Genotoxic aldehyde stress prematurely ages hematopoietic stem cells in a p53-driven manner.

Aged hematopoietic stem cells (HSCs) display diminished self-renewal and a myeloid differentiation bias. However, the drivers and mechanisms that underpin this fundamental switch are not understood. HSCs produce genotoxic formaldehyde that requires protection by the detoxification enzymes ALDH2 and ADH5 and the Fanconi anemia (FA) DNA repair pathway. We find that the HSCs in young Aldh2-/-Fancd2-/- mice harbor a transcriptomic signature equivalent to aged wild-type HSCs, along with increased epigenetic age, telomere attrition, and myeloid-biased differentiation quantified by single HSC transplantation. In addition, the p53 response is vigorously activated in Aldh2-/-Fancd2-/- HSCs, while p53 deletion rescued this aged HSC phenotype. To further define the origins of the myeloid differentiation bias, we use a GFP genetic reporter to find a striking enrichment of Vwf+ myeloid and megakaryocyte-lineage-biased HSCs. These results indicate that metabolism-derived formaldehyde-DNA damage stimulates the p53 response in HSCs to drive accelerated aging.

Interactions between lineage-associated transcription factors govern haematopoietic progenitor states.

Recent advances in molecular profiling provide descriptive datasets of complex differentiation landscapes including the haematopoietic system, but the molecular mechanisms defining progenitor states and lineage choice remain ill-defined. Here, we employed a cellular model of murine multipotent haematopoietic progenitors (Hoxb8-FL) to knock out 39 transcription factors (TFs) followed by RNA-Seq analysis, to functionally define a regulatory network of 16,992 regulator/target gene links. Focussed analysis of the subnetworks regulated by the B-lymphoid TF Ebf1 and T-lymphoid TF Gata3 revealed a surprising role in common activation of an early myeloid programme. Moreover, Gata3-mediated repression of Pax5 emerges as a mechanism to prevent precocious B-lymphoid differentiation, while Hox-mediated activation of Meis1 suppresses myeloid differentiation. To aid interpretation of large transcriptomics datasets, we also report a new method that visualises likely transitions that a progenitor will undergo following regulatory network perturbations. Taken together, this study reveals how molecular network wiring helps to establish a multipotent progenitor state, with experimental approaches and analysis tools applicable to dissecting a broad range of both normal and perturbed cellular differentiation landscapes.

Notch ligand Dll4 impairs cell recruitment to aortic clusters and limits blood stem cell generation.

Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium in cluster structures that protrude into the embryonic aortic lumen. Although much is known about the molecular characteristics of the developing hematopoietic cells, we lack a complete understanding of their origin and the three-dimensional organization of the niche. Here, we use advanced live imaging techniques of organotypic slice cultures, clonal analysis, and mathematical modeling to show the two-step process of intra-aortic hematopoietic cluster (IACH) formation. First, a hemogenic progenitor buds up from the endothelium and undergoes division forming the monoclonal core of the IAHC. Next, surrounding hemogenic cells are recruited into the IAHC, increasing their size and heterogeneity. We identified the Notch ligand Dll4 as a negative regulator of the recruitment phase of IAHC. Blocking of Dll4 promotes the entrance of new hemogenic Gfi1+ cells into the IAHC and increases the number of cells that acquire HSC activity. Mathematical modeling based on our data provides estimation of the cluster lifetime and the average recruitment time of hemogenic cells to the cluster under physiologic and Dll4-inhibited conditions.

STAT1 is essential for HSC function and maintains MHCIIhi stem cells that resist myeloablation and neoplastic expansion.

Adult hematopoietic stem cells (HSCs) are predominantly quiescent and can be activated in response to acute stress such as infection or cytotoxic insults. STAT1 is a pivotal downstream mediator of interferon (IFN) signaling and is required for IFN-induced HSC proliferation, but little is known about the role of STAT1 in regulating homeostatic hematopoietic stem/progenitor cells (HSPCs). Here, we show that loss of STAT1 altered the steady state HSPC landscape, impaired HSC function in transplantation assays, delayed blood cell regeneration following myeloablation, and disrupted molecular programs that protect HSCs, including control of quiescence. Our results also reveal STAT1-dependent functional HSC heterogeneity. A previously unrecognized subset of homeostatic HSCs with elevated major histocompatibility complex class II (MHCII) expression (MHCIIhi) displayed molecular features of reduced cycling and apoptosis and was refractory to 5-fluorouracil-induced myeloablation. Conversely, MHCIIlo HSCs displayed increased megakaryocytic potential and were preferentially expanded in CALR mutant mice with thrombocytosis. Similar to mice, high MHCII expression is a feature of human HSCs residing in a deeper quiescent state. Our results therefore position STAT1 at the interface of stem cell heterogeneity and the interplay between stem cells and the adaptive immune system, areas of broad interest in the wider stem cell field.

Reconstructing blood stem cell regulatory network models from single-cell molecular profiles.

Adult blood contains a mixture of mature cell types, each with specialized functions. Single hematopoietic stem cells (HSCs) have been functionally shown to generate all mature cell types for the lifetime of the organism. Differentiation of HSCs toward alternative lineages must be balanced at the population level by the fate decisions made by individual cells. Transcription factors play a key role in regulating these decisions and operate within organized regulatory programs that can be modeled as transcriptional regulatory networks. As dysregulation of single HSC fate decisions is linked to fatal malignancies such as leukemia, it is important to understand how these decisions are controlled on a cell-by-cell basis. Here we developed and applied a network inference method, exploiting the ability to infer dynamic information from single-cell snapshot expression data based on expression profiles of 48 genes in 2,167 blood stem and progenitor cells. This approach allowed us to infer transcriptional regulatory network models that recapitulated differentiation of HSCs into progenitor cell types, focusing on trajectories toward megakaryocyte-erythrocyte progenitors and lymphoid-primed multipotent progenitors. By comparing these two models, we identified and subsequently experimentally validated a difference in the regulation of nuclear factor, erythroid 2 (Nfe2) and core-binding factor, runt domain, alpha subunit 2, translocated to, 3 homolog (Cbfa2t3h) by the transcription factor Gata2. Our approach confirms known aspects of hematopoiesis, provides hypotheses about regulation of HSC differentiation, and is widely applicable to other hierarchical biological systems to uncover regulatory relationships.

Integrated genome-scale analysis of the transcriptional regulatory landscape in a blood stem/progenitor cell model.

Comprehensive study of transcriptional control processes will be required to enhance our understanding of both normal and malignant hematopoiesis. Modern sequencing technologies have revolutionized our ability to generate genome-scale expression and histone modification profiles, transcription factor (TF)-binding maps, and also comprehensive chromatin-looping information. Many of these technologies, however, require large numbers of cells, and therefore cannot be applied to rare hematopoietic stem/progenitor cell (HSPC) populations. The stem cell factor-dependent multipotent progenitor cell line HPC-7 represents a well-recognized cell line model for HSPCs. Here we report genome-wide maps for 17 TFs, 3 histone modifications, DNase I hypersensitive sites, and high-resolution promoter-enhancer interactomes in HPC-7 cells. Integrated analysis of these complementary data sets revealed TF occupancy patterns of genomic regions involved in promoter-anchored loops. Moreover, preferential associations between pairs of TFs bound at either ends of chromatin loops led to the identification of 4 previously unrecognized protein-protein interactions between key blood stem cell regulators. All HPC-7 data sets are freely available both through standard repositories and a user-friendly Web interface. Together with previously generated genome-wide data sets, this study integrates HPC-7 data into a genomic resource on par with ENCODE tier 1 cell lines and, importantly, is the only current model with comprehensive genome-scale data that is relevant to HSPC biology.

ConBind: motif-aware cross-species alignment for the identification of functional transcription factor binding sites.

Eukaryotic gene expression is regulated by transcription factors (TFs) binding to promoter as well as distal enhancers. TFs recognize short, but specific binding sites (TFBSs) that are located within the promoter and enhancer regions. Functionally relevant TFBSs are often highly conserved during evolution leaving a strong phylogenetic signal. While multiple sequence alignment (MSA) is a potent tool to detect the phylogenetic signal, the current MSA implementations are optimized to align the maximum number of identical nucleotides. This approach might result in the omission of conserved motifs that contain interchangeable nucleotides such as the ETS motif (IUPAC code: GGAW). Here, we introduce ConBind, a novel method to enhance alignment of short motifs, even if their mutual sequence similarity is only partial. ConBind improves the identification of conserved TFBSs by improving the alignment accuracy of TFBS families within orthologous DNA sequences. Functional validation of the Gfi1b + 13 enhancer reveals that ConBind identifies additional functionally important ETS binding sites that were missed by all other tested alignment tools. In addition to the analysis of known regulatory regions, our web tool is useful for the analysis of TFBSs on so far unknown DNA regions identified through ChIP-sequencing.

Constrained transcription factor spacing is prevalent and important for transcriptional control of mouse blood cells.

Combinatorial transcription factor (TF) binding is essential for cell-type-specific gene regulation. However, much remains to be learned about the mechanisms of TF interactions, including to what extent constrained spacing and orientation of interacting TFs are critical for regulatory element activity. To examine the relative prevalence of the 'enhanceosome' versus the 'TF collective' model of combinatorial TF binding, a comprehensive analysis of TF binding site sequences in large scale datasets is necessary. We developed a motif-pair discovery pipeline to identify motif co-occurrences with preferential distance(s) between motifs in TF-bound regions. Utilizing a compendium of 289 mouse haematopoietic TF ChIP-seq datasets, we demonstrate that haematopoietic-related motif-pairs commonly occur with highly conserved constrained spacing and orientation between motifs. Furthermore, motif clustering revealed specific associations for both heterotypic and homotypic motif-pairs with particular haematopoietic cell types. We also showed that disrupting the spacing between motif-pairs significantly affects transcriptional activity in a well-known motif-pair-E-box and GATA, and in two previously unknown motif-pairs with constrained spacing-Ets and Homeobox as well as Ets and E-box. In this study, we provide evidence for widespread sequence-specific TF pair interaction with DNA that conforms to the 'enhanceosome' model, and furthermore identify associations between specific haematopoietic cell-types and motif-pairs.

Genome-wide analysis of transcriptional regulators in human HSPCs reveals a densely interconnected network of coding and noncoding genes.

Genome-wide combinatorial binding patterns for key transcription factors (TFs) have not been reported for primary human hematopoietic stem and progenitor cells (HSPCs), and have constrained analysis of the global architecture of molecular circuits controlling these cells. Here we provide high-resolution genome-wide binding maps for a heptad of key TFs (FLI1, ERG, GATA2, RUNX1, SCL, LYL1, and LMO2) in human CD34(+) HSPCs, together with quantitative RNA and microRNA expression profiles. We catalog binding of TFs at coding genes and microRNA promoters, and report that combinatorial binding of all 7 TFs is favored and associated with differential expression of genes and microRNA in HSPCs. We also uncover a previously unrecognized association between FLI1 and RUNX1 pairing in HSPCs, we establish a correlation between the density of histone modifications that mark active enhancers and the number of overlapping TFs at a peak, we demonstrate bivalent histone marks at promoters of heptad target genes in CD34(+) cells that are poised for later expression, and we identify complex relationships between specific microRNAs and coding genes regulated by the heptad. Taken together, these data reveal the power of integrating multifactor sequencing of chromatin immunoprecipitates with coding and noncoding gene expression to identify regulatory circuits controlling cell identity.

Genome-scale expression and transcription factor binding profiles reveal therapeutic targets in transgenic ERG myeloid leukemia.

The ETS transcription factor ERG plays a central role in definitive hematopoiesis, and its overexpression in acute myeloid leukemia (AML) is associated with a stem cell signature and poor prognosis. Yet how ERG causes leukemia is unclear. Here we show that pan-hematopoietic ERG expression induces an early progenitor myeloid leukemia in transgenic mice. Integrated genome-scale analysis of gene expression and ERG binding profiles revealed that ERG activates a transcriptional program similar to human AML stem/progenitor cells and to human AML with high ERG expression. This transcriptional program was associated with activation of RAS that was required for leukemia cells growth in vitro and in vivo. We further show that ERG induces expression of the Pim1 kinase oncogene through a novel hematopoietic enhancer validated in transgenic mice and human CD34(+) normal and leukemic cells. Pim1 inhibition disrupts growth and induces apoptosis of ERG-expressing leukemic cells. The importance of the ERG/PIM1 axis is further underscored by the poorer prognosis of AML highly expressing ERG and PIM1. Thus, integrative genomic analysis demonstrates that ERG causes myeloid progenitor leukemia characterized by an induction of leukemia stem cell transcriptional programs. Pim1 and the RAS pathway are potential therapeutic targets of these high-risk leukemias.

Hard-wired heterogeneity in blood stem cells revealed using a dynamic regulatory network model.

MOTIVATION: Combinatorial interactions of transcription factors with cis-regulatory elements control the dynamic progression through successive cellular states and thus underpin all metazoan development. The construction of network models of cis-regulatory elements, therefore, has the potential to generate fundamental insights into cellular fate and differentiation. Haematopoiesis has long served as a model system to study mammalian differentiation, yet modelling based on experimentally informed cis-regulatory interactions has so far been restricted to pairs of interacting factors. Here, we have generated a Boolean network model based on detailed cis-regulatory functional data connecting 11 haematopoietic stem/progenitor cell (HSPC) regulator genes. RESULTS: Despite its apparent simplicity, the model exhibits surprisingly complex behaviour that we charted using strongly connected components and shortest-path analysis in its Boolean state space. This analysis of our model predicts that HSPCs display heterogeneous expression patterns and possess many intermediate states that can act as 'stepping stones' for the HSPC to achieve a final differentiated state. Importantly, an external perturbation or 'trigger' is required to exit the stem cell state, with distinct triggers characterizing maturation into the various different lineages. By focusing on intermediate states occurring during erythrocyte differentiation, from our model we predicted a novel negative regulation of Fli1 by Gata1, which we confirmed experimentally thus validating our model. In conclusion, we demonstrate that an advanced mammalian regulatory network model based on experimentally validated cis-regulatory interactions has allowed us to make novel, experimentally testable hypotheses about transcriptional mechanisms that control differentiation of mammalian stem cells. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

DNA methylation restricts coordinated germline and neural fates in embryonic stem cell differentiation.

As embryonic stem cells (ESCs) transition from naive to primed pluripotency during early mammalian development, they acquire high DNA methylation levels. During this transition, the germline is specified and undergoes genome-wide DNA demethylation, while emergence of the three somatic germ layers is preceded by acquisition of somatic DNA methylation levels in the primed epiblast. DNA methylation is essential for embryogenesis, but the point at which it becomes critical during differentiation and whether all lineages equally depend on it is unclear. Here, using culture modeling of cellular transitions, we found that DNA methylation-free mouse ESCs with triple DNA methyltransferase knockout (TKO) progressed through the continuum of pluripotency states but demonstrated skewed differentiation abilities toward neural versus other somatic lineages. More saliently, TKO ESCs were fully competent for establishing primordial germ cell-like cells, even showing temporally extended and self-sustained capacity for the germline fate. By mapping chromatin states, we found that neural and germline lineages are linked by a similar enhancer dynamic upon exit from the naive state, defined by common sets of transcription factors, including methyl-sensitive ones, that fail to be decommissioned in the absence of DNA methylation. We propose that DNA methylation controls the temporality of a coordinated neural-germline axis of the preferred differentiation route during early development.

A time- and single-cell-resolved model of murine bone marrow hematopoiesis.

The paradigmatic hematopoietic tree model is increasingly recognized to be limited, as it is based on heterogeneous populations largely defined by non-homeostatic assays testing cell fate potentials. Here, we combine persistent labeling with time-series single-cell RNA sequencing to build a real-time, quantitative model of in vivo tissue dynamics for murine bone marrow hematopoiesis. We couple cascading single-cell expression patterns with dynamic changes in differentiation and growth speeds. The resulting explicit linkage between molecular states and cellular behavior reveals widely varying self-renewal and differentiation properties across distinct lineages. Transplanted stem cells show strong acceleration of differentiation at specific stages of erythroid and neutrophil production, illustrating how the model can quantify the impact of perturbations. Our reconstruction of dynamic behavior from snapshot measurements is akin to how a kinetoscope allows sequential images to merge into a movie. We posit that this approach is generally applicable to understanding tissue-scale dynamics at high resolution.

The transcriptional programme controlled by Runx1 during early embryonic blood development.

Transcription factors have long been recognised as powerful regulators of mammalian development yet it is largely unknown how individual key regulators operate within wider regulatory networks. Here we have used a combination of global gene expression and chromatin-immunoprecipitation approaches during the early stages of haematopoietic development to define the transcriptional programme controlled by Runx1, an essential regulator of blood cell specification. Integrated analysis of these complementary genome-wide datasets allowed us to construct a global regulatory network model, which suggested that key regulators are activated sequentially during blood specification, but will ultimately collaborate to control many haematopoietically expressed genes. Using the CD41/integrin alpha 2b gene as a model, cellular and in vivo studies showed that CD41 is controlled by both Scl/Tal1 and Runx1 in fully specified blood cells, and initiation of CD41 expression in E7.5 embryos is severely compromised in the absence of Runx1. Taken together, this study represents the first global analysis of the transcriptional programme controlled by any key haematopoietic regulator during the process of early blood cell specification. Moreover, the concept of interplay between sequentially deployed core regulators is likely to represent a design principle widely applicable to the transcriptional control of mammalian development.

ERG promotes T-acute lymphoblastic leukemia and is transcriptionally regulated in leukemic cells by a stem cell enhancer.

The Ets-related gene (ERG) is an Ets-transcription factor required for normal blood stem cell development. ERG expression is down-regulated during early T-lymphopoiesis but maintained in T-acute lymphoblastic leukemia (T-ALL), where it is recognized as an independent risk factor for adverse outcome. However, it is unclear whether ERG is directly involved in the pathogenesis of T-ALL and how its expression is regulated. Here we demonstrate that transgenic expression of ERG causes T-ALL in mice and that its knockdown reduces the proliferation of human MOLT4 T-ALL cells. We further demonstrate that ERG expression in primary human T-ALL cells is mediated by the binding of other T-cell oncogenes SCL/TAL1, LMO2, and LYL1 in concert with ERG, FLI1, and GATA3 to the ERG +85 enhancer. This enhancer is not active in normal T cells but in transgenic mice targets expression to fetal liver c-kit(+) cells, adult bone marrow stem/progenitors and early CD4(-)CD8(-) double-negative thymic progenitors. Taken together, these data illustrate that ERG promotes T-ALL and that failure to extinguish activity of stem cell enhancers associated with regulatory transcription factors such as ERG can contribute to the development of leukemia.

Yolk sac cell atlas reveals multiorgan functions during human early development.

The extraembryonic yolk sac (YS) ensures delivery of nutritional support and oxygen to the developing embryo but remains ill-defined in humans. We therefore assembled a comprehensive multiomic reference of the human YS from 3 to 8 postconception weeks by integrating single-cell protein and gene expression data. Beyond its recognized role as a site of hematopoiesis, we highlight roles in metabolism, coagulation, vascular development, and hematopoietic regulation. We reconstructed the emergence and decline of YS hematopoietic stem and progenitor cells from hemogenic endothelium and revealed a YS-specific accelerated route to macrophage production that seeds developing organs. The multiorgan functions of the YS are superseded as intraembryonic organs develop, effecting a multifaceted relay of vital functions as pregnancy proceeds.

Transcriptional regulation of Elf-1: locus-wide analysis reveals four distinct promoters, a tissue-specific enhancer, control by PU.1 and the importance of Elf-1 downregulation for erythroid maturation.

Ets transcription factors play important roles during the development and maintenance of the haematopoietic system. One such factor, Elf-1 (E74-like factor 1) controls the expression of multiple essential haematopoietic regulators including Scl/Tal1, Lmo2 and PU.1. However, to integrate Elf-1 into the wider regulatory hierarchies controlling haematopoietic development and differentiation, regulatory elements as well as upstream regulators of Elf-1 need to be identified. Here, we have used locus-wide comparative genomic analysis coupled with chromatin immunoprecipitation (ChIP-chip) assays which resulted in the identification of five distinct regulatory regions directing expression of Elf-1. Further, ChIP-chip assays followed by functional validation demonstrated that the key haematopoietic transcription factor PU.1 is a major upstream regulator of Elf-1. Finally, overexpression studies in a well-characterized erythroid differentiation assay from primary murine fetal liver cells demonstrated that Elf-1 downregulation is necessary for terminal erythroid differentiation. Given the known activation of PU.1 by Elf-1 and our newly identified reciprocal activation of Elf-1 by PU.1, identification of an inhibitory role for Elf-1 has significant implications for our understanding of how PU.1 controls myeloid-erythroid differentiation. Our findings therefore not only represent the first report of Elf-1 regulation but also enhance our understanding of the wider regulatory networks that control haematopoiesis.

Relaxin/insulin-like family peptide receptor 4 (Rxfp4) expressing hypothalamic neurons modulate food intake and preference in mice.

OBJECTIVE: Insulin-like peptide 5 (INSL5) signalling, through its cognate receptor relaxin/insulin-like family peptide receptor 4 (RXFP4), has been reported to be orexigenic, and the high fat diet (HFD) preference observed in wildtype mice is altered in Rxfp4 knock-out mice. In this study, we used a new Rxfp4-Cre mouse model to investigate the mechanisms underlying these observations. METHODS: We generated transgenic Rxfp4-Cre mice and investigated central expression of Rxfp4 by RT-qPCR, RNAscope and intraparenchymal infusion of INSL5. Rxfp4-expressing cells were chemogenetically manipulated in global Cre-reporter mice using designer receptors exclusively activated by designer drugs (DREADDs) or after stereotactic injection of a Cre-dependent AAV-DIO-Dq-DREADD targeting a population located in the ventromedial hypothalamus (RXFP4VMH). Food intake and feeding motivation were assessed in the presence and absence of a DREADD agonist. Rxfp4-expressing cells in the hypothalamus were characterised by single-cell RNA-sequencing (scRNAseq) and the connectivity of RXFP4VMH cells was investigated using viral tracing. RESULTS: Rxfp4-Cre mice displayed Cre-reporter expression in the hypothalamus. Active expression of Rxfp4 in the adult mouse brain was confirmed by RT-qPCR and RNAscope. Functional receptor expression was supported by cyclic AMP-responses to INSL5 application in ex vivo brain slices and increased HFD and highly palatable liquid meal (HPM), but not chow, intake after intra-VMH INSL5 infusion. scRNAseq of hypothalamic RXFP4 neurons defined a cluster expressing VMH markers, alongside known appetite-modulating neuropeptide receptors (Mc4r, Cckar and Nmur2). Viral tracing demonstrated RXFP4VMH neural projections to nuclei implicated in hedonic feeding behaviour. Whole body chemogenetic inhibition (Di-DREADD) of Rxfp4-expressing cells, mimicking physiological INSL5-RXFP4 Gi-signalling, increased intake of the HFD and HPM, but not chow, whilst activation (Dq-DREADD), either at whole body level or specifically within the VMH, reduced HFD and HPM intake and motivation to work for the HPM. CONCLUSION: These findings identify RXFP4VMH neurons as regulators of food intake and preference, and hypothalamic RXFP4 signalling as a target for feeding behaviour manipulation.

Expression of the leukemia oncogene Lmo2 is controlled by an array of tissue-specific elements dispersed over 100 kb and bound by Tal1/Lmo2, Ets, and Gata factors.

The Lmo2 gene encodes a transcriptional cofactor critical for the development of hematopoietic stem cells. Ectopic LMO2 expression causes leukemia in T-cell acute lymphoblastic leukemia (T-ALL) patients and severe combined immunodeficiency patients undergoing retroviral gene therapy. Tightly controlled Lmo2 expression is therefore essential, yet no comprehensive analysis of Lmo2 regulation has been published so far. By comparative genomics, we identified 17 highly conserved noncoding elements, 9 of which revealed specific acetylation marks in chromatin-immunoprecipitation and microarray (ChIP-chip) assays performed across 250 kb of the Lmo2 locus in 11 cell types covering different stages of hematopoietic differentiation. All candidate regulatory regions were tested in transgenic mice. An extended LMO2 proximal promoter fragment displayed strong endothelial activity, while the distal promoter showed weak forebrain activity. Eight of the 15 distal candidate elements functioned as enhancers, which together recapitulated the full expression pattern of Lmo2, directing expression to endothelium, hematopoietic cells, tail, and forebrain. Interestingly, distinct combinations of specific distal regulatory elements were required to extend endothelial activity of the LMO2 promoter to yolk sac or fetal liver hematopoietic cells. Finally, Sfpi1/Pu.1, Fli1, Gata2, Tal1/Scl, and Lmo2 were shown to bind to and transactivate Lmo2 hematopoietic enhancers, thus identifying key upstream regulators and positioning Lmo2 within hematopoietic regulatory networks.

The transcriptional program controlled by the stem cell leukemia gene Scl/Tal1 during early embryonic hematopoietic development.

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 liver cells and comprehensive transgenic mouse assays. Transgenic analysis provided in vivo validation of multiple new direct Scl target genes and allowed us to reconstruct an in vivo validated network consisting of 17 factors and their respective regulatory elements. By coupling ChIP-Seq in model cell lines with in vivo transgenic validation and sophisticated bioinformatic analysis, we have identified a widely applicable strategy for the reconstruction of stem cell regulatory networks in which biologic material is otherwise limiting. Moreover, in addition to revealing multiple previously unrecognized links to known HSC regulators, as well as novel links to genes not previously implicated in HSC function, comprehensive transgenic analysis of regulatory elements provided substantial new insights into the transcriptional control of several important hematopoietic regulators, including Cbfa2t3h/Eto2, Cebpe, Nfe2, Zfpm1/Fog1, Erg, Mafk, Gfi1b, and Myb.