Claus Nerlov

Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy

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.

Regulation of eosinophil‐specific gene expression by a C/EBP–Ets complex and GATA‐1

The EOS47 antigen is an early and specific marker of eosinophil differentiation in the chicken haematopoietic system. To elucidate the transciptional events controlling commitment to the eosinophil lineage, we studied the regulation of the eosinophil‐specific EOS47 promoter. This promoter is TATA‐less, and binds trancription factors of the Ets, C/EBP, GATA and Myb families. These sites are contained within a 309 bp promoter fragment which is sufficient for specific high level transcription in an eosinophil cell line. Co‐transfection experiments in Q2bn fibroblasts showed cooperative activation of the EOS47 proximal promoter by c‐Myb, Ets‐1/Fli‐1, GATA‐1 and C/EBPα. The Ets‐1/Fli‐1 and C/EBPα proteins were the most potent activators, and acted with high synergy through juxtaposed binding sites located ∼60 bp upstream of the transcription start site. The Ets‐1 and C/EBPα proteins were found to associate physically via their DNA‐binding domains and to bind their combined binding site cooperatively. GATA‐1 showed biphasic regulation of the EOS47 promoter, activating at low and repressing at high protein concentrations. These results demonstrate combinatorial activation of an eosinophil‐specific promoter by ubiquitous and lineage‐restricted haematopoietic transcription factors. They also indicate that direct interactions between C/EBPs and specific Ets family members, together with GATA‐1, are important for eosinophil lineage determination.

Hematopoietic Stem Cell Expansion Precedes the Generation of Committed Myeloid Leukemia-Initiating Cells in C/EBPα Mutant AML

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.

Cdk6 blocks myeloid differentiation by interfering with Runx1 DNA binding and Runx1‐C/EBPα interaction

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/EBPα or PU.1. Cdk6 inhibits Runx1 activity by binding to the runt domain of Runx1, interfering with Runx1 DNA binding and Runx1‐C/EBPα 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.

FOG-1 and GATA-1 act sequentially to specify definitive megakaryocytic and erythroid progenitors

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.

Distinct C/EBPα motifs regulate lipogenic and gluconeogenic gene expression in vivo

The C/EBPα 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/EBPα 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/EBPα, and the PHR was required for C/EBPα‐SREBP transcriptional synergy. In contrast, the highly conserved C/EBPα 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‐1α, 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/EBPα phosphorylation as a PGC‐1α‐independent mechanism for regulating hepatic gluconeogenesis.

Impact of gene dosage, loss of wild-type allele, and FLT3 ligand on Flt3-ITD-induced myeloproliferation

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.

Transcriptional and translational control of C/EBPs: The case for “deep” genetics to understand physiological function

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.

Pontin is essential for murine hematopoietic stem cell survival

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.

Mutation of C/EBPalpha predisposes to the development of myeloid leukemia in a retroviral insertional mutagenesis screen.

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.