To test if loss of affected chromatin structure we performed replicate ChIP-seq analysis for the presence of two important histone marks, H3K4me3 and H3K27me3 using were marginally downregulated (1.4-fold in all three probesets) in leads to a 2-fold reduction of immunophenotypic HSCs and that deficient HSCs were severely compromised. features are regulated in part by transcription factors, which take action by controlling the expression of genes important for the functional properties of HSCs. C/EBP is usually a well-known inducer of myeloid differentiation. It is lowly expressed in HSCs ST 2825 and its potential function in these cells has been extensively debated. Here, we demonstrate that deletion impacts on HSC self-renewal, differentiation, quiescence and survival. Through gene expression and ChIP-seq analyses of stem and progenitor cell-enriched cell populations, we further show that C/EBP binds to regulatory regions of genes that are induced during granulocytic differentiation, suggesting that C/EBP functions to primary HSCs for differentiation along the myeloid lineage. Finally, we demonstrate that C/EBP loss prospects to epigenetic changes at genes central to HSC biology, which implies that it may take action to recruit chromatin writers/erasers through mechanisms that remain to be characterized. In conclusion, our work identifies C/EBP as a central hub for HSC function and highlights how a single transcription factor may coordinate several HSC fate options. Introduction Hematopoietic stem cells (HSCs) are responsible for the maintenance of a constant production of blood cells throughout life. To achieve this, HSCs have to tightly regulate their different fate options including self-renewal, proliferation, differentiation and apoptosis, as alterations in any of these may lead to HSC exhaustion, expansion or leukemia [1]. HSC fate options are controlled by a number of different pathways and are influenced both by the microenvironment and by the actions of cell-autonomous regulators such as transcription factors (TFs) and chromatin-interacting proteins [2]. Given their impact on gene expression, the influence of TFs on HSC properties has been the focus of several studies. Indeed, factors such as C-MYB, ERG, and PU.1 are all essential for preserving HSC self-renewal and their deletion have dramatic impact on hematopoietic Rabbit Polyclonal to POLE4 maintenance both during fetal and adult life [3], [4], [5], [6]. Other factors, as exemplified by SOX17, are required exclusively for the maintenance of fetal HSCs, whereas GFI-1 and ETV6 only appear to play a role in an adult setting [7], [8], [9]. TF function is usually interpreted in a chromatin context and, accordingly, chromatin readers and writers have been shown to be important for HSC function and maintenance. Examples include the PRC1 component BMI-1 [10], [11], the maintenance DNA methyltransferase DNMT1 [12], [13] as well as the H3K4 methyltransferase MLL1 [14]. Despite the importance of both TFs and chromatin context for HSC function, our knowledge on how TF binding is usually interpreted within an epigenetic landscape, and how they may influence epigenetic configurations remains limited. Importantly, given their inherent ST 2825 developmental plasticity, stem cells have been reported to exhibit unique epigenetic signatures of which the so-called bivalent configuration is the best characterized. Work in ES cells has shown that bivalently marked genes are lowly expressed, enriched in genes involved in development/differentiation, and display active (H3K4me3) as well as repressive (H3K27me3) histone marks [15], [16]. As stem cells progress along the path of differentiation the bivalent configuration is resolved into an active or repressed state with a concomitant upregulation or downregulation, respectively, of the expression of previously marked genes [15], [16]. To what extent the bivalent signature is influenced by loss of TFs in HSCs has not been characterized. C/EBP is an important myeloid TF that functions not only by binding to regulatory DNA elements and directing transcription, but also through its ability to constrain proliferation by inhibiting the transcriptional activity of E2F-complexes [17], [18], [19], [20]. In the hematopoietic system loss of C/EBP prospects to a differentiation block upstream of the Granulocytic Monocytic Progenitor (GMP) ST 2825 accompanied by an accumulation of earlier stem and myeloid progenitor populations [17], [21]. In acute myeloid leukemia (AML), is found mutated in approximately 10% of cases, and studies in mouse have shown that this tumor-suppressive functions of C/EBP can be ascribed to its ability to balance the proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs) populations [18], [22]. Indeed, HSCs from mice harboring tumor-prone variants of C/EBP displayed altered cell cycle kinetics, but how this impacts on HSC function was hard to assess due to the leukemic transformation in these animals. Furthermore, complete loss of C/EBP has been reported to endow fetal HSC-enriched populations with a minor competitive advantage in a transplantation setting [21] and C/EBP has recently.