The platelet surface is poorly characterized due to the low abundance of many membrane proteins and the lack of specialist tools for their investigation. In this study we identified novel human platelet and mouse megakaryocyte membrane proteins using specialist proteomics and genomics approaches. Three separate methods were used to enrich platelet surface proteins prior to identification by liquid chromatography and tandem mass spectrometry: lectin affinity chromatography, biotin/NeutrAvidin affinity chromatography, and free flow electrophoresis. Many known, abundant platelet surface transmembrane proteins and several novel proteins were identified using each receptor enrichment strategy. In total, two or more unique peptides were identified for 46, 68, and 22 surface membrane, intracellular membrane, and membrane proteins of unknown subcellular localization, respectively. The majority of these were single transmembrane proteins. To complement the proteomics studies, we analyzed the transcriptome of a highly purified preparation of mature primary mouse megakaryocytes using serial analysis of gene expression in view of the increasing impor-

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The basic helix-loop-helix transcription factor Scl/Tal1 controls the development and subsequent differentiation of hematopoietic stem cells (HSCs). However, because few Scl target genes have been validated to date, the underlying mechanisms have remained largely unknown. In this study, we have used ChIP-Seq technology (coupling chromatin immunoprecipitation with deep sequencing) to generate a genome-wide catalog of Scl-binding events in a stem/progenitor cell line, followed by validation using primary fetal IntroductionHematopoiesis has long served as a model system for adult stem cells, with many paradigms of stem cell biology first being established as a result of studying hematopoietic stem cells (HSCs). A large body of work over the past 25 years has established that transcription factors (TFs) play critical roles during the specification, maintenance, and/or differentiation of HSCs. However, the underlying mechanisms have remained largely obscure because of a lack of comprehensive data on target genes, as well as very limited information on the way key TFs interact to form the regulatory networks that control blood stem cell development and subsequent behavior.The basic helix-loop-helix (bHLH) TF Scl (also known as Tal1) is required for the specification of HSCs as well as their subsequent differentiation into erythroid and megakaryocytic lineages. 1,2 Sclnull embryos do not survive beyond embryonic day (E) 9.5 due to a complete absence of hematopoiesis, 3,4 a more striking phenotype than seen with other important regulators of early hematopoiesis such as Runx1 or Gata2. [5][6][7] Moreover, together with its paralogue Lyl1, Scl was recently shown to be essential for the survival of adult HSCs, thus emphasizing critical functions for Scl at multiple stages of hematopoietic ontogeny. 8 In addition to its pleiotropic roles in hematopoiesis, Scl is also required for vascular and central nervous system development. [9][10][11] Within the blood system, Scl is thought to be a key component of the regulatory networks controlling the specification and subsequent differentiation of HSCs. 12,13 Studies on the transcriptional regulation of the murine Scl gene identified Ets and Gata factors as well as an autoregulatory loop as key upstream inputs. [14][15][16] However, to fully understand how Scl functions within hematopoietic regulatory networks, comprehensive information on downstream target genes will also be required. Scl has been found to regulate a handful of genes, including Gata1, 17 Runx1, 18 c-kit, 19 and ␣-globin 20 in different hematopoietic lineages. However, to date, no systematic genome-scale approach has been taken to interrogate Scl target genes at early developmental time points in which Scl function is critical.Together with bHLH class I proteins, such as E47, Scl binds DNA as a heterodimer to the so-called E-box sequence motif CANNTG. In addition to its bHLH DNA-binding partners, Scl can interact with various proteins, including the lim-only protein Lmo2 and Gata factors Gata1/Gata2 in multimeric ...

A common feature of early embryo cells from the inner cell mass (ICM) and of ESCs is an absolute dependence on an atypical cell cycle in which the G1 phase is shortened to preserve their self-renewing and pluripotent nature. The transcription factor B-Myb has been attributed a role in proliferation, in particular during the G2/M phases of the cell cycle. Intriguingly, B-Myb levels in ICM/ESCs are greater than 100 times compared with those in normal proliferating cells, suggesting a particularly important function for this transcription factor in pluripotent stem cells. B-Myb is essential for embryo development beyond the preimplantation stage, but its role in ICM/ESCs remains unclear. Using a combination of mouse genetics, single DNA fiber analyses and high-resolution three-dimensional (3D) imaging, we demonstrate that B-Myb has no influence on the expression of pluripotency factors, but instead B-Myb ablation leads to stalling of replication forks and superactivation of replication factories that result in disorganization of the replication program and an increase in double-strand breaks. These effects are partly due to aberrant transcriptional regulation of cell cycle proliferation factors, namely c-Myc and FoxM1, which dictate normal S phase progression. We conclude that B-Myb acts crucially during the S phase in ESCs by facilitating proper progression of replication, thereby protecting the cells from genomic damage. Our findings have particular relevance in the light of the potential therapeutic application of ESCs and the need to maintain their genomic integrity. STEM CELLS

STAT5A and STAT5B are two highly related transcription factors encoded by two distinct genes. STAT5A and STAT5B are activated by a broad range of cytokines and growth factors. Although they can be differentially activated, the functional difference between these two molecules relative to their structure is not known. Here we demonstrated that STAT5A and STAT5B homodimers have distinct DNA binding preferences. Chimeric STAT5 molecules allowed us to identify a region between amino acid 420 and 545 responsible for the DNA binding specificity. This region is located in the previously characterized DNA binding region of STAT proteins. Sequence comparison between STAT5A and STAT5B from different species showed a difference of 5 amino acids in the region 420 -545 between STAT5A and STAT5B. Substitution of these amino acids demonstrated that a glycine residue at position 433 in STAT5B and a glutamic residue at a similar position in STAT5A determined the DNA binding specificity. These data indicate that STAT5A and STAT5B homodimers may have distinct function and probably regulate the expression of common as well as distinct genes.Cytokines activate intracellular signaling pathways during growth and differentiation responses. One of the signal transduction pathways activated by these ligands involves the family of STAT (signal transducer and activator of transcription) proteins. STAT proteins are latent transcription factors that transmit signals from activated receptors to the nucleus (1, 2). All STAT members contain a SH2 domain that allows their selective recruitment to the activated receptor kinase complexes (3-5). After phosphorylation on a single tyrosine residue present in the COOH part of the protein (6, 7), STAT proteins dimerize (homodimerize and/or heterodimerize) through an interaction between the SH2 domain of one STAT and the phosphotyrosine residue of another STAT molecule (8). Dimers of STAT migrate to the nucleus, bind to specific DNA sequences, and activate or repress the transcription of target genes. Functional domains involved in DNA binding and activation of transcription have been defined. The DNA binding region is located in the center of the STAT proteins (from amino acids 350 to 500), and the transactivation domain is present at their COOH-terminal end (9 -13). Isoforms of STAT proteins that lack the COOH-terminal transactivation domain still bind to DNA but do not induce transcriptional activation of responsive genes (14). In addition to tyrosine phosphorylation, STAT proteins are phosphorylated on serine residues. This phosphorylation has been shown to modulate transcriptional activity and the DNA binding of STAT proteins (15-17). The NH 2 -terminal region has been found to be required for the cooperative binding of STAT dimers (18,19). STAT5 also forms tetramers on the IL-2 1 receptor enhancer element that contains two STAT binding sites (20, 21). Similar observations were reported for the cis gene and for the hepatic serine protease inhibitor 2.1 (spi2.1) gene promoter elements (22,23).At pre...

The involvement of the cytokine signaling pathway in oncogenesis has long been postulated. Recently, rearrangements of the gene encoding the tyrosine Janus kinase 2 (JAK2) have been reported in human leukemias indicating a direct JAK-signal transduction and activator of transcription (STAT)-mediated leukemic process. The leukemia-associated TEL-JAK2 fusion protein is formed by the oligomerization domain of the translocated ets leukemia (TEL) protein fused to the catalytic domain of JAK2. TEL-mediated oligomerization results in a constitutive tyrosine kinase activity that, in turn, is able to confer growth factor independence to the murine hematopoietic interleukin-3 (IL-3)-dependent Ba/F3 cell line. Results of the present study indicate that fusion proteins containing the oligomerization domain of TEL and the tyrosine kinase domains of Jak1, Jak2, JAK3, or TYK2 share similar properties and are able to efficiently substitute for the survival and mitogenic signals controlled by IL-3, without concomitant activation of the IL-3 receptor. Electrophoretic mobility shift assays demonstrated Stat5 as the only activated Stat factor in TEL-Jak2- and TEL-Jak1-expressing cells, whereas other Stats, namely Stat1 and Stat3, could be detected in TEL-JAK3-, TEL-TYK2-, and also in TEL-ABL-expressing Ba/F3 cells. High levels of expression of the Stat5-target genes pim-1, osm, and Cis were observed in all the cytokine-independent cell lines. Furthermore, the expression of a dominant negative form of Stat5A markedly interfered with the growth factor independence process mediated by TEL-Jak2 in Ba/F3 cells. Because the BCR-ABL and TEL-PDGFβR oncoproteins also activate Stat5, activation of this factor should be a crucial step in activated tyrosine kinase-mediated leukemogenesis.

Cytokine-dependent activation of distinct signaling pathways is a common scheme thought to be required for the subsequent programmation into cell proliferation and survival. The PI 3-kinase/Akt, Ras/MAP kinase, Ras/ NFIL3 and JAK/STAT pathways have been shown to participate in cytokine mediated suppression of apoptosis in various cell types. However the relative importance of these signaling pathways seems to depend on the cellular context. In several cases, individual inhibition of each pathway is not su cient to completely abrogate cytokine mediated cell survival suggesting that cooperation between these pathways is required. Here we showed that individual inhibition of STAT5, PI 3-kinase or MEK activities did not or weakly a ected the IL-3 dependent survival of the bone marrow derived Ba/F3 cell line. However, the simultaneous inhibition of STAT5 and PI 3-kinase activities but not that of STAT5 and MEK reduced the IL-3 dependent survival of Ba/F3. Analysis of the expression of the Bcl-2 members indicated that phosphorylation of Bad and Bcl-x expression which are respectively regulated by the PI 3-kinase/Akt pathway and STAT5 probably explain this cooperation. Furthermore, we showed by co-immunoprecipitation studies and pull down experiments with fusion proteins encoding the GST-SH2 domains of p85 that STAT5 in its phosphorylated form interacts with the p85 subunit of the PI 3-kinase. These results indicate that the activations of STAT5 and the PI 3-kinase by IL-3 in Ba/F3 cells are tightly connected and cooperate to mediate IL-3-dependent suppression of apoptosis by modulating Bad phosphorylation and Bcl-x expression.

The interaction between the receptor FLT3 (FMS-like tyrosine kinase-3) and its ligand FL leads to crucial signalling during the early stages of the commitment of haematopoietic stem cells. Mutation or over-expression of the FLT3 gene, leading to constitutive signalling, enhances the survival and expansion of a variety of leukaemias and is associated with an unfavourable clinical outcome for acute myeloid leukaemia (AML) patients. In this study, we used a murine cellular model for AML and primary leukaemic cells from AML patients to investigate the molecular mechanisms underlying the regulation of FLT3 gene expression and identify its key cis-and transregulators. By assessing DNA accessibility and epigenetic markings, we defined regulatory domains in the FLT3 promoter and first intron. These elements permit in vivo binding of several AML-related transcription factors, including the proto-oncogene MYB and the CCAAT/enhancer binding protein C/EBPα, which are recruited to the FLT3 promoter and intronic module, respectively. Substantiating their relevance to the human disease, our analysis of gene expression profiling arrays from AML patients uncovered significant correlations between FLT3 expression level and that of MYB and CEBPA. The latter relationship permits discrimination between patients with CEBPA mono-and bi-allelic mutations, and thus connects two major prognostic factors for AML.