target for Cdk6. We propose that Cdk6 downregulation in myeloid progenitors releases Runx1 from Cdk6 inhibition, thereby allowing terminal differentiation. Since Runx transcription factors play central roles in hematopoietic, neuronal and osteogenic lineages, this novel, noncanonical Cdk6 function may control terminal differentiation in multiple tissues and cell types. Hide abstract

Pedersen TA, Bereshchenko O, Garcia-Silva S, Ermakova O, Kurz E, Mandrup S, Porse BT, Nerlov C. 2007. Distinct C/EBPalpha motifs regulate lipogenic and gluconeogenic gene expression in vivo. EMBO J, 26 (4), pp. 1081-1093. Read abstract | Read more

The C/EBPalpha transcription factor regulates hepatic nitrogen, glucose, lipid and iron metabolism. However, how it is able to independently control these processes is not known. Here, we use mouse knock-in mutagenesis to identify C/EBPalpha domains that specifically regulate hepatic gluconeogenesis and lipogenesis. In vivo deletion of a proline-histidine rich domain (PHR), dephosphorylated at S193 by insulin signaling, dysregulated genes involved in the generation of acetyl-CoA and NADPH for triglyceride synthesis and led to increased hepatic lipogenesis. These promoters bound SREBP-1 as well as C/EBPalpha, and the PHR was required for C/EBPalpha-SREBP transcriptional synergy. In contrast, the highly conserved C/EBPalpha CR4 domain was found to undergo liver-specific dephosphorylation of residues T222 and T226 upon fasting, and alanine mutation of these residues upregulated the hepatic expression of the gluconeogenic G6Pase and PEPCK mRNAs, but not PGC-1alpha, leading to glucose intolerance. Our results show that pathway-specific metabolic regulation can be achieved through a single transcription factor containing context-sensitive regulatory domains, and indicate C/EBPalpha phosphorylation as a PGC-1alpha-independent mechanism for regulating hepatic gluconeogenesis. Hide abstract

Kirstetter P, Anderson K, Porse BT, Jacobsen SE, Nerlov C. 2006. Activation of the canonical Wnt pathway leads to loss of hematopoietic stem cell repopulation and multilineage differentiation block. Nat Immunol, 7 (10), pp. 1048-1056. Read abstract | Read more

Wnt signaling increases hematopoietic stem cell self-renewal and is activated in both myeloid and lymphoid malignancies, indicating involvement in both normal and malignant hematopoiesis. We report here activated canonical Wnt signaling in the hematopoietic system through conditional expression of a stable form of beta-catenin. This enforced expression led to hematopoietic failure associated with loss of myeloid lineage commitment at the granulocyte-macrophage progenitor stage; blocked erythrocyte differentiation; disruption of lymphoid development; and loss of repopulating stem cell activity. Loss of hematopoietic stem cell function was associated with decreased expression of Cdkn1a (encoding the cell cycle inhibitor p21(cdk)), Sfpi1, Hoxb4 and Bmi1 (encoding the transcription factors PU.1, HoxB4 and Bmi-1, respectively) and altered integrin expression in Lin(-)Sca-1(+)c-Kit(+) cells, whereas PU.1 was upregulated in erythroid progenitors. Constitutive activation of canonical Wnt signaling therefore causes multilineage differentiation block and compromised hematopoietic stem cell maintenance. Hide abstract

Porse BT, Pedersen TA, Hasemann MS, Schuster MB, Kirstetter P, Luedde T, Damgaard I, Kurz E, Schjerling CK, Nerlov C. 2006. The proline-histidine-rich CDK2/CDK4 interaction region of C/EBPalpha is dispensable for C/EBPalpha-mediated growth regulation in vivo. Mol Cell Biol, 26 (3), pp. 1028-1037. Read abstract | Read more

The C/EBPalpha transcription factor regulates growth and differentiation of several tissues during embryonic development. Several hypotheses as to how C/EBPalpha inhibits cellular growth in vivo have been derived, mainly from studies of tissue culture cells. In fetal liver it has been proposed that a short, centrally located, 15-amino-acid proline-histidine-rich region (PHR) of C/EBPalpha is responsible for the growth-inhibitory function of the protein through its ability to interact with CDK2 and CDK4, thereby inhibiting their activities. Homozygous Cebpa(DeltaPHR/DeltaPHR) (DeltaPHR) mice, carrying a modified cebpa allele lacking amino acids 180 to 194, were born at the Mendelian ratio, reached adulthood, and displayed no apparent adverse phenotypes. When fetal livers from the DeltaPHR mice were analyzed for their expression of cell cycle markers, bromodeoxyuridine incorporation, cyclin-dependent kinase 2 kinase activity, and global gene expression, we failed to detect any cell cycle or developmental differences between the DeltaPHR mice and their control littermates. These in vivo data demonstrate that any C/EBPalpha-mediated growth repression via the PHR as well as the basic region is dispensable for proper embryonic development of, and cell cycle control in, the liver. Surprisingly, control experiments performed in C/EBPalpha null fetal livers yielded similar results. Hide abstract

Porse BT, Bryder D, Theilgaard-Mönch K, Hasemann MS, Anderson K, Damgaard I, Jacobsen SE, Nerlov C. 2005. Loss of C/EBP alpha cell cycle control increases myeloid progenitor proliferation and transforms the neutrophil granulocyte lineage. J Exp Med, 202 (1), pp. 85-96. Read abstract | Read more

CCAAT/enhancer binding protein (C/EBP)alpha is a myeloid-specific transcription factor that couples lineage commitment to terminal differentiation and cell cycle arrest, and is found mutated in 9% of patients who have acute myeloid leukemia (AML). We previously showed that mutations which dissociate the ability of C/EBP alpha to block cell cycle progression through E2F inhibition from its function as a transcriptional activator impair the in vivo development of the neutrophil granulocyte and adipose lineages. We now show that such mutations increase the capacity of bone marrow (BM) myeloid progenitors to proliferate, and predispose mice to a granulocytic myeloproliferative disorder and transformation of the myeloid compartment of the BM. Both of these phenotypes were transplantable into lethally irradiated recipients. BM transformation was characterized by a block in granulocyte differentiation, accumulation of myeloblasts and promyelocytes, and expansion of myeloid progenitor populations--all characteristics of AML. Circulating myeloblasts and hepatic leukocyte infiltration were observed, but thrombocytopenia, anemia, and elevated leukocyte count--normally associated with AML-were absent. These results show that disrupting the cell cycle regulatory function of C/EBP alpha is sufficient to initiate AML-like transformation of the granulocytic lineage, but only partially the peripheral pathology of AML. Hide abstract

Nerlov C. 2004. C/EBPalpha mutations in acute myeloid leukaemias. Nat Rev Cancer, 4 (5), pp. 394-400. Read abstract | Read more

Specific mutations in the gene that encodes the multifunctional transcription factor C/EBPα are frequently associated with acute myeloid leukaemias. Are only a specific subset of the functions of C/EBPα therefore involved in leukaemogenesis? Hide abstract

Pedersen-Bjergaard J, Andersen MK, Christiansen DH, Nerlov C. 2002. Genetic pathways in therapy-related myelodysplasia and acute myeloid leukemia. Blood, 99 (6), pp. 1909-1912. Read abstract | Read more

Therapy-related acute myeloid leukemia (t-AML) in most cases develops after chemotherapy of other malignancies and shows characteristic chromosome aberrations. Two general types of t-AML have previously been identified. One type is observed after therapy with alkylating agents and characteristically presents as therapy-related myelodysplasia with deletions or loss of the long arms of chromosomes 5 and 7 or loss of the whole chromosomes. The other type is observed after therapy with topoisomerase II inhibitors and characteristically presents as overt t-AML with recurrent balanced chromosome aberrations. Recent research suggests that these 2 general types of t-AML can now be subdivided into at least 8 genetic pathways with a different etiology and different biologic characteristics. Hide abstract

Pedersen TA, Kowenz-Leutz E, Leutz A, Nerlov C. 2001. Cooperation between C/EBPalpha TBP/TFIIB and SWI/SNF recruiting domains is required for adipocyte differentiation. Genes Dev, 15 (23), pp. 3208-3216. Read abstract | Read more

Chromatin remodeling is an important step in promoter activation during cellular lineage commitment and differentiation. We show that the ability of the C/EBPalpha transcription factor to direct adipocyte differentiation of uncommitted fibroblast precursors and to activate SWI/SNF-dependent myeloid-specific genes depends on a domain, C/EBPalpha transactivation element III (TE-III), that binds the SWI/SNF chromatin remodeling complex. TE-III collaborates with C/EBPalpha TBP/TFIIB interaction motifs during induction of adipogenesis and adipocyte-specific gene expression. These results indicate that C/EBPalpha acts as a lineage-instructive transcription factor through SWI/SNF-dependent modification of the chromatin structure of lineage-specific genes, followed by direct promoter activation via recruitment of the basal transcription-initiation complex, and provide a mechanism by which C/EBPalpha can mediate differentiation along multiple cellular lineages. Hide abstract

Porse BT, Pedersen TA, Xu X, Lindberg B, Wewer UM, Friis-Hansen L, Nerlov C. 2001. E2F repression by C/EBPalpha is required for adipogenesis and granulopoiesis in vivo. Cell, 107 (2), pp. 247-258. Read abstract | Read more

The C/EBPalpha transcription factor is required for differentiation of adipocytes and neutrophil granulocytes, and controls cellular proliferation in vivo. To address the molecular mechanisms of C/EBPalpha action, we have identified C/EBPalpha mutants defective in repression of E2F-dependent transcription and found them to be impaired in their ability to suppress cellular proliferation, and to induce adipocyte differentiation in vitro. Using targeted mutagenesis of the mouse germline, we show that E2F repression-deficient C/EBPalpha alleles failed to support adipocyte and granulocyte differentiation in vivo. These results indicate that E2F repression by C/EBPalpha is critical for its ability to induce terminal differentiation, and thus provide genetic evidence that direct cell cycle control by a mammalian lineage-instructive transcription factor couples cellular growth arrest and differentiation. Hide abstract

Querfurth E, Schuster M, Kulessa H, Crispino JD, Döderlein G, Orkin SH, Graf T, Nerlov C. 2000. Antagonism between C/EBPbeta and FOG in eosinophil lineage commitment of multipotent hematopoietic progenitors. Genes Dev, 14 (19), pp. 2515-2525. Read abstract | Read more

The commitment of multipotent cells to particular developmental pathways requires specific changes in their transcription factor complement to generate the patterns of gene expression characteristic of specialized cell types. We have studied the role of the GATA cofactor Friend of GATA (FOG) in the differentiation of avian multipotent hematopoietic progenitors. We found that multipotent cells express high levels of FOG mRNA, which were rapidly down-regulated upon their C/EBPbeta-mediated commitment to the eosinophil lineage. Expression of FOG in eosinophils led to a loss of eosinophil markers and the acquisition of a multipotent phenotype, and constitutive expression of FOG in multipotent progenitors blocked activation of eosinophil-specific gene expression by C/EBPbeta. Our results show that FOG is a repressor of the eosinophil lineage, and that C/EBP-mediated down-regulation of FOG is a critical step in eosinophil lineage commitment. Furthermore, our results indicate that maintenance of a multipotent state in hematopoiesis is achieved through cooperation between FOG and GATA-1. We present a model in which C/EBPbeta induces eosinophil differentiation by the coordinate direct activation of eosinophil-specific promoters and the removal of FOG, a promoter of multipotency as well as a repressor of eosinophil gene expression. Hide abstract

Nerlov C, Querfurth E, Kulessa H, Graf T. 2000. GATA-1 interacts with the myeloid PU.1 transcription factor and represses PU.1-dependent transcription. Blood, 95 (8), pp. 2543-2551. Read abstract

The GATA-1 transcription factor is capable of suppressing the myeloid gene expression program when ectopically expressed in myeloid cells. We examined the ability of GATA-1 to repress the expression and function of the PU.1 transcription factor, a central regulator of myeloid differentiation. We found that GATA-1 is capable of suppressing the myeloid phenotype without interfering with PU.1 gene expression, but instead was capable of inhibiting the activity of the PU.1 protein in a dose-dependent manner. This inhibition was independent of the ability of GATA-1 to bind DNA, suggesting that it is mediated by protein-protein interaction. We examined the ability of PU.1 to interact with GATA-1 and found a direct interaction between the PU.1 ETS domain and the C-terminal finger region of GATA-1. Replacing the PU.1 ETS domain with the GAL4 DNA-binding domain removed the ability of GATA-1 to inhibit PU.1 activity, indicating that the PU.1 DNA-binding domain, rather than the transactivation domain, is the target for GATA-1-mediated repression. We therefore propose that GATA-1 represses myeloid gene expression, at least in part, through its ability to directly interact with the PU.1 ETS domain and thereby interfere with PU.1 function. (Blood. 2000;95:2543-2551) Hide abstract

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